@article {pmid35748558,
year = {2022},
author = {Mohammad-Rafiei, F and Safdarian, E and Adel, B and Vandchali, NR and Navashenaq, JG and Gheibihayat, SM},
title = {CRISPR: A Promising Tool for Cancer Therapy.},
journal = {Current molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566524022666220624111311},
pmid = {35748558},
issn = {1875-5666},
abstract = {The clustered regularly interspaced short palindromic repeats system, called CRISPR, as one of the major technological advances, allows geneticists and researchers to perform genome editing. This remarkable technology is quickly eclipsing zinc-finger nucleases (ZFNs) and other editing tools, and its ease of use and accuracy have thus far revolutionized genome editing, from fundamental science projects to medical research and treatment options. This system consists of two key components: a CRISPR-associated (Cas) nuclease, which binds and cuts deoxyribonucleic acid (DNA) and a guide ribonucleic acid (gRNA) sequence, directing the Cas nuclease to its target site. In the research arena, CRISPR has been up to now exploited in various ways alongside gene editing, such as epigenome modifications, genome-wide screening, targeted cancer therapies, and so on. This article reviews the current perceptions of the CRISPR/Cas systems with special attention to studies reflecting on the relationship between the CRISPR/Cas systems and their role in cancer therapy.},
}
@article {pmid35743007,
year = {2022},
author = {Thomson, MJ and Biswas, S and Tsakirpaloglou, N and Septiningsih, EM},
title = {Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
doi = {10.3390/ijms23126565},
pmid = {35743007},
issn = {1422-0067},
support = {2017-67013-26194; 2022-67013-36210; 2020-67013-31811//United States Department of Agriculture/ ; NA//Texas A&M AgriLife Research/ ; NA//Texas A&M University X-Grant/ ; },
abstract = {Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.},
}
@article {pmid35741761,
year = {2022},
author = {Wang, HQ and Wang, T and Gao, F and Ren, WZ},
title = {Application of CRISPR/Cas Technology in Spermatogenesis Research and Male Infertility Treatment.},
journal = {Genes},
volume = {13},
number = {6},
pages = {},
doi = {10.3390/genes13061000},
pmid = {35741761},
issn = {2073-4425},
support = {31972570//National Natural Science Foundation of China/ ; },
abstract = {As the basis of animal reproductive activity, normal spermatogenesis directly determines the efficiency of livestock production. An in-depth understanding of spermatogenesis will greatly facilitate animal breeding efforts and male infertility treatment. With the continuous development and application of gene editing technologies, they have become valuable tools to study the mechanism of spermatogenesis. Gene editing technologies have provided us with a better understanding of the functions and potential mechanisms of action of factors that regulate spermatogenesis. This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis-related genes and disease treatment. The problems of gene editing technologies in the field of spermatogenesis research are also discussed.},
}
@article {pmid35741243,
year = {2022},
author = {Hernandez-Garcia, A and Morales-Moreno, MD and Valdés-Galindo, EG and Jimenez-Nieto, EP and Quezada, A},
title = {Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
doi = {10.3390/diagnostics12061434},
pmid = {35741243},
issn = {2075-4418},
support = {proyectos de investigación y desarrollo para hacer frente a la COVID-19//AMEXCID/ ; IV200820//DGAPA-PAPIIT/ ; },
abstract = {The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.},
}
@article {pmid35741144,
year = {2022},
author = {Selvam, K and Ahmad Najib, M and Khalid, MF and Ozsoz, M and Aziah, I},
title = {CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
doi = {10.3390/diagnostics12061335},
pmid = {35741144},
issn = {2075-4418},
support = {311/CIPPM/4401005//Higher Institution Centre of Excellence (HICoE), Ministry of Higher Education, Malaysia/ ; },
abstract = {Recently, CRISPR-Cas system-based assays for bacterial detection have been developed. The aim of this scoping review is to map existing evidence on the utilization of CRISPR-Cas systems in the development of bacterial detection assays. A literature search was conducted using three databases (PubMed, Scopus, and Cochrane Library) and manual searches through the references of identified full texts based on a PROSPERO-registered protocol (CRD42021289140). Studies on bacterial detection using CRISPR-Cas systems that were published before October 2021 were retrieved. The Critical Appraisal Skills Programme (CASP) qualitative checklist was used to assess the risk of bias for all the included studies. Of the 420 studies identified throughout the search, 46 studies that met the inclusion criteria were included in the final analysis. Bacteria from 17 genera were identified utilising CRISPR-Cas systems. Most of the bacteria came from genera such as Staphylococcus, Escherichia, Salmonella, Listeria, Mycobacterium and Streptococcus. Cas12a (64%) is the most often used Cas enzyme in bacterial detection, followed by Cas13a (13%), and Cas9 (11%). To improve the signal of detection, 83% of the research exploited Cas enzymes' trans-cleavage capabilities to cut tagged reporter probes non-specifically. Most studies used the extraction procedure, whereas only 17% did not. In terms of amplification methods, isothermal reactions were employed in 66% of the studies, followed by PCR (23%). Fluorescence detection (67%) was discovered to be the most commonly used method, while lateral flow biosensors (13%), electrochemical biosensors (11%), and others (9%) were found to be less commonly used. Most of the studies (39) used specific bacterial nucleic acid sequences as a target, while seven used non-nucleic acid targets, including aptamers and antibodies particular to the bacteria under investigation. The turnaround time of the 46 studies was 30 min to 4 h. The limit of detection (LoD) was evaluated in three types of concentration, which include copies per mL, CFU per mL and molarity. Most of the studies used spiked samples (78%) rather than clinical samples (22%) to determine LoD. This review identified the gap in clinical accuracy evaluation of the CRISPR-Cas system in bacterial detection. More research is needed to assess the diagnostic sensitivity and specificity of amplification-free CRISPR-Cas systems in bacterial detection for nucleic acid-based tests.},
}
@article {pmid35739111,
year = {2022},
author = {Lainšček, D and Forstnerič, V and Mikolič, V and Malenšek, Š and Pečan, P and Benčina, M and Sever, M and Podgornik, H and Jerala, R},
title = {Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3604},
pmid = {35739111},
issn = {2041-1723},
support = {P4-0176//Javna Agencija za Raziskovalno Dejavnost RS (Slovenian Research Agency)/ ; },
abstract = {The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.},
}
@article {pmid35551512,
year = {2022},
author = {Lei, Z and Meng, H and Liu, L and Zhao, H and Rao, X and Yan, Y and Wu, H and Liu, M and He, A and Yi, C},
title = {Mitochondrial base editor induces substantial nuclear off-target mutations.},
journal = {Nature},
volume = {606},
number = {7915},
pages = {804-811},
pmid = {35551512},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Cytosine/metabolism ; DNA, Mitochondrial/genetics ; *Gene Editing/methods ; Mitochondria/genetics/metabolism ; Mutation ; },
abstract = {DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.},
}
@article {pmid35736067,
year = {2022},
author = {Devanna, BN and Jain, P and Solanke, AU and Das, A and Thakur, S and Singh, PK and Kumari, M and Dubey, H and Jaswal, R and Pawar, D and Kapoor, R and Singh, J and Arora, K and Saklani, BK and AnilKumar, C and Maganti, SM and Sonah, H and Deshmukh, R and Rathour, R and Sharma, TR},
title = {Understanding the Dynamics of Blast Resistance in Rice-Magnaporthe oryzae Interactions.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {8},
number = {6},
pages = {},
doi = {10.3390/jof8060584},
pmid = {35736067},
issn = {2309-608X},
support = {NA//Department of Science and Technology/ ; },
abstract = {Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional security. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice-M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally, we review the demonstrated examples and potential applications of the latest genome-editing tools in understanding and managing blast disease in rice.},
}
@article {pmid35735623,
year = {2022},
author = {Li, X and Xu, S and Fuhrmann-Aoyagi, MB and Yuan, S and Iwama, T and Kobayashi, M and Miura, K},
title = {CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.},
journal = {Current issues in molecular biology},
volume = {44},
number = {6},
pages = {2664-2682},
doi = {10.3390/cimb44060182},
pmid = {35735623},
issn = {1467-3045},
support = {JPMJOP1851//Japan Science and Technology Agency/ ; },
abstract = {Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.},
}
@article {pmid35732990,
year = {2022},
author = {Kobelt, D and Pahle, J and Walther, W},
title = {A Brief Introduction to Current Cancer Gene Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2521},
number = {},
pages = {1-21},
pmid = {35732990},
issn = {1940-6029},
abstract = {Gene therapy has started in the late 1980s as novel, clinically applicable therapeutic option. It revolutionized the treatment of genetic diseases with the initial intent to repair or replace defective genes. Gene therapy has been adapted for treatment of malignant diseases to improve the outcome of cancer patients. In fact, cancer gene therapy has rapidly gained great interest and evolved into a research field with highest proportion of research activities in gene therapy. In this context, cancer gene therapy has long entered translation into clinical trials and therefore more than two-thirds of all gene therapy trials worldwide are aiming at the treatment of cancer disease using different therapeutic strategies. During the decades in cancer gene therapy, tremendous knowledge has accumulated. This led to significant improvements in vector design, transgene repertoire, more targeted interventions, use of novel gene therapeutic technologies such as CRISPR/Cas, sleeping beauty vectors, and development of effective cancer immunogene therapies. In this chapter, a brief overview of current key developments in cancer gene therapy is provided to gain insights into the recent directions in research as well as in clinical application of cancer gene therapy.},
}
@article {pmid35727982,
year = {2022},
author = {Hu, M and Qiu, Z and Bi, Z and Tian, T and Jiang, Y and Zhou, X},
title = {Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {26},
pages = {e2202034119},
doi = {10.1073/pnas.2202034119},
pmid = {35727982},
issn = {1091-6490},
support = {32150019//National Natural Science Foundation of China (NSFC)/ ; 91959128//National Natural Science Foundation of China (NSFC)/ ; 21874049//National Natural Science Foundation of China (NSFC)/ ; 2020BCA090//The Key Research and Development Plan of Hubei Province/ ; 2021A1515220164//GDSTC | Basic and Applied Basic Research Foundation of Guangdong Province ()/ ; },
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; RNA ; Recombinases/genetics ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.},
}
@article {pmid35727555,
year = {2022},
author = {Ametrano, A and Coscia, MR},
title = {Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2498},
number = {},
pages = {337-350},
pmid = {35727555},
issn = {1940-6029},
mesh = {Animals ; Antibodies, Monoclonal/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Fishes/metabolism ; *Gene Editing/methods ; Hybridomas/metabolism ; Mice ; RNA, Guide/genetics ; Technology ; },
abstract = {The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.},
}
@article {pmid35727554,
year = {2022},
author = {Russo, MT and Santin, A and Rogato, A and Ferrante, MI},
title = {Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2498},
number = {},
pages = {327-336},
pmid = {35727554},
issn = {1940-6029},
mesh = {Biolistics/methods ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Cell Nucleus/genetics ; *Diatoms/genetics ; },
abstract = {The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.},
}
@article {pmid35727450,
year = {2022},
author = {Chen, W and She, W and Li, A and Zhai, C and Ma, L},
title = {Site-Directed Mutagenesis Method Mediated by Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2461},
number = {},
pages = {165-174},
pmid = {35727450},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Mutagenesis ; Mutagenesis, Site-Directed ; Plasmids/genetics ; Polymerase Chain Reaction ; },
abstract = {This study presents an in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants or site-saturation mutagenesis libraries in a PCR-independent manner. The plasmid DNA is double digested with Cas9 bearing specific single guide RNAs to remove the target nucleotides. Next, T5 exonuclease excises both 5'-ends of the linearized plasmid to generate homologous regions of approximately 15 nt. Subsequently, a short dsDNA of approximately 30-50 bp containing the desired mutation cyclizes the plasmid through base pairing and introduces the mutation into the plasmid. The gaps are repaired in Escherichia coli host cells after transformation. This method is highly efficient and accurate. Both single and multiple site-directed mutagenesis can be successfully performed, especially to large sized plasmids. This method demonstrates the great potential for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.},
}
@article {pmid35695482,
year = {2022},
author = {Zhang, RX and Li, BB and Yang, ZG and Huang, JQ and Sun, WH and Bhanbhro, N and Liu, WT and Chen, KM},
title = {Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {24},
pages = {7343-7359},
doi = {10.1021/acs.jafc.2c01754},
pmid = {35695482},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; *Genes, Plant ; Genome, Plant ; Plant Breeding ; },
abstract = {The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.},
}
@article {pmid33945142,
year = {2022},
author = {Thongsin, N and Wattanapanitch, M},
title = {CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {607-624},
pmid = {33945142},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing ; HLA Antigens/genetics ; Histocompatibility Antigens Class I/metabolism ; Humans ; *Induced Pluripotent Stem Cells ; Ribonucleoproteins/metabolism ; },
abstract = {Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.},
}
@article {pmid33834266,
year = {2022},
author = {Kratzer, K and Getz, LJ and Peterlini, T and Masson, JY and Dellaire, G},
title = {Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.},
journal = {Human genetics},
volume = {141},
number = {6},
pages = {1175-1193},
pmid = {33834266},
issn = {1432-1203},
support = {PJT-156017/CAPMC/CIHR/Canada ; PJT-156017/CAPMC/CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genetic Therapy/methods ; Germ Cells ; Humans ; },
abstract = {Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.},
}
@article {pmid33830454,
year = {2022},
author = {Thamodaran, V and Rani, S and Velayudhan, SR},
title = {Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {755-773},
pmid = {33830454},
issn = {1940-6029},
support = {IA/S/17/1/503118/WTDBT_/DBT-Wellcome Trust India Alliance/India ; },
mesh = {CRISPR-Cas Systems/genetics ; Doxycycline/pharmacology ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. Due to the ease of programming, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-based gene editing tools have gained pace in gene manipulation studies, including investigating complex diseases like cancer. An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.},
}
@article {pmid33755904,
year = {2022},
author = {Brandão, KO and Grandela, C and Yiangou, L and Mummery, CL and Davis, RP},
title = {CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {531-557},
pmid = {33755904},
issn = {1940-6029},
support = {638030/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.},
}
@article {pmid33755901,
year = {2022},
author = {Sanjurjo-Soriano, C and Erkilic, N and Mamaeva, D and Kalatzis, V},
title = {CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {589-606},
pmid = {33755901},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mammals/genetics ; },
abstract = {The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.},
}
@article {pmid33750926,
year = {2022},
author = {Chen, Y and Wen, R and Yang, Z and Chen, Z},
title = {Genome editing using CRISPR/Cas9 to treat hereditary hematological disorders.},
journal = {Gene therapy},
volume = {29},
number = {5},
pages = {207-216},
pmid = {33750926},
issn = {1476-5462},
support = {81971886//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Hematologic Diseases/genetics/therapy ; Humans ; RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. Induced pluripotent stem cells (iPSCs) can be generated from a variety of somatic cells and hold great promise for personalized cell-based therapies. CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.},
}
@article {pmid35727866,
year = {2022},
author = {Lee, YY and Park, R and Miller, SM and Li, Y},
title = {Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.15874},
pmid = {35727866},
issn = {1365-313X},
abstract = {Genetic compensation has been proposed to explain phenotypic differences between gene knockouts and knockdowns in several metazoan and plant model systems. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in the model alga Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, e.g., CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δvtc1 mutant, in which PDAT was up-regulated under stress, resulting in a 90% increase in TAG content. Knockout of PDAT in the Δvtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δvtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis under stress, and provides guidance for future genetic engineering and mutant characterization efforts.},
}
@article {pmid35715750,
year = {2022},
author = {Vaghari-Tabari, M and Hassanpour, P and Sadeghsoltani, F and Malakoti, F and Alemi, F and Qujeq, D and Asemi, Z and Yousefi, B},
title = {CRISPR/Cas9 gene editing: a new approach for overcoming drug resistance in cancer.},
journal = {Cellular & molecular biology letters},
volume = {27},
number = {1},
pages = {49},
pmid = {35715750},
issn = {1689-1392},
mesh = {CRISPR-Cas Systems/genetics ; Drug Resistance ; *Gene Editing/methods ; Humans ; *Neoplasms/drug therapy/genetics ; RNA ; },
abstract = {The CRISPR/Cas9 system is an RNA-based adaptive immune system in bacteria and archaea. Various studies have shown that it is possible to target a wide range of human genes and treat some human diseases, including cancers, by the CRISPR/Cas9 system. In fact, CRISPR/Cas9 gene editing is one of the most efficient genome manipulation techniques. Studies have shown that CRISPR/Cas9 technology, in addition to having the potential to be used as a new therapeutic approach in the treatment of cancers, can also be used to enhance the effectiveness of existing treatments. Undoubtedly, the issue of drug resistance is one of the main obstacles in the treatment of cancers. Cancer cells resist anticancer drugs by a variety of mechanisms, such as enhancing anticancer drugs efflux, enhancing DNA repair, enhancing stemness, and attenuating apoptosis. Mutations in some proteins of different cellular signaling pathways are associated with these events and drug resistance. Recent studies have shown that the CRISPR/Cas9 technique can be used to target important genes involved in these mechanisms, thereby increasing the effectiveness of anticancer drugs. In this review article, studies related to the applications of this technique in overcoming drug resistance in cancer cells will be reviewed. In addition, we will give a brief overview of the limitations of the CRISP/Cas9 gene-editing technique.},
}
@article {pmid35725215,
year = {2022},
author = {Khademi, Z and Ramezani, M and Alibolandi, M and Zirak, MR and Salmasi, Z and Abnous, K and Taghdisi, SM},
title = {A novel dual-targeting delivery system for specific delivery of CRISPR/Cas9 using hyaluronic acid, chitosan and AS1411.},
journal = {Carbohydrate polymers},
volume = {292},
number = {},
pages = {119691},
doi = {10.1016/j.carbpol.2022.119691},
pmid = {35725215},
issn = {1879-1344},
mesh = {Aptamers, Nucleotide ; *CRISPR-Cas Systems/genetics ; *Chitosan ; Gene Transfer Techniques ; HEK293 Cells ; Humans ; Hyaluronic Acid ; Oligodeoxyribonucleotides ; },
abstract = {A facile method was designed that can specifically deliver CRISPR/Cas9 into target cells nuclei and reduce the off-target effects. A multifunctional delivery vector for FOXM1 knockout was composed by integration of cell targeting polymer (hyaluronic acid) and cell and nuclear targeting group (AS1411 aptamer) on the surface of nanoparticles formed by genome editing plasmid and chitosan (CS) as the core (Apt-HA-CS-CRISPR/Cas9). The data of cytotoxicity experiment and western blot confirmed this issue. The results of flow cytometry analysis and fluorescence imaging demonstrated that Apt-HA-CS-CRISPR/Cas9 was significantly internalized into target cells (MCF-7, SK-MES-1, HeLa) but not into nontarget cells (HEK293). Furthermore, the in vivo studies displayed that the Apt-HA-CS-CRISPR/Cas9 was strongly rendered tumor inhibitory effect and delivered efficiently CRISPR/Cas9 into the tumor with no detectable distribution in other organs compared with naked plasmid. This approach provides an avenue for specific in vivo gene editing therapeutics with the lowest side effect.},
}
@article {pmid35724001,
year = {2022},
author = {Montano Gomez, P},
title = {.},
journal = {Journal international de bioethique et d'ethique des sciences},
volume = {33},
number = {1},
pages = {85-101},
doi = {10.3917/jibes.331.0085},
pmid = {35724001},
issn = {2608-1008},
mesh = {*Gene Editing ; Humans ; },
abstract = {There are many reasons why we might think that the human species might be in danger. We talk about global warming which produces major unusual natural disasters; increasingly destructive weapons; serious terrorist attacks with chemical weapons, etc. But we cannot ignore one of the threats hanging over the human species, which is transhumanism or posthumanism. It is from the point of view of this last subject, on which much has already been written, that we will try to focus our analysis, paying particular attention to the question relating to gene editing (CRISPR/Cas 9).},
}
@article {pmid35723482,
year = {2022},
author = {Rathbone, T and Ates, I and Stuart, C and Parker, T and Cottle, RN},
title = {Electroporation-mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {184},
pages = {},
doi = {10.3791/63828},
pmid = {35723482},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation/methods ; Hepatocytes/metabolism ; Mice ; RNA, Messenger/genetics ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 × 106 cells per liver and cell viability of >85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of >35% in mouse hepatocytes. To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.},
}
@article {pmid35722725,
year = {2022},
author = {Zhou, J and Liu, Y and Guo, X and Birchler, JA and Han, F and Su, H},
title = {Centromeres: from chromosome biology to biotechnology applications and synthetic genomes in plants.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13875},
pmid = {35722725},
issn = {1467-7652},
abstract = {Centromeres are the genomic regions that organize and regulate chromosome behaviors during cell cycle, and their variations are associated with genome instability, karyotype evolution, and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.},
}
@article {pmid35722296,
year = {2022},
author = {Parra-Flores, J and Holý, O and Acuña, S and Lepuschitz, S and Pietzka, A and Contreras-Fernández, A and Chavarría-Sepulveda, P and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Castillo, A and Ruppitsch, W and Forsythe, S},
title = {Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {884721},
doi = {10.3389/fmicb.2022.884721},
pmid = {35722296},
issn = {1664-302X},
abstract = {This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, β-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.},
}
@article {pmid35722276,
year = {2022},
author = {Panahi, B and Majidi, M and Hejazi, MA},
title = {Genome Mining Approach Reveals the Occurrence and Diversity Pattern of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Systems in Lactobacillus brevis Strains.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {911706},
doi = {10.3389/fmicb.2022.911706},
pmid = {35722276},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) together with their CRISPR-associated (Cas) genes are widely distributed in prokaryotes that provide an adaptive defense mechanism against foreign invasive DNA. There is relatively little knowledge about the CRISPR-Cas diversity and evolution in Lactobacillus brevis strains. Therefore, in this study, a genome-mining approach was employed to investigate the diversity and occurrence of the CRISPR-Cas system in 83 L. brevis strains. Moreover, trans-activating CRISPR RNA (tracrRNA) and protospacer adjacent motif (PAM) as pivotal elements for the successful targeting and inference of phages by the subtype II CRISPR-Cas systems were surveyed. Finally, evolutionary paths of L. brevis strains under selective pressure from foreign invasive DNA such as plasmids and phages of studied strains were surveyed using acquisition and deletion events analysis of spacers. A total of 127 confirmed CRISPRs were identified, which were distributed in 69 strains. Among strains with confirmed CRISPRs, 35 strains only contained one CRISPR locus, 23 strains contained two CRISPR loci, and 12 strains contained three to six CRISPR loci. L. brevis strains frequently harbor more than one CRISPR system. Analysis of confirmed CRISPR arrays showed that 31 out of 127 confirmed CRISPRs included Cas genes which were categorized as one of the II-A, II-C, and I-E subtypes. Analysis of subtype II-A spacers reflected divergent evolution for 18 strains into 16 unique groups. Additional analysis of spacer sequences also confirmed the implication of characterizing CRISPR-Cas systems in targeting of phages and plasmids. The current study highlighted the potential of utilizing CRISPR spacer polymorphism in genotyping lactobacillus strains. Moreover, it provides deep insights into the occurrence, diversity, and functional impacts of the CRISPR-Cas system in L. brevis strains.},
}
@article {pmid35721864,
year = {2022},
author = {Zhang, Y and Ge, H and Marchisio, MA},
title = {A Mutated Nme1Cas9 Is a Functional Alternative RNase to Both LwaCas13a and RfxCas13d in the Yeast S. cerevisiae.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {922949},
doi = {10.3389/fbioe.2022.922949},
pmid = {35721864},
issn = {2296-4185},
abstract = {CRISPR-Cas systems provide powerful biological tools for genetic manipulation and gene expression regulation. Class 2 systems, comprising type II, type V, and type VI, have the significant advantage to require a single effector Cas protein (Cas9, Cas12, and Cas13 respectively) to cleave nucleic acids upon binding the crRNA. Both Cas9 and Cas12 recognize DNA and induce a double-strand break in it. In contrast, Cas13 bind and cleave RNA exclusively. However, some Cas9 homologs have shown RNase activity as well. Here, we harnessed Nme1Cas9, LwaCas13a, and RfxCas13d to carry out gene downregulation in Saccharomyces cerevisiae by triggering mRNA degradation. To avoid potential DNA damage, we mutated Nme1Cas9 into d16ANme1Cas9 that lost the nuclease activity of the RuvC domain but retained the active HNH domain, able to act on the target DNA strand and, therefore, on the corresponding transcript. Our results showed that d16ANme1Cas9 is a functional RNase in vivo, although with moderate activity since it provoked a fluorescence reduction from 21% to 32%. Interestingly, d16ANme1Cas9 works in a PAM-independent way nor demands helper PAMmer molecules. LwaCas13a and RfxCas13d appeared substantially unfunctional in S. cerevisiae, though they were shown to perform well in mammalian cells. To the best of our knowledge, this is the first report about the working in vivo of a variant of Nme1Cas9 as an RNase and the issues connected with the usage of Cas13 proteins in S. cerevisiae.},
}
@article {pmid35719725,
year = {2022},
author = {Chilian, M and Vargas Parra, K and Sandoval, A and Ramirez, J and Yoon, WH},
title = {CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101465},
doi = {10.1016/j.xpro.2022.101465},
pmid = {35719725},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA, Complementary/genetics ; Drosophila/genetics ; *Drosophila melanogaster/genetics ; Exons/genetics ; Introns ; },
abstract = {In this protocol, we take CRISPR/Cas9 and Gal4/UAS approaches to achieve tissue-specific knockout in parallel with rescue of the knockout by cDNA expression in Drosophila. We demonstrate that guide RNAs targeting the exon-intron junction of target genes cleave the genomic locus of the genes, but not UAS-cDNA transgenes, in a tissue where Gal4 drives Cas9 expression. The efficiency of this approach enables the determination of pathogenicity of disease-associated variants in human genes in a tissue-specific manner in Drosophila. For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).},
}
@article {pmid35719360,
year = {2022},
author = {Qiu, X and Xu, S and Liu, X and Ren, H and Han, L and Li, Z},
title = {CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {884411},
doi = {10.3389/fcimb.2022.884411},
pmid = {35719360},
issn = {2235-2988},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Nocardia ; *Nocardia Infections ; *Nucleic Acids ; Pandemics ; },
abstract = {Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.},
}
@article {pmid35717416,
year = {2022},
author = {Velimirovic, M and Zanetti, LC and Shen, MW and Fife, JD and Lin, L and Cha, M and Akinci, E and Barnum, D and Yu, T and Sherwood, RI},
title = {Peptide fusion improves prime editing efficiency.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3512},
pmid = {35717416},
issn = {2041-1723},
support = {1R01HG008754//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R21HG010391//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Fusion ; Peptides/genetics ; },
abstract = {Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.},
}
@article {pmid35716656,
year = {2022},
author = {Ali, S and Khan, N and Tang, Y},
title = {Epigenetic marks for mitigating abiotic stresses in plants.},
journal = {Journal of plant physiology},
volume = {275},
number = {},
pages = {153740},
doi = {10.1016/j.jplph.2022.153740},
pmid = {35716656},
issn = {1618-1328},
abstract = {Abiotic stressors are one of the major factors affecting agricultural output. Plants have evolved adaptive systems to respond appropriately to various environmental cues. These responses can be accomplished by modulating or fine-tuning genetic and epigenetic regulatory mechanisms. Understanding the response of plants' molecular features to abiotic stress is a priority in the current period of continued environmental changes. Epigenetic modifications are necessary that control gene expression by changing chromatin status and recruiting various transcription regulators. The present study summarized the current knowledge on epigenetic modifications concerning plant responses to various environmental stressors. The functional relevance of epigenetic marks in regulating stress tolerance has been revealed, and epigenetic changes impact the effector genes. This study looks at the epigenetic mechanisms that govern plant abiotic stress responses, especially DNA methylation, histone methylation/acetylation, chromatin remodeling, and various metabolites. Plant breeders will benefit from a thorough understanding of these processes to create alternative crop improvement approaches. Genome editing with clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) provides genetic tools to make agricultural genetic engineering more sustainable and publicly acceptable.},
}
@article {pmid35715136,
year = {2022},
author = {Taghdisi, SM and Ramezani, M and Alibolandi, M and Khademi, Z and Hajihasani, MM and Alinezhad Nameghi, M and Khakshour Abdolabadi, A and Rahimi, H and Abnous, K and Danesh, NM},
title = {A highly sensitive fluorescent aptasensor for detection of prostate specific antigen based on the integration of a DNA structure and CRISPR-Cas12a.},
journal = {Analytica chimica acta},
volume = {1219},
number = {},
pages = {340031},
doi = {10.1016/j.aca.2022.340031},
pmid = {35715136},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; DNA, Cruciform ; Humans ; Male ; *Prostate-Specific Antigen ; },
abstract = {Herein, a facile fluorescent CRISPR-Cas12a-based sensing strategy is presented for prostate specific antigen (PSA), as a prostate cancer biomarker, with the assistance of a cruciform DNA nanostructure and PicoGreen (PG) as a fluorochrome. Highly sensitive recognition of PSA is one of the virtues of the proposed method which comes from the use of unique features of both CRISPR-Cas12a and DNA structure in the design of the aptasensor. The presence of PSA creates a cruciform DNA nanostructure in the sample which can be loaded by PG and make sharp fluorescence emission. While, when there is no PSA, the CRISPR-Cas12a digests sequences 1 and 3 as single-stranded DNAs, causing no DNA structure and a negligible fluorescence is detected after addition of PG. This aptasensor presents a sensitive recognition performance with detection limit of 4 pg/mL and a practical use for determination of PSA in serum samples. So, this analytical strategy introduces a convenient and highly sensitive approach for detection of disease biomarkers.},
}
@article {pmid35713718,
year = {2022},
author = {Rather, GA and Ayzenshtat, D and Teper-Bamnolker, P and Kumar, M and Forotan, Z and Eshel, D and Bocobza, S},
title = {Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.},
journal = {Planta},
volume = {256},
number = {1},
pages = {14},
pmid = {35713718},
issn = {1432-2048},
support = {20-01-0209//Office of the Chief Scientist, Ministry of Health/ ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA/metabolism ; *Gene Editing/methods ; Genome, Plant ; Kanamycin/metabolism ; Plant Breeding/methods ; Protoplasts/metabolism ; *Solanum tuberosum/genetics/metabolism ; Tetraploidy ; Transcription Factors/genetics ; Transfection ; },
abstract = {MAIN CONCLUSION: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.},
}
@article {pmid35713220,
year = {2022},
author = {Yang, H and Wei, Y and Zhang, Q and Yang, Y and Bi, X and Yang, L and Xiao, N and Zang, A and Ren, L and Li, X},
title = {CRISPR/Cas9‑induced saturated mutagenesis identifies Rad51 haplotype as a marker of PARP inhibitor sensitivity in breast cancer.},
journal = {Molecular medicine reports},
volume = {26},
number = {2},
pages = {},
doi = {10.3892/mmr.2022.12774},
pmid = {35713220},
issn = {1791-3004},
mesh = {*Antineoplastic Agents/pharmacology ; *Breast Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Female ; Haplotypes ; Humans ; Mutagenesis ; Phthalazines/pharmacology/therapeutic use ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *Rad51 Recombinase/genetics/metabolism ; },
abstract = {Breast cancer treatment with poly(ADP‑ribose)polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination repair (HRR) pathway. The chemical inhibition of many HRR deficiency genes may sensitize cancer cells to PARP inhibitors. In the present study, Rad51, a central player in the HRR pathway, was selected to explore additional low variation and highly representative markers for PARP inhibitor activity. A CRISPR/Cas9‑based saturated mutation approach for the Rad51 WALKER domain was used to evaluate the sensitivity of the PARP inhibitor olaparib. Five amino acid mutation sites were identified in olaparib‑resistant cells. Two Rad51 haplotypes were assembled from the mutations, and may represent useful pharmacogenomic markers of PARP inhibitor sensitivity.},
}
@article {pmid35712010,
year = {2022},
author = {Novarina, D and Koutsoumpa, A and Milias-Argeitis, A},
title = {A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101358},
doi = {10.1016/j.xpro.2022.101358},
pmid = {35712010},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Saccharomyces cerevisiae/genetics ; *Saccharomycetales/genetics ; },
abstract = {CRISPR/Cas9 technology allows accurate, marker-less genome editing. We report a detailed, robust, and streamlined protocol for CRISPR/Cas9 genome editing in Saccharomyces cerevisiae, based on the widely used MoClo-Yeast Toolkit (https://www.addgene.org/kits/moclo-ytk/). This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group. For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021).},
}
@article {pmid35710827,
year = {2022},
author = {Ryu, J and Statz, JP and Chan, W and Burch, FC and Brigande, JV and Kempton, B and Porsov, EV and Renner, L and McGill, T and Burwitz, BJ and Hanna, CB and Neuringer, M and Hennebold, JD},
title = {CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {10036},
pmid = {35710827},
issn = {2045-2322},
support = {P51 OD011092/GF/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Editing ; Humans ; Macaca mulatta/genetics/metabolism ; RNA, Guide/metabolism ; RNA, Messenger ; *Usher Syndromes/genetics ; },
abstract = {Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.},
}
@article {pmid35708998,
year = {2022},
author = {Gemperle, J and Harrison, TS and Flett, C and Adamson, AD and Caswell, PT},
title = {On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
doi = {10.7554/eLife.76651},
pmid = {35708998},
issn = {2050-084X},
support = {DCRPGF\100002/CRUK_/Cancer Research UK/United Kingdom ; 836212//Horizon 2020 Framework Programme/ ; C147/A25254/CRUK_/Cancer Research UK/United Kingdom ; MR/R009376/1/MRC_/Medical Research Council/United Kingdom ; 203128/A/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Doxycycline ; Indoleacetic Acids ; Methacrylates ; *rab GTP-Binding Proteins/genetics/metabolism ; },
abstract = {CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.},
}
@article {pmid35687910,
year = {2022},
author = {Zuo, Q and Xu, W and Wan, Y and Feng, D and He, C and Lin, C and Huang, D and Chen, F and Han, L and Sun, Q and Chen, D and Du, H and Huang, L},
title = {Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {152},
number = {},
pages = {113243},
doi = {10.1016/j.biopha.2022.113243},
pmid = {35687910},
issn = {1950-6007},
mesh = {*CRISPR-Cas Systems/genetics ; *Cytochrome P-450 CYP3A/genetics ; Gene Editing/methods ; Gene Knock-In Techniques ; Luciferases/genetics ; },
abstract = {CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.},
}
@article {pmid35670376,
year = {2022},
author = {Zhang, W and Shi, R and Dong, K and Hu, H and Shu, W and Mu, Y and Yan, B and Li, L and Xiao, X and Wang, H},
title = {The Off-Target Effect of CRISPR-Cas12a System toward Insertions and Deletions between Target DNA and crRNA Sequences.},
journal = {Analytical chemistry},
volume = {94},
number = {24},
pages = {8596-8604},
doi = {10.1021/acs.analchem.1c05499},
pmid = {35670376},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Mutation ; RNA/genetics ; },
abstract = {The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41-42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.},
}
@article {pmid35666940,
year = {2022},
author = {Hou, Y and Wang, D and Lu, S and Guo, D and Li, M and Cui, M and Zhang, XE},
title = {Optogenetic Control of Background Fluorescence Reduction for CRISPR-Based Genome Imaging.},
journal = {Analytical chemistry},
volume = {94},
number = {24},
pages = {8724-8731},
doi = {10.1021/acs.analchem.2c01113},
pmid = {35666940},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Nucleus ; Genome ; Microscopy, Fluorescence ; *Optogenetics ; },
abstract = {The CRISPR/dCas9 system has become an essential tool for live-cell imaging of genomic loci, but it has limited applications in imaging low-/non-repetitive genomic loci due to the strong nuclear background noise emerging from many untargeted fluorescent modules. Here, we propose an optogenetically controlled background fluorescence reduction strategy that combines the CRISPR-SunTag system with a light-inducible nuclear export tag (LEXY). Utilizing the SunTag system, multiple copies of LEXY-tagged sfGFP were recruited to the C-terminal dCas9, recognizing the target genomic loci. As the nuclear export sequence at the C-terminal LEXY could be exposed to pulsed blue light irradiation, the untargeted nuclear labeling modules were light controllably transferred to the cytoplasm. Consequently, genomic loci containing as few as nine copies of repeats were clearly visualized, and a significant increase in the signal-to-noise ratio was achieved. This simple and controllable method is expected to have a wide range of applications in cell biology.},
}
@article {pmid35623274,
year = {2022},
author = {Chen, H and Li, ZY and Chen, J and Yu, H and Zhou, W and Shen, F and Chen, Q and Wu, L},
title = {CRISPR/Cas12a-based electrochemical biosensor for highly sensitive detection of cTnI.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {146},
number = {},
pages = {108167},
doi = {10.1016/j.bioelechem.2022.108167},
pmid = {35623274},
issn = {1878-562X},
mesh = {*Aptamers, Nucleotide ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; Troponin I ; },
abstract = {The successful fabrication of the cTnI detection platform is very meaningful for instant diagnosis of the myocardialinjury and related cardiovascular diseases (CVDs). In this research work, the magnetic nanoparticles and aptamer collaboration with the Cas12a/crRNA are used for the electrochemical detection of cTnI. The aptamer is hybridized with its partially complementary DNA (probe 2, P2) and then is modified on the magnetic nanoparticles. In the presence of cTnI, the cTnI combines with the aptamer and P2 is released. The released P2 is hybridized with the crRNA and the trans-cleavage activity of CRISPR/Cas12a is triggered. Therefore, the methylene blue-modified DNA (probe1, P1) on the surface of the electrode is cleaved, resulting in the decrease of the electrochemical signal. Based on the synergy effect of the high specific target recognition of aptamer, target-specifically triggering trans-cleavage activity of CRISPR/Cas12a, as well as good separation ability of magnetic nanoparticles, the developed electrochemical biosensor enables to detect cTnI with high specificity and sensitivity. The detection limit is low down to 10 pg/mL with a linear range from 100 pg/mL to 50000 pg/mL. The developed sensing platform was successfully applied for the detection of cTnI in human serum. This fabricated CRISPR/Cas12a-based electrochemical biosensor can offer a valuable tool for the diagnosis, prognosis, and treatment of patient with CVDs.},
}
@article {pmid35618221,
year = {2022},
author = {Yu, H and Iqbal, A and Fang, X and Jiang, P and Zhao, Z},
title = {Transcriptome analysis of CRISPR/Cas9-mediated GPAM-/- in bovine mammary epithelial cell-line unravelled the effects of GPAM gene on lipid metabolism.},
journal = {Gene},
volume = {834},
number = {},
pages = {146574},
doi = {10.1016/j.gene.2022.146574},
pmid = {35618221},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Epithelial Cells/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; Gene Expression Profiling ; Glycerol-3-Phosphate O-Acyltransferase/genetics ; *Lipid Metabolism/genetics ; *Mammary Glands, Animal/metabolism ; Milk/metabolism ; Phosphates/metabolism ; },
abstract = {Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is an enzyme in animal lipid metabolism pathways that catalyzes the initial and most committed step of glycerolipid biosynthesis. The present study mainly focused on exploring the relationship between the GPAM gene and the lipid metabolism of mammary epithelial cells and the effect of GPAM on the related pathways of lipid metabolism. The GPAM gene was knocked out entirely in bovine mammary epithelial cells(BMECs) using CRISPR/Cas9 technology, and the mechanism by which the GPAM gene regulates lipid metabolism in BMECs was confirmed. Furthermore, after the complete loss of GPAM, BMECs' triglycerides (TGs) and cholesterol (CHOL) levels were significantly decreased (p < 0.05). Concurrently, the content of octanoic acid, a medium-chain saturated fatty acid, increased substantially in BMECs. RNA-seq of GPAM-/- BMECs revealed that GPAM could affect the expression of genes related to lipid metabolism, downregulated the expression of Acyl-CoA synthetase long-chain family member 5 (ACSL5), Fatty Acid Binding Protein 3 (FABP3), Hormone-sensitive lipase (HSL), Protease, serine-2 (PRSS2), 1-Acylglycerol-3-Phosphate O Acyltransferase 4 (AGPAT4), and regulated the milk synthesis metabolism pathway.The findings revealed that a number of genes were expressed, a number of genes were differentially expressed genes (DEGs), and a number of GO terms were enriched, with a number of GO terms considerably increased. Further, the differentially expressed genes (DEGs) were significantly enriched in Fat digestion and absorption pathway, Fatty acid metabolic pathway, Biosynthesis of unsaturated fatty acids, Biosynthesis of unsaturated fatty acids and steroids, NF-kappa B signalling pathway, MAPK signalling pathway. In conclusion, the current research results show that GPAM is a crucial regulator of BMEC lipid metabolism. GPAM-/- BMEC may also become useful genetic materials and tools for future research on gene functions related to lipid and fatty acid metabolism. This study will contribute to the discovery of gene regulation and molecular mechanisms in milk fat synthesis.},
}
@article {pmid35617843,
year = {2022},
author = {He, Z and Feng, K and Sun, H and Yu, T and Zhu, D and Yang, Y},
title = {Generation of a human extended pluripotent stem cell line (SKLRMe002-A) carrying a doxycycline-inducible Cas9 expression cassette.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102816},
doi = {10.1016/j.scr.2022.102816},
pmid = {35617843},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; *Doxycycline/pharmacology ; Humans ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Human extended pluripotent stem cell (hEPS) is a novel type of pluripotent stem cell, which possesses bi-potency towards both embryonic and extraembryonic lineages. Here, we generated a hEPS cell line (hEPS1-iCas9-B) from the cell line named hEPS1, carrying a doxycycline-inducible Cas9 expression cassette along with a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette at the AAVS1 locus, thus we could efficiently generate genetically modified hEPS for studies. This cell lined remained self-renewal, differentiation potential and normal karyotype. Meanwhile, it showed robust transcriptional expression of Cas9 with doxycycline induction and could target the site where the sgRNA guided.},
}
@article {pmid35614226,
year = {2022},
author = {Nusser, A and Sagar, and Swann, JB and Krauth, B and Diekhoff, D and Calderon, L and Happe, C and Grün, D and Boehm, T},
title = {Developmental dynamics of two bipotent thymic epithelial progenitor types.},
journal = {Nature},
volume = {606},
number = {7912},
pages = {165-171},
pmid = {35614226},
issn = {1476-4687},
support = {/ERC_/European Research Council/International ; },
mesh = {Aging ; Animals ; Autocrine Communication ; CRISPR-Cas Systems ; Cellular Microenvironment ; *Epithelial Cells/cytology/metabolism ; Epithelium ; Fibroblast Growth Factor 7 ; Mice ; RNA-Seq ; Single-Cell Analysis ; *Stem Cells/cytology ; *T-Lymphocytes/cytology/metabolism ; *Thymus Gland/cytology ; },
abstract = {T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age1-10. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice11-18; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved1,12,16,17,19-27. Here we combine scRNA-seq and a new CRISPR-Cas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity.},
}
@article {pmid35588782,
year = {2022},
author = {Tripathi, R and Sinha, NR and Kempuraj, D and Balne, PK and Landreneau, JR and Juneja, A and Webel, AD and Mohan, RR},
title = {Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro.},
journal = {Experimental eye research},
volume = {220},
number = {},
pages = {109113},
doi = {10.1016/j.exer.2022.109113},
pmid = {35588782},
issn = {1096-0007},
mesh = {Actins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Co-Repressor Proteins/genetics/metabolism ; Collagen/metabolism ; *Corneal Diseases/pathology ; Fibroblasts/metabolism ; Fibrosis ; *Gene Editing ; Homeodomain Proteins ; Humans ; Myofibroblasts/metabolism ; Repressor Proteins/metabolism ; Transcription Factors/genetics ; Transforming Growth Factor beta1/pharmacology ; },
abstract = {Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-β1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.},
}
@article {pmid35581476,
year = {2022},
author = {Tringe, SG},
title = {A toolkit for microbial community editing.},
journal = {Nature reviews. Microbiology},
volume = {20},
number = {7},
pages = {383},
pmid = {35581476},
issn = {1740-1534},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *Microbiota ; },
}
@article {pmid35576811,
year = {2022},
author = {Gulimiheranmu, M and Li, S and Zhou, J},
title = {Generation of a MIR5004 knockout cell line from human induced pluripotent stem cells by CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102805},
doi = {10.1016/j.scr.2022.102805},
pmid = {35576811},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {MIR5004 is located in the intronic region of SYNGAP1, a genetic risk factor for Autism Spectrum Disorders (ASD), and co-expressed with SYNGAP1 in brain tissue, which indicates that MIR5004 may play an important role in ASD pathogenesis through the regulation of SYNGAP1. Here, we generated a MIR5004 knockout human induced pluripotent stem cell (iPSC) line SHCDCLi001-B. SHCDCLi001-B shows expression of pluripotent markers, three lineage differentiation capacity, normal morphology and karyotypes, the same DNA origin with wild type iPSC (iPSC-WT) and no off-target effects, making it as a valuable tool for studying the interplay between MIR5004 and SYNGAP1 in ASD pathogenesis.},
}
@article {pmid35567848,
year = {2022},
author = {Zhang, D and Zhou, M and Zhang, Y and Shan, Y and Pan, G},
title = {Generation of an RNF1-deficient human pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102809},
doi = {10.1016/j.scr.2022.102809},
pmid = {35567848},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/physiology ; Cell Line ; *Human Embryonic Stem Cells/metabolism ; Humans ; *Pluripotent Stem Cells ; Technology ; },
abstract = {RNF1 (RING1A) is a catalytic component of the polycomb repressive complex 1 (PRC1) involved in regulation of, among others, embryonic development and disease progression. However, the exact role of RNF1 in self-renewal and differentiation of human embryonic stem cells (ESCs) remains unknown. Here, we derive one RNF1 knockout human ESC line using CRISPR/Cas9 system. The cell line retains the canonical stem cell morphology and normal karyotype. Moreover, the cell line highly expresses pluripotency genes and has three germ-layer differentiation potential. The RNF1 -/- cell line will be useful for studies on the function and role of RNF1 in human embryonic stem cell fate decisions.},
}
@article {pmid35561459,
year = {2022},
author = {Fu, J and Jiang, L and Yu, B and Liu, Y and Wei, R and Hu, Y and Ho, WI and Yang, B and Chu, M and Tse, HF and Yang, J},
title = {Generation of a human iPSC line CIBi010-A with a reporter for ASGR1 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102800},
doi = {10.1016/j.scr.2022.102800},
pmid = {35561459},
issn = {1876-7753},
mesh = {Asialoglycoprotein Receptor/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Hepatocytes/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells/metabolism ; },
abstract = {ASGR1 is a liver-specific surface marker that has been used to purify human pluripotent stem cell (PSC)-derived hepatocytes (iHeps). Furthermore, ASGR1+ iHeps represents a more mature subpopulation of iHeps. To utilize this marker for optimizing iHep differentiation and purification, we substituted the stop coden of ASGR1 with a fluorescent reporter protein mCherry in a human iPSC line iPSN0052 via CRISPR/Cas9-mediated homologus recombination. The generated CIBi010-A enableds us to monitor ASGR1 expression during hepatic differentiation and thus can be used to optimize our hepatic differentiation procedures.},
}
@article {pmid35561458,
year = {2022},
author = {Chen, CX and You, Z and Abdian, N and Sirois, J and Shlaifer, I and Tabatabaei, M and Boivin, MN and Gaborieau, L and Karamchandani, J and Beitel, LK and Fon, EA and Durcan, TM},
title = {Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102806},
doi = {10.1016/j.scr.2022.102806},
pmid = {35561458},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mitophagy/genetics ; *Parkinson Disease/genetics ; Protein Kinases/genetics ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.},
}
@article {pmid35537243,
year = {2022},
author = {Zhang, J and Zhou, T and Shan, Y and Pan, G},
title = {Generation of RYBP FLAG-HA knock-in human embryonic stem cell line through CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102803},
doi = {10.1016/j.scr.2022.102803},
pmid = {35537243},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Embryonic Stem Cells/metabolism ; Homologous Recombination ; *Human Embryonic Stem Cells/metabolism ; Humans ; Mice ; Polycomb-Group Proteins ; Repressor Proteins/metabolism ; },
abstract = {RYBP, a critical component of polycomb repressive complex1 (PRC1), is required for the pluripotency and differentiation of mouse embryonic stem cells(mESCs). However, its function and mechanism to regulate human embryonic stem cells(hESCs) remain unknown. Here, to investigate the role of RYBP in hESCs, we generate an hESC line with FLAG-HA tag knock-in to RYBP locus through CRISPR/Cas9-mediated homologous recombination. hESC with RYBP_FLAG-HA knock-in maintains normal morphology and karyotype, while it maintains pluripotency to differentiate into three germ layers.},
}
@article {pmid35533514,
year = {2022},
author = {Xu, Q and Zhao, T and Zhang, Y and Fan, W and Yan, Y and Lan, F},
title = {Generation of a human embryonic stem cell line (WAe009-A-78) carrying homozygous TBX18 knockout.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102804},
doi = {10.1016/j.scr.2022.102804},
pmid = {35533514},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells/metabolism ; Female ; Homozygote ; *Human Embryonic Stem Cells/metabolism ; Humans ; Pregnancy ; Sinoatrial Node ; T-Box Domain Proteins/genetics/metabolism ; },
abstract = {T-Box Transcription Factor 18 is a member of the T-box family, encoding TBX18 protein. As a transcriptional repressor, it related to developmental processes of a majority of tissues and organs and plays crucial part in the embryonic development of sinoatrial node. Using an episomal vector-based CRISPR/Cas9 system, we have established a homozygous TBX18 knockout (TBX18-KO) human embryonic stem cell (hESC) line. This newly TBX18-/- hESC line display normal pluripotency, morphology, karyotype and trilineage differentiating capacity. This cell line may provide a powerful tool to investigate the role of TBX18 gene in sinoatrial node development in future.},
}
@article {pmid35367933,
year = {2022},
author = {Liu, N and Liu, R and Zhang, J},
title = {CRISPR-Cas12a-mediated label-free electrochemical aptamer-based sensor for SARS-CoV-2 antigen detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {146},
number = {},
pages = {108105},
doi = {10.1016/j.bioelechem.2022.108105},
pmid = {35367933},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; DNA ; Humans ; Nucleic Acid Amplification Techniques/methods ; SARS-CoV-2/genetics ; },
abstract = {Serological antigen testing has emerged as an important diagnostic paradigm in COVID-19, but often suffers from potential cross-reactivity. To address this limitation, we herein report a label-free electrochemical aptamer-based sensor for the detection of SARS-CoV-2 antigen by integrating aptamer-based specific recognition with CRISPR-Cas12a-mediated signal amplification. The sensing principle is based on the competitive binding of antigen and the preassembled Cas12a-crRNA complex to the antigen-specific aptamer, resulting in a change in the collateral cleavage activity of Cas12a. To further generate an electrochemical signal, a DNA architecture was fabricated by in situ rolling circle amplification on a gold electrode, which serves as a novel substrate for Cas12a. Upon Cas12a-based collateral DNA cleavage, the DNA architecture was degraded, leading to a significant decrease in impedance that can be measured spectroscopically. Using SARS-CoV-2 nucleocapsid antigen as the model, the proposed CRISPR-Cas12a-based electrochemical sensor (CRISPR-E) showed excellent analytical performance for the quantitative detection of nucleocapsid antigen. Since in vitro selection can obtain aptamers selective for many SARS-CoV-2 antigens, the proposed strategy can expand this powerful CRISPR-E system significantly for quantitative monitoring of a wide range of COVID-19 biomarkers.},
}
@article {pmid35169967,
year = {2022},
author = {Widjaya, MA and Ju, JC and Lee, SD},
title = {CRISPR-Edited Stem Cell Transplantation for HIV-Related Gene Modification In Vivo: A Systematic Review.},
journal = {Stem cell reviews and reports},
volume = {18},
number = {5},
pages = {1743-1755},
pmid = {35169967},
issn = {2629-3277},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *HIV Infections/genetics/therapy ; *Hematopoietic Stem Cell Transplantation ; Humans ; Stem Cell Transplantation ; },
abstract = {BACKGROUND: CRISPR is a novel genomic editing technology which can be useful for the treatment of immune diseases such as HIV. However, the application of CRISPR in stem cells for HIV-related research was not effective, and most of the research was done in vivo. This systematic review is to identify a new research idea about increase CRISPR-editing efficiencies in stem cell transplantation for HIV treatment, as well as its future perspective.<br><br>METHOD: Four databases were searched for articles published during 1952 to 2020. PRISMA method was used to select appropriate research papers. CAMARADES was used to identify the paper quality. The outcome was engraftment efficiency, gene disruption percentage, differentiation ability, HIV-resistant efficiency.<br><br>RESULT: Screening method showed 196 papers mentioned the topic. However, only 5 studies were reliable with the research objective. We found that (1) Two research ideas which was double gene knockout and knockout-knockin method to provide HIV-resistant cells, engraftment support and avoid cardiac disease as an HIV disease side effect. (2) Ribonucleoprotein (RNP) delivery was the best way to deliver the CRISPR/Cas9 and Adeno-Associated Virus (AAV) would be effective for knockin purpose. (3) CRISPR/SaCas9 could replace CRISPR/Cas9 role in editing HIV-related gene.<br><br>CONCLUSION: Potential genes to increase HIV resistance and stem cell engraftment should be explored more in the future. Double knockout and knock-in procedures should be applied to set up a better engraftment for improving HIV treatment or resistance of patients. CRISPR/SaCas9 and RNP delivery should be explored more in the future.<br><br>PROSPERO CRD42020203312.},
}
@article {pmid35165384,
year = {2022},
author = {Sánchez-Rivera, FJ and Diaz, BJ and Kastenhuber, ER and Schmidt, H and Katti, A and Kennedy, M and Tem, V and Ho, YJ and Leibold, J and Paffenholz, SV and Barriga, FM and Chu, K and Goswami, S and Wuest, AN and Simon, JM and Tsanov, KM and Chakravarty, D and Zhang, H and Leslie, CS and Lowe, SW and Dow, LE},
title = {Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {862-873},
pmid = {35165384},
issn = {1546-1696},
support = {P01 CA087497/CA/NCI NIH HHS/United States ; R01 CA229773/CA/NCI NIH HHS/United States ; R01CA229773-01A1//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 5T32CA160001-08//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P01 CA129243/CA/NCI NIH HHS/United States ; F31-CA192835//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31-CA261061-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31-CA247351-02//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P01 CA087497/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Neoplasms/genetics ; Nucleotides ; RNA, Guide/genetics ; },
abstract = {Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.},
}
@article {pmid35089463,
year = {2022},
author = {Fu, J and Fu, YW and Zhao, JJ and Yang, ZX and Li, SA and Li, GH and Quan, ZJ and Zhang, F and Zhang, JP and Zhang, XB and Sun, CK},
title = {Improved and Flexible HDR Editing by Targeting Introns in iPSCs.},
journal = {Stem cell reviews and reports},
volume = {18},
number = {5},
pages = {1822-1833},
pmid = {35089463},
issn = {2629-3277},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Introns/genetics ; Pyridazines ; Quinazolines ; *Recombinational DNA Repair ; },
abstract = {Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.},
}
@article {pmid34813932,
year = {2022},
author = {Hu, M and Zhu, D and Zhou, X},
title = {M-CDC: Magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {259-267},
doi = {10.1016/j.ymeth.2021.11.009},
pmid = {34813932},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; Centers for Disease Control and Prevention, U.S. ; Colorimetry ; DNA ; Magnetic Phenomena ; *Nucleic Acid Amplification Techniques/methods ; United States ; },
abstract = {The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M-CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M-CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M-CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium-Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.},
}
@article {pmid34767923,
year = {2022},
author = {Okafor, IC and Choi, J and Ha, T},
title = {Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.},
journal = {Methods (San Diego, Calif.)},
volume = {204},
number = {},
pages = {319-326},
doi = {10.1016/j.ymeth.2021.11.003},
pmid = {34767923},
issn = {1095-9130},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/chemistry ; *DNA Cleavage ; RNA, Guide/genetics/metabolism ; },
abstract = {Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9's HNH domain before and after cleavage.},
}
@article {pmid34112918,
year = {2022},
author = {Fan, W and Yu, M and Wang, X and Xie, W and Tian, R and Cui, Z and Jin, Z and Huang, Z and Das, BC and Severinov, K and Hitzeroth, II and Debata, PR and Tian, X and Xie, H and Lang, B and Tan, J and Xu, H and Hu, Z},
title = {Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells.},
journal = {Cancer gene therapy},
volume = {29},
number = {6},
pages = {758-769},
pmid = {34112918},
issn = {1476-5500},
mesh = {CRISPR-Cas Systems/genetics ; Female ; Humans ; Mutagens ; Oligodeoxyribonucleotides ; Oncogenes ; *Papillomavirus Infections/genetics ; *Uterine Cervical Neoplasms/genetics/therapy ; },
abstract = {Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.},
}
@article {pmid35712599,
year = {2022},
author = {Mattiello, L and Rütgers, M and Sua-Rojas, MF and Tavares, R and Soares, JS and Begcy, K and Menossi, M},
title = {Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {868027},
doi = {10.3389/fpls.2022.868027},
pmid = {35712599},
issn = {1664-462X},
abstract = {The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.},
}
@article {pmid35711363,
year = {2022},
author = {Wang, Q and Park, KH and Geng, B and Chen, P and Yang, C and Jiang, Q and Yi, F and Tan, T and Zhou, X and Bian, Z and Ma, J and Zhu, H},
title = {MG53 Inhibits Necroptosis Through Ubiquitination-Dependent RIPK1 Degradation for Cardiac Protection Following Ischemia/Reperfusion Injury.},
journal = {Frontiers in cardiovascular medicine},
volume = {9},
number = {},
pages = {868632},
doi = {10.3389/fcvm.2022.868632},
pmid = {35711363},
issn = {2297-055X},
abstract = {Rationale: While reactive oxygen species (ROS) has been recognized as one of the main causes of cardiac injury following myocardial infarction, the clinical application of antioxidants has shown limited effects on protecting hearts against ischemia-reperfusion (I/R) injury. Thus, the precise role of ROS following cardiac injury remains to be fully elucidated.<br><br>Objective: We investigated the role of mitsugumin 53 (MG53) in regulating necroptosis following I/R injury to the hearts and the involvement of ROS in MG53-mediated cardioprotection.<br><br>Methods and Results: Antioxidants were used to test the role of ROS in MG53-mediated cardioprotection in the mouse model of I/R injury and induced human pluripotent stem cells (hiPSCs)-derived cardiomyocytes subjected to hypoxia or re-oxygenation (H/R) injury. Western blotting and co-immunoprecipitation were used to identify potential cell death pathways that MG53 was involved in. CRISPR/Cas 9-mediated genome editing and mutagenesis assays were performed to further identify specific interaction amino acids between MG53 and its ubiquitin E3 ligase substrate. We found that MG53 could protect myocardial injury via inhibiting the necroptosis pathway. Upon injury, the generation of ROS in the infarct zone of the hearts promoted interaction between MG53 and receptor-interacting protein kinase 1 (RIPK1). As an E3 ubiquitin ligase, MG53 added multiple ubiquitin chains to RIPK1 at the sites of K316, K604, and K627 for proteasome-mediated RIPK1 degradation and inhibited necroptosis. The application of N-acetyl cysteine (NAC) disrupted the interaction between MG53 and RIPK1 and abolished MG53-mediated cardioprotective effects.<br><br>Conclusions: Taken together, this study provided a molecular mechanism of a potential beneficial role of ROS following acute myocardial infarction. Thus, fine-tuning ROS levels might be critical for cardioprotection.},
}
@article {pmid35711292,
year = {2022},
author = {Bhattacharjee, R and Nandi, A and Mitra, P and Saha, K and Patel, P and Jha, E and Panda, PK and Singh, SK and Dutt, A and Mishra, YK and Verma, SK and Suar, M},
title = {Theragnostic application of nanoparticle and CRISPR against food-borne multi-drug resistant pathogens.},
journal = {Materials today. Bio},
volume = {15},
number = {},
pages = {100291},
doi = {10.1016/j.mtbio.2022.100291},
pmid = {35711292},
issn = {2590-0064},
abstract = {Foodborne infection is one of the leading sources of infections spreading across the world. Foodborne pathogens are recognized as multidrug-resistant (MDR) pathogens posing a significant problem in the food industry and healthy consumers resulting in enhanced economic burden, and nosocomial infections. The continued search for enhanced microbial detection tools has piqued the interest of the CRISPR-Cas system and Nanoparticles. CRISPR-Cas system is present in the bacterial genome of some prokaryotes and is repurposed as a theragnostic tool against MDR pathogens. Nanoparticles and composites have also emerged as an efficient tool in theragnostic applications against MDR pathogens. The diagnostic limitations of the CRISPR-Cas system are believed to be overcome by a synergistic combination of the nanoparticles system and CRISPR-Cas using nanoparticles as vehicles. In this review, we have discussed the diagnostic application of CRISPR-Cas technologies along with their potential usage in applications like phage resistance, phage vaccination, strain typing, genome editing, and antimicrobial. we have also elucidated the antimicrobial and detection role of nanoparticles against foodborne MDR pathogens. Moreover, the novel combinatorial approach of CRISPR-Cas and nanoparticles for their synergistic effects in pathogen clearance and drug delivery vehicles has also been discussed.},
}
@article {pmid35705772,
year = {2022},
author = {Wang, Y and Huang, C and Zhao, W},
title = {Recent advances of the biological and biomedical applications of CRISPR/Cas systems.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {35705772},
issn = {1573-4978},
support = {2020M683751//Postdoctoral Research Foundation of China/ ; 2021JQ-559//Natural Science Foundation of Shaanxi Province/ ; },
abstract = {The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas) system, referred to as CRISPR/Cas system, has attracted significant interest in scientific community due to its great potential in translating into versatile therapeutic tools in biomedical field. For instance, a myriad of studies has demonstrated that the CRISPR/Cas system is capable of detecting various types of viruses, killing antibiotic-resistant bacteria, treating inherited genetic diseases, and providing new strategies for cancer therapy. Furthermore, CRISPR/Cas systems are also exploited as research tools such as genome engineering tool that allows researchers to interrogate the biological roles of unexplored genes or uncover novel functions of known genes. Additionally, the CRISPR/Cas system has been employed to edit the genome of a wide range of eukaryotic, prokaryotic organisms and experimental models, including but not limited to mammalian cells, mice, zebrafish, plants, yeast, and Escherichia coli. The present review mainly focuses on summarizing recent discoveries regarding the type II CRISPR/Cas9 and type VI CRISPR/Cas13a systems to give researchers a glimpse of their potential applications in the biological and biomedical field.},
}
@article {pmid35705709,
year = {2022},
author = {},
title = {Caribou's first CRISPR CAR-T impresses.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {807},
doi = {10.1038/s41587-022-01371-6},
pmid = {35705709},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Immunotherapy, Adoptive ; Receptors, Antigen, T-Cell/genetics ; *Receptors, Chimeric Antigen/metabolism ; *Reindeer/metabolism ; },
}
@article {pmid35705705,
year = {2022},
author = {},
title = {CRISPR technology.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {832},
doi = {10.1038/s41587-022-01359-2},
pmid = {35705705},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Technology ; },
}
@article {pmid35707386,
year = {2022},
author = {Zhang, YY and Li, SQ and Song, Y and Wang, P and Song, XG and Zhu, WF and Wang, DM},
title = {Silencing the ADAM9 Gene through CRISPR/Cas9 Protects Mice from Alcohol-Induced Acute Liver Injury.},
journal = {BioMed research international},
volume = {2022},
number = {},
pages = {5110161},
doi = {10.1155/2022/5110161},
pmid = {35707386},
issn = {2314-6141},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Gene Editing ; Liver ; Mice ; *RNA, Guide/genetics ; },
abstract = {Alcoholic liver injury is a major global public health concern at present. The ADAM9 gene plays a crucial role in the occurrence and development of various liver diseases, but its role in acute alcoholic liver injury remains ambiguous. In this study, a chimeric single-guide RNA targeting the genomic regions of mouse ADAM9 was designed using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. Next, the role of ADAM9 in acute alcoholic liver injury in vitro in cultured mouse cells and in vivo in a hydrodynamic injection-based alcoholic liver injury mouse model was documented. The findings of this study suggest that ADAM9 induces by regulating cell proliferation, apoptosis, and stress metabolism in mice. Thus, inhibiting the expression of ADAM9 gene using CRISPR/Cas9 can attenuate alcohol-induced acute liver injury in mice.},
}
@article {pmid35703314,
year = {2022},
author = {Inwood, SL and Tian, L and Parratt, K and Maragh, S and Wang, L},
title = {Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing.},
journal = {BioTechniques},
volume = {72},
number = {6},
pages = {279-286},
doi = {10.2144/btn-2022-0015},
pmid = {35703314},
issn = {1940-9818},
support = {//Material Measurement Laboratory/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Flow Cytometry ; *Gene Editing/methods ; },
abstract = {Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.},
}
@article {pmid35701478,
year = {2022},
author = {Rosello, M and Serafini, M and Mignani, L and Finazzi, D and Giovannangeli, C and Mione, MC and Concordet, JP and Del Bene, F},
title = {Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3435},
pmid = {35701478},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome/genetics ; Zebrafish/genetics/metabolism ; },
abstract = {Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.},
}
@article {pmid35701417,
year = {2022},
author = {Teufel, M and Klein, CA and Mager, M and Sobetzko, P},
title = {A multifunctional system for genome editing and large-scale interspecies gene transfer.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3430},
pmid = {35701417},
issn = {2041-1723},
support = {SO 1447/5-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Escherichia coli/genetics ; *Gene Editing ; Genetic Therapy ; Genome, Bacterial/genetics ; },
abstract = {CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA extraction. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. The modular character facilitates DNA library applications, and recycling of standardized parts. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process.},
}
@article {pmid35701408,
year = {2022},
author = {Kosicki, M and Allen, F and Steward, F and Tomberg, K and Pan, Y and Bradley, A},
title = {Cas9-induced large deletions and small indels are controlled in a convergent fashion.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3422},
pmid = {35701408},
issn = {2041-1723},
support = {098051/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA Repair/genetics ; INDEL Mutation ; Mice ; },
abstract = {Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.},
}
@article {pmid35696571,
year = {2022},
author = {Simonetti, B and Daly, JL and Simón-Gracia, L and Klein, K and Weeratunga, S and Antón-Plágaro, C and Tobi, A and Hodgson, L and Lewis, PA and Heesom, KJ and Shoemark, DK and Davidson, AD and Collins, BM and Teesalu, T and Yamauchi, Y and Cullen, PJ},
title = {ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {25},
pages = {e2201980119},
doi = {10.1073/pnas.2201980119},
pmid = {35696571},
issn = {1091-6490},
support = {104568/Z/14/Z//Wellcome Trust (WT)/ ; 20260/Z/20/Z//Wellcome Trust (WT)/ ; MR/L007363/1//UKRI | Medical Research Council (MRC)/ ; MR/P018807/1//UKRI | Medical Research Council (MRC)/ ; RSRP/R1/211004//Royal Society (The Royal Society)/ ; 203959/Z/16/Z//Wellcome Trust (WT)/ ; BB/L01386X/1//UKRI | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; APP1136021//Department of Health | National Health and Medical Research Council (NHMRC)/ ; APP1156493//Department of Health | National Health and Medical Research Council (NHMRC)/ ; No 856581 - CHUbVi).//EC | H2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC)/ ; MR/W005611/1//UKRI | Medical Research Council (MRC)/ ; Project No. 2014-2020.4.01.15-0012//EC | H2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC)/ ; PRG230 and EAG79//Eesti Teadusagentuur (ETAg)/ ; },
mesh = {*COVID-19/metabolism/virology ; CRISPR-Cas Systems ; *Endosomes/virology ; Gene Deletion ; *Host-Pathogen Interactions ; Humans ; Nanoparticles ; *Neuropilin-1/genetics/metabolism ; Proteomics ; *SARS-CoV-2/metabolism ; Sorting Nexins/metabolism ; Spike Glycoprotein, Coronavirus/metabolism ; },
abstract = {Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.},
}
@article {pmid35695893,
year = {2022},
author = {Coon, BG and Timalsina, S and Astone, M and Zhuang, ZW and Fang, J and Han, J and Themen, J and Chung, M and Yang-Klingler, YJ and Jain, M and Hirschi, KK and Yamamato, A and Trudeau, LE and Santoro, M and Schwartz, MA},
title = {A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.},
journal = {The Journal of cell biology},
volume = {221},
number = {7},
pages = {},
doi = {10.1083/jcb.202109144},
pmid = {35695893},
issn = {1540-8140},
support = {AHA#13POST16720007//American Heart Association/ ; R01 HL75092/NH/NIH HHS/United States ; //Leducq Trans-Atlantic Network/ ; ERC-CoG 647057/ERC_/European Research Council/International ; 20119//Associazione Italiana Ricerca sul Cancro/ ; //Krembil Foundation/ ; //Fondation Brain Canada/ ; /CAPMC/CIHR/Canada ; },
mesh = {Atherosclerosis/pathology ; CRISPR-Cas Systems ; Calcium Signaling ; *Endothelial Cells/metabolism ; Humans ; Inflammation ; *Kruppel-Like Transcription Factors/genetics/metabolism ; MAP Kinase Kinase 5 ; MAP Kinase Kinase Kinase 2 ; MAP Kinase Kinase Kinase 3 ; *Mitochondria/metabolism ; Mitogen-Activated Protein Kinase 7/genetics/metabolism ; Reactive Oxygen Species ; *Stress, Mechanical ; },
abstract = {Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.},
}
@article {pmid35597342,
year = {2022},
author = {Liu, L and Duan, JJ and Wei, XY and Hu, H and Wang, YB and Jia, PP and Pei, DS},
title = {Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.},
journal = {The Science of the total environment},
volume = {838},
number = {Pt 2},
pages = {156048},
doi = {10.1016/j.scitotenv.2022.156048},
pmid = {35597342},
issn = {1879-1026},
mesh = {Animals ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; *Recombinases/genetics ; *Staphylococcal Infections ; Staphylococcus aureus/genetics ; },
abstract = {Staphylococcus aureus (S. aureus) is an important opportunistic human and animal pathogen that can cause a wide diversity of infections. Due to its environmental health risks, it is crucial to establish a time-saving, high-throughput, and highly sensitive technique for water quality surveillance. In this study, we developed a novel method to detect S. aureus in the water environment based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a. This method utilizes isothermal amplification of nucleic acids and the trans-cleavage activity of the CRISPR/Cas12a system to generate fluorescence signals with a single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter and a naked-eye detected lateral flow assay (LFA). Our RPA-CRISPR/Cas12a detection system can reduce the detection time to 35 min and enhance the high-throughput detection threshold to ≥5 copies of pathogen DNA, which is more sensitive than that of reported. Moreover, in the lower reaches of the Jialing River in Chongqing, China, 10 water samples from the mainstream and 7 ones from tributaries were successfully monitored S. aureus for less than 35 min using RPA-CRISPR/Cas12a detection system. Taken together, a novel high-throughput RPA-CRISPR detection was established and firstly applied for sensitively monitoring S. aureus in the natural water environment.},
}
@article {pmid35588272,
year = {2022},
author = {Meliawati, M and May, T and Eckerlin, J and Heinrich, D and Herold, A and Schmid, J},
title = {Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {11},
pages = {e0016422},
doi = {10.1128/aem.00164-22},
pmid = {35588272},
issn = {1098-5336},
mesh = {Bacillus subtilis/genetics ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Paenibacillus polymyxa/genetics/metabolism ; Point Mutation ; },
abstract = {Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.},
}
@article {pmid35586918,
year = {2022},
author = {He, D and Liu, G and Yang, J and Jiang, X and Wang, H and Fan, Y and Gong, S and Wei, F and Diao, Y and Tang, Y},
title = {Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics.},
journal = {Bioconjugate chemistry},
volume = {33},
number = {6},
pages = {1232-1240},
doi = {10.1021/acs.bioconjchem.2c00200},
pmid = {35586918},
issn = {1520-4812},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Flavivirus/genetics ; RNA ; Recombinases ; },
abstract = {In China, drastic losses in the economy have been caused by the Tembusu virus (TMUV), the causative agent of the egg-drop syndrome, to the duck-raising industry. To succeed in preventing and controlling infections, extant techniques must be upgraded to achieve fast detection of viruses. This work is the first attempt to present the development of a recombinase polymerase amplification (RPA)-based clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas13a approach for the TMUV infection diagnosis, where the CRISPR-Cas13a system is exploited, i.e., the programmability of CRISPR RNA (crRNA) and the promiscuous RNase collateral cleavage of Cas13a upon recognition of target RNAs. A prokaryotic expression system was utilized for the expression of LwCas13a soluble protein, while its purification was accomplished by nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the design of a particular crRNA, the target used was the TMUV NS3 RNA transcribed in vitro. The signals used for the Cas13a activity validation were an RNA-bound fluorescent group (single-stranded) and a quenching fluorophore. In the present work, a specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a was established by combining Cas13a with T7 transcription and RPA for sensitive detection of TMUV at room temperature. This system can detect 102 copies of the target TMUV DNA standard/μL within 50 min. A comparison revealed that the specificity was superior to that for other avian viruses. Furthermore, the RPA-based CRISPR-Cas13a detection system was successfully applied for clinical samples, and its performance is comparable to the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR). Being satisfyingly reliable, simple, specific, and sensitive, our RPA-based CRISPR-Cas13a detection system could be expanded and universalized for identifying other viruses, enabling quick detection in the field with a portable lateral flow dipstick.},
}
@article {pmid35578739,
year = {2022},
author = {Mu, K and Ren, X and Yang, H and Zhang, T and Yan, W and Yuan, F and Wu, J and Kang, Z and Han, D and Deng, R and Zeng, Q},
title = {CRISPR-Cas12a-Based Diagnostics of Wheat Fungal Diseases.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {23},
pages = {7240-7247},
doi = {10.1021/acs.jafc.1c08391},
pmid = {35578739},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Fusarium/metabolism ; *Mycoses/genetics ; Plant Diseases/microbiology ; RNA, Guide/metabolism ; Triticum/genetics/microbiology ; },
abstract = {Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum (F. graminearum) infection, reduces crop yield and contaminates grain with mycotoxins. We report a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based nucleic acid assay for an early and rapid diagnosis of wheat FHB. Guide RNA (gRNA) was screened for highly specific recognition of polymerase chain reaction (PCR) amplicon of the internal transcribed spacer (ITS) region and the transcription elongation factor 1α (EF1α) of F. graminearum. The trans-activation of Cas12a protein cleaves the single-stranded DNA probes with the terminal fluorophore and quencher groups, thus allowing us to report the presence of ITS and EF1α of F. graminearum. Owing to the dual recognition process through PCR primers and gRNA hybridization, the approach realized specific discrimination of F. graminearum from other pathogenic fungi. It also allowed us to detect as low as 1 fg/μL total DNA from F. graminearum, which is sufficient to diagnose a 4 day F. graminearum infection. CRISPR-Cas12a-based nucleic acid assay promises the molecular diagnosis of crop diseases and broadens the application of CRISPR tools.},
}
@article {pmid35561628,
year = {2022},
author = {Cai, G and Lin, Z and Shi, S},
title = {Development and expansion of the CRISPR/Cas9 toolboxes for powerful genome engineering in yeast.},
journal = {Enzyme and microbial technology},
volume = {159},
number = {},
pages = {110056},
doi = {10.1016/j.enzmictec.2022.110056},
pmid = {35561628},
issn = {1879-0909},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Guide/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Yeasts represent a group of the microorganisms most frequently seen in biotechnology. Recently, the class 2 type II CRISPR system (CRISPR/Cas9) has become the principal toolbox for genome editing. By efficiently implementing genetic manipulations such as gene integration/knockout, base editor, and transcription regulation, the development of biotechnological applications in yeasts has been extensively promoted. The genome-level tools based on CRISPR/Cas9, used for screening and identifying functional genes/gene clusters, are also advancing. In general, CRISPR/Cas9-assisted editing tools have gradually become standardized and function as host-orthogonal genetic systems, which results in time-saving for strain engineering and biotechnological application processes. In this review, we summarize the key points of the basic elements in the CRISPR/Cas9 system, including Cas9 variants, guide RNA, donors, and effectors. With a focus on yeast, we have also introduced the development of various CRISPR/Cas9 systems and discussed their future possibilities.},
}
@article {pmid35523179,
year = {2022},
author = {Breau, KA and Ok, MT and Gomez-Martinez, I and Burclaff, J and Kohn, NP and Magness, ST},
title = {Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.},
journal = {Stem cell reports},
volume = {17},
number = {6},
pages = {1493-1506},
doi = {10.1016/j.stemcr.2022.04.005},
pmid = {35523179},
issn = {2213-6711},
support = {P30 DK034987/DK/NIDDK NIH HHS/United States ; R01 DK109559/DK/NIDDK NIH HHS/United States ; R01 DK115806/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Targeting ; Humans ; Organoids ; Stem Cells ; Transfection ; },
abstract = {Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.},
}
@article {pmid35411116,
year = {2022},
author = {Kim, Y and Lee, S and Cho, S and Park, J and Chae, D and Park, T and Minna, JD and Kim, HH},
title = {High-throughput functional evaluation of human cancer-associated mutations using base editors.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {874-884},
pmid = {35411116},
issn = {1546-1696},
support = {the MD-PhD/Medical Scientist Training Program//Korea Health Industry Development Institute (KHIDI)/ ; 2017R1A2B3004198//National Research Foundation of Korea (NRF)/ ; 2017M3A9B4062403//National Research Foundation of Korea (NRF)/ ; 2018R1A5A2025079//National Research Foundation of Korea (NRF)/ ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mutation/genetics ; *Neoplasms/genetics ; RNA, Guide/genetics ; },
abstract = {Comprehensive phenotypic characterization of the many mutations found in cancer tissues is one of the biggest challenges in cancer genomics. In this study, we evaluated the functional effects of 29,060 cancer-related transition mutations that result in protein variants on the survival and proliferation of non-tumorigenic lung cells using cytosine and adenine base editors and single guide RNA (sgRNA) libraries. By monitoring base editing efficiencies and outcomes using surrogate target sequences paired with sgRNA-encoding sequences on the lentiviral delivery construct, we identified sgRNAs that induced a single primary protein variant per sgRNA, enabling linking those mutations to the cellular phenotypes caused by base editing. The functions of the vast majority of the protein variants (28,458 variants, 98%) were classified as neutral or likely neutral; only 18 (0.06%) and 157 (0.5%) variants caused outgrowing and likely outgrowing phenotypes, respectively. We expect that our approach can be extended to more variants of unknown significance and other tumor types.},
}
@article {pmid35388425,
year = {2022},
author = {Ezura, H},
title = {Letter to the Editor: The World's First CRISPR Tomato Launched to a Japanese Market: The Social-Economic Impact of its Implementation on Crop Genome Editing.},
journal = {Plant &amp; cell physiology},
volume = {63},
number = {6},
pages = {731-733},
doi = {10.1093/pcp/pcac048},
pmid = {35388425},
issn = {1471-9053},
support = {//Japan Science and Technology Agency/ ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Plant/genetics ; Japan ; *Lycopersicon esculentum/genetics ; },
}
@article {pmid35704073,
year = {2022},
author = {Dash, HR and Arora, M},
title = {CRISPR-CasB technology in forensic DNA analysis: challenges and solutions.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35704073},
issn = {1432-0614},
abstract = {CRISPR-Cas technology has revolutionized the field of biotechnology with its precise therapeutic use from genetic as well as infectious diseases point of view. This technology is rapidly evolving to single tool enabling site-directed cut in the genome and highly specific activation or inhibition of gene expression or the exchange of single bases. Besides clinical applications, CRISPR-Cas technology has also shown promising use in the field of forensic DNA analysis. Enrichment of targeted genetic marker for identification followed by sequencing and non-PCR-dependent technique ensures the use of CRISPR-Cas technology in challenging forensic biological samples. The use of this advanced technology is also deemed helpful in mixed profile attribution, mostly in LCN contributors and the generation of a useful DNA profile in degraded samples. Besides its useful applications in forensic DNA analysis, CRISPR-Cas technology poses a huge threat from the generation of ghost DNA profiles by modification/alteration of target genetic markers. Forensic DNA analysts should carry out analysis of additional markers such as non-CODIS markers, Y-, X-chromosome markers, and mitochondrial DNA sequencing in a suspected ghost DNA profile case. KEY POINTS: • CRISPR-Cas9 technique is useful in analyzing LCN, mixed and degraded samples • Alteration of DNA using this technique can lead to generation of ghost DNA profiles • Alternative genetic markers and methylation pattern may detect a ghost DNA profile.},
}
@article {pmid35703039,
year = {2022},
author = {Kurushima, J and Tomita, H},
title = {Advances of genetic engineering in Streptococci and Enterococci.},
journal = {Microbiology and immunology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1348-0421.13015},
pmid = {35703039},
issn = {1348-0421},
abstract = {In the post-genome era, reverse genetic engineering is an indispensable methodology for experimental molecular biology to provide a deeper understanding of the principal relationship between genomic features and biological phenotypes. Technically, genetic engineering is carried out through allele replacement of a target genomic locus with a designed nucleotide sequence, so called site-directed mutagenesis. To artificially manipulate allele replacement through homologous recombination, researchers have improved various methodologies that are optimized to the bacterial species of interest. Here, we review widely used genetic engineering technologies, particularly for streptococci and enterococci, and recent advances that enable more effective and flexible manipulation. The development of genetic engineering has been promoted by synthetic biology approaches based on basic biology knowledge of horizontal gene transfer systems, such as natural conjugative transfer, natural transformation, and the CRISPR/Cas system. Therefore, this review also describes basic insights into molecular biology that underlie improvements in genetic engineering technology. This article is protected by copyright. All rights reserved.},
}
@article {pmid35701838,
year = {2022},
author = {Liu, Z and Yan, Q and Jiang, C and Li, J and Jian, H and Fan, L and Zhang, R and Xiao, X and Meng, D and Liu, X and Wang, J and Yin, H},
title = {Growth rate determines prokaryote-provirus network modulated by temperature and host genetic traits.},
journal = {Microbiome},
volume = {10},
number = {1},
pages = {92},
pmid = {35701838},
issn = {2049-2618},
support = {91851206//National Natural Science Foundation of China/ ; 91851117//National Natural Science Foundation of China/ ; 41877345//National Nature Science Foundation of China/ ; 2018YFE0110200//National Key Research and Development Program by the Ministry of Science and Technology of China/ ; S2020GCZDYF1057//Key Research and Development Program of Hunan Province/ ; },
abstract = {BACKGROUND: Prokaryote-virus interactions play key roles in driving biogeochemical cycles. However, little is known about the drivers shaping their interaction network structures, especially from the host features. Here, we compiled 7656 species-level genomes in 39 prokaryotic phyla across environments globally and explored how their interaction specialization is constrained by host life history traits, such as growth rate.<br><br>RESULTS: We first reported that host growth rate indicated by the reverse of minimal doubling time was negatively related to interaction specialization for host in host-provirus network across various ecosystems and taxonomy groups. Such a negative linear growth rate-specialization relationship (GrSR) was dependent on host optimal growth temperature (OGT), and stronger toward the two gradient ends of OGT. For instance, prokaryotic species with an OGT ≥ 40 °C showed a stronger GrSR (Pearson's r = -0.525, P < 0.001). Significant GrSRs were observed with the presences of host genes in promoting the infection cycle at stages of adsorption, establishment, and viral release, but nonsignificant with the presence of immune systems, such as restriction-modification systems and CRISPR-Cas systems. Moreover, GrSR strength was increased with the presence of temperature-dependent lytic switches, which was also confirmed by mathematical modeling.<br><br>CONCLUSIONS: Together, our results advance our understanding of the interactions between prokaryotes and proviruses and highlight the importance of host growth rate in interaction specialization during lysogenization. Video Abstract.},
}
@article {pmid35701400,
year = {2022},
author = {Liang, F and Zhang, Y and Li, L and Yang, Y and Fei, JF and Liu, Y and Qin, W},
title = {SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3421},
pmid = {35701400},
issn = {2041-1723},
abstract = {Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis.},
}
@article {pmid35696040,
year = {2022},
author = {Kalds, P and Crispo, M and Li, C and Tesson, L and Anegón, I and Chen, Y and Wang, X and Menchaca, A},
title = {Generation of Double-Muscled Sheep and Goats by CRISPR /Cas9-Mediated Knockout of the Myostatin Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {295-323},
pmid = {35696040},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Goats/genetics/metabolism ; Muscle, Skeletal/metabolism ; *Myostatin/genetics ; Sheep/genetics ; },
abstract = {The myostatin (MSTN) gene has shown to play a critical role in the regulation of skeletal muscle mass, and the translational inhibition of this gene has shown increased muscle mass, generating what is known as "double-muscling phenotype." Disruption of the MSTN gene expression using the CRISPR/Cas9 genome-editing system has shown improved muscle development and growth rates in livestock species, including sheep and goats. Here, we describe procedures for the generation of MSTN knockout sheep and goats using the microinjection approach of the CRISPR/Cas9 system, including the selection of targeting sgRNAs, the construction of CRISPR/Cas9 targeting vector, the in vitro examination of system efficiency, the in vivo targeting to generate MSTN knockout founders, the genomic and phenotypic characterization of the generated offspring, and the assessment of off-target effects in gene-edited founders through targeted validation of predicted off-target sites, as well as genome-wide off-target analysis by whole-genome sequencing. Editing the MSTN gene using the CRISPR/Cas9 system might be a rapid and promising alternative to promote meat production in livestock.},
}
@article {pmid35696038,
year = {2022},
author = {Liu, J and Verma, PJ},
title = {Generating a Heat-Tolerance Mouse Model.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {259-272},
pmid = {35696038},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Female ; Gene Editing ; *Hot Temperature ; Mice ; Recombinational DNA Repair ; Zygote/metabolism ; },
abstract = {Creating mouse models of human genetic disease (Gurumurthy and Lloyd, Dis Models Mech 12(1):dmm029462, 2019) and livestock trait (Schering et al. Arch Physiol Biochem 121(5):194-205, 2015; Habiela et al. J Gen Virol 95 (Pt 11):2329-2345, 2014) have been proven to be a useful tool for understanding the mechanism behind the phenotypes and fundamental and applied research in livestock. A single base pair deletion of prolactin receptor (PRLR) has an impact on hair morphology phenotypes beyond its classical roles in lactation in cattle, the so-called slick cattle (Littlejohn et al. Nat Commun 5:5861, 2014). Here, we generate a knock-in mouse model by targeting the specific locus of PRLR gene using Cas9-mediated genome editing via homology-directed repair (HDR) in mouse zygotes. The mouse model carrying the identical PRLR mutation in slick cattle may provide a useful animal model to study the pathway of thermoregulation and the mechanism of heat-tolerance in the livestock.},
}
@article {pmid35696037,
year = {2022},
author = {Goldsmith, T and Bondareva, A and Webster, D and Voigt, AL and Su, L and Carlson, DF and Dobrinski, I},
title = {Targeted Gene Editing in Porcine Germ Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {245-258},
pmid = {35696037},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Male ; Mutation ; Spermatogonia ; Swine ; },
abstract = {As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive. Germline genetic modification through gene edited spermatogonia provides an effective alternative to how these models are developed. Here, we report an improved technique of gene editing in spermatogonia of pigs using CRISPR-Cas9 to generate different edits that reflect the genotypes of human diseases.},
}
@article {pmid35696035,
year = {2022},
author = {Bunting, MD and Pfitzner, C and Gierus, L and White, M and Piltz, S and Thomas, PQ},
title = {Generation of Gene Drive Mice for Invasive Pest Population Suppression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {203-230},
pmid = {35696035},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Gene Editing/methods ; Introduced Species ; Mice ; Mice, Transgenic ; Transgenes ; },
abstract = {Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquitos, flies, and yeast. For example, studies in Anopheles mosquitos have demonstrated >95% transmission of CRISPR-engineered gene drive constructs, providing a possible strategy for malaria control. Recently published studies have also indicated that it may be possible to develop gene drive technology in invasive rodents such as mice. Here, we discuss the prospects for gene drive development in mice, including synthetic "homing drive" and X-shredder strategies as well as modifications of the naturally occurring t haplotype. We also provide detailed protocols for generation of gene drive mice through incorporation of plasmid-based transgenes in a targeted and non-targeted manner. Importantly, these protocols can be used for generating transgenic mice for any project that requires insertion of kilobase-scale transgenes such as knock-in of fluorescent reporters, gene swaps, overexpression/ectopic expression studies, and conditional "floxed" alleles.},
}
@article {pmid35696034,
year = {2022},
author = {Paulo, DF and Williamson, ME and Scott, MJ},
title = {CRISPR/Cas9 Genome Editing in the New World Screwworm and Australian Sheep Blowfly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {173-201},
pmid = {35696034},
issn = {1940-6029},
mesh = {Animals ; Australia ; CRISPR-Cas Systems/genetics ; Calliphoridae ; *Diptera/genetics ; *Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Blowflies are of interest for medical applications (maggot therapy), forensic investigations, and for evolutionary developmental studies such as the evolution of parasitism. It is because of the latter that some blowflies such as the New World screwworm and the Australian sheep blowfly are considered major economic pests of livestock. Due to their importance, annotated assembled genomes for several species are now available. Here, we present a detailed guide for using the Streptococcus pyogenes Cas9 RNA-guided nuclease to efficiently generate both knockout and knock-in mutations in screwworm and sheep blowfly. These methods should accelerate genetic investigations in these and other closely related species and lead to a better understanding of the roles of selected genes in blowfly development and behavior.},
}
@article {pmid35696032,
year = {2022},
author = {Alberio, V and Savy, V and Salamone, DF},
title = {CRISPR-on for Endogenous Activation of SMARCA4 Expression in Bovine Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {129-148},
pmid = {35696032},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Cell Differentiation ; Cell Line ; Cellular Reprogramming/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR-on system is a programmable, simple, and versatile gene activator that has proven to be efficient in cultured cells from several species and in bovine embryos. This technology allows for the precise and specific activation of single endogenous gene expression and also multiplexed gene expression in a simple fashion. Therefore, CRISPR-on has unique advantages over other activator systems and a wide adaptability for studies in basic and applied science, such as cell reprogramming and cell fate differentiation for regenerative medicine.In this chapter, we describe the materials and methods of the CRISPR-on system for activation of the endogenous SMARCA4 expression in bovine embryos.},
}
@article {pmid35696031,
year = {2022},
author = {Delerue, F and Ittner, LM},
title = {Microinjection of Zygotes for CRISPR/Cas9-Mediated Insertion of Transgenes into the Murine Rosa26 Safe Harbor.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {115-128},
pmid = {35696031},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Microinjections ; Transgenes ; *Zygote ; },
abstract = {Genetically modified (GM) mice are widely used in biomedical research because they can address complex questions in an in-vivo setting that could not otherwise be addressed in-vitro. Microinjection of zygotes remains the most common technique to generate GM animals to date. Here, we describe the targeted insertion (knock-in) of transgenes by microinjection of 1-cell or 2-cell stage embryos into the murine Rosa26 safe harbor.},
}
@article {pmid35696030,
year = {2022},
author = {Gu, J and Rollo, B and Sumer, H and Cromer, B},
title = {Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {99-114},
pmid = {35696030},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells ; Transgenes ; },
abstract = {CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.},
}
@article {pmid35696029,
year = {2022},
author = {Malaver-Ortega, LF and Rosenbluh, J},
title = {Immortalised Cas9-expressing Cell lines for Gene interrogation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {91-97},
pmid = {35696029},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Genome ; },
abstract = {The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.},
}
@article {pmid35635176,
year = {2022},
author = {Lin, M and Yue, H and Tian, T and Xiong, E and Zhu, D and Jiang, Y and Zhou, X},
title = {Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.},
journal = {Analytical chemistry},
volume = {94},
number = {23},
pages = {8277-8284},
doi = {10.1021/acs.analchem.2c00616},
pmid = {35635176},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics ; Animals ; *COVID-19 ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Glycerol ; Nucleic Acid Amplification Techniques/methods ; Recombinases ; SARS-CoV-2 ; Sensitivity and Specificity ; Swine ; },
abstract = {CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.},
}
@article {pmid35635022,
year = {2022},
author = {Hu, T and Ke, X and Ou, Y and Lin, Y},
title = {CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.},
journal = {Analytical chemistry},
volume = {94},
number = {23},
pages = {8506-8513},
doi = {10.1021/acs.analchem.2c01507},
pmid = {35635022},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Hydrogen Peroxide ; Luminescence ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Tyramine ; },
abstract = {CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.},
}
@article {pmid35700604,
year = {2022},
author = {Kang, W and Liu, L and Yu, P and Zhang, T and Lei, C and Nie, Z},
title = {A switchable Cas12a enabling CRISPR-based direct histone deacetylase activity detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {213},
number = {},
pages = {114468},
doi = {10.1016/j.bios.2022.114468},
pmid = {35700604},
issn = {1873-4235},
abstract = {The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by anti-CRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics.},
}
@article {pmid35699339,
year = {2022},
author = {Ahator, SD and Sagar, S and Zhu, M and Wang, J and Zhang, LH},
title = {Nutrient Availability and Phage Exposure Alter the Quorum-Sensing and CRISPR-Cas-Controlled Population Dynamics of Pseudomonas aeruginosa.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0009222},
doi = {10.1128/msystems.00092-22},
pmid = {35699339},
issn = {2379-5077},
abstract = {Quorum sensing (QS) coordinates bacterial communication and cooperation essential for virulence and dominance in polymicrobial settings. QS also regulates the CRISPR-Cas system for targeted defense against parasitic genomes from phages and horizontal gene transfer. Although the QS and CRISPR-Cas systems are vital for bacterial survival, they undergo frequent selection in response to biotic and abiotic factors. Using the opportunistic Pseudomonas aeruginosa with well-established QS and CRISPR-Cas systems, we show how the social interactions between the acyl-homoserine lactone (AHL)-QS signal-blind mutants (ΔlasRrhlR) and the CRISPR-Cas mutants are affected by phage exposure and nutrient availability. We demonstrate that media conditions and phage exposure alter the resistance and relative fitness of ΔlasRrhlR and CRISPR-Cas mutants while tipping the fitness advantage in favor of the QS signal-blind mutants under nutrient-limiting conditions. We also show that the AHL signal-blind mutants are less selected by phages under QS-inducing conditions than the CRISPR-Cas mutants, whereas the mixed population of the CRISPR-Cas and AHL signal-blind mutants reduce phage infectivity, which can improve survival during phage exposure. Our data reveal that phage exposure and nutrient availability reshape the population dynamics between the ΔlasRrhlR QS mutants and CRISPR-Cas mutants, with key indications for cooperation and conflict between the strains. IMPORTANCE The increase in antimicrobial resistance has created the need for alternative interventions such as phage therapy. However, as previously observed with antimicrobial resistance, phage therapy will not be effective if bacteria evolve resistance and persist in the presence of the phages. The QS is commonly known as an arsenal for bacteria communication, virulence, and regulation of the phage defense mechanism, the CRISPR-Cas system. The QS and CRISPR-Cas systems are widespread in bacteria. However, they are known to evolve rapidly under the influence of biotic and abiotic factors in the bacterial environment, resulting in alteration in bacterial genotypes, which enhance phage resistance and fitness. We believe that adequate knowledge of the influence of environmental factors on the bacterial community lifestyle and phage defense mechanisms driven by the QS and CRISPR-Cas system is necessary for developing effective phage therapy.},
}
@article {pmid35698891,
year = {2022},
author = {Uranga, M and Daròs, JA},
title = {Tools and targets: The dual role of plant viruses in CRISPR-Cas genome editing.},
journal = {The plant genome},
volume = {},
number = {},
pages = {e20220},
doi = {10.1002/tpg2.20220},
pmid = {35698891},
issn = {1940-3372},
support = {FPU17/05503//"Ministerio de Ciencia e Innovaciòn (Spain)"/ ; PID2020-114691RB-I00//"Ministerio de Ciencia e Innovaciòn (Spain)"/ ; },
abstract = {The recent emergence of tools based on the clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized targeted genome editing, thus holding great promise to both basic plant science and precision crop breeding. Conventional approaches for the delivery of editing components rely on transformation technologies or transient delivery to protoplasts, both of which are time-consuming, laborious, and can raise legal concerns. Alternatively, plant RNA viruses can be used as transient delivery vectors of CRISPR-Cas reaction components, following the so-called virus-induced genome editing (VIGE). During the last years, researchers have been able to engineer viral vectors for the delivery of CRISPR guide RNAs and Cas nucleases. Considering that each viral vector is limited to its molecular biology properties and a specific host range, here we review recent advances for improving the VIGE toolbox with a special focus on strategies to achieve tissue-culture-free editing in plants. We also explore the utility of CRISPR-Cas technology to enhance biotic resistance with a special focus on plant virus diseases. This can be achieved by either targeting the viral genome or modifying essential host susceptibility genes that mediate in the infection process. Finally, we discuss the challenges and potential that VIGE holds in future breeding technologies.},
}
@article {pmid35693210,
year = {2022},
author = {Liu, Y and Chen, Q and Song, C and Xu, Z and Yang, S and Li, X},
title = {Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101436},
doi = {10.1016/j.xpro.2022.101436},
pmid = {35693210},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Embryonic Stem Cells ; Mice ; *Mouse Embryonic Stem Cells ; Puromycin/pharmacology ; Transfection ; },
abstract = {Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.},
}
@article {pmid35692096,
year = {2022},
author = {Wang, Q and Li, J and Zhu, J and Mao, J and Duan, C and Liang, X and Zhu, L and Zhu, M and Zhang, Z and Lin, F and Guo, R},
title = {Genome-wide CRISPR/Cas9 screening for therapeutic targets in NSCLC carrying wild-type TP53 and receptor tyrosine kinase genes.},
journal = {Clinical and translational medicine},
volume = {12},
number = {6},
pages = {e882},
doi = {10.1002/ctm2.882},
pmid = {35692096},
issn = {2001-1326},
support = {NSFC 81972188//National Natural Science Foundation of China/ ; NSFC 81672962//National Natural Science Foundation of China/ ; NSFC 3217050420//National Natural Science Foundation of China/ ; ZDRCA2016024//Medical Important Talents/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology ; Pemetrexed/therapeutic use ; Receptor Protein-Tyrosine Kinases/genetics/therapeutic use ; Tumor Suppressor Protein p53/genetics/therapeutic use ; },
abstract = {BACKGROUND: Targeted drugs have greatly improved the therapeutic outcome of non-small cell lung cancer (NSCLC) patients compared with conventional chemotherapy, whereas about one-third of patients are so far not suitable for targeted therapy due to lack of known driver oncogenes such as a mutated receptor tyrosine kinase (RTK) genes. In this study, we aimed to identify therapeutic targets for this subgroup of NSCLC patients.<br><br>METHODS: We performed genome-wide CRISPR/Cas9 screens in two NSCLC cell lines carrying wild-type TP53 and receptor tyrosine kinase (wtTP53-RTK) genes using a GeCKO v2.0 lentiviral library (containing 123411 sgRNAs and targeting 19050 genes). MAGeCKFlute was used to analyse and identify candidate genes. Genetic perturbation and pharmacological inhibition were used to validate the result in vitro and in vivo.<br><br>RESULTS: The Genome-wide CRISPR/Cas9 screening identified MDM2 as a potential therapeutic target for wtTP53-RTK NSCLC. Genetic and pharmacological inhibition of MDM2 reduced cell proliferation and impaired tumour growth in the xenograft model, thus confirming the finding of the CRISPR/Cas9 screening. Moreover, treatment by a selective MDM2 inhibitor RG7388 triggered both cell cycle arrest and apoptosis in several NSCLC cell lines. Additionally, RG7388 and pemetrexed synergistically blocked the cell proliferation and growth of wtTP53-RTK tumours but had limited effects for other genotypes.<br><br>CONCLUSIONS: We identified MDM2 as an essential gene and a potential therapeutic target in wtTP53-RTK NSCLC via a genome-wide CRISPR/Cas9 screening. For this subgroup, treatment by RG7388 alone or by its combination with pemetrexed resulted in significant tumour inhibition.},
}
@article {pmid35690427,
year = {2022},
author = {Zeng, H and Zhang, P and Jiang, X and Duan, C and Yu, Y and Wu, Q and Yang, X},
title = {Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification.},
journal = {Analytica chimica acta},
volume = {1217},
number = {},
pages = {340009},
doi = {10.1016/j.aca.2022.340009},
pmid = {35690427},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide/metabolism ; RNA, Viral/genetics ; },
abstract = {The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This "mix &amp; read" RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the "mix &amp; read" mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections.},
}
@article {pmid35688932,
year = {2022},
author = {Harmsen, TJW and Pritchard, CEJ and Riepsaame, J and van de Vrugt, HJ and Huijbers, IJ and Te Riele, H},
title = {HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {9606},
pmid = {35688932},
issn = {2045-2322},
support = {ZonMw-TOP 40-00812-98-10033//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; ZonMw-TOP 40-00812-98-10033//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; ZonMw-TOP 40-00812-98-10033//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; ALW 822.02.010//Aard- en Levenswetenschappen, Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Endonucleases/genetics ; *Gene Editing/methods ; Mice ; RNA, Guide/genetics ; },
abstract = {Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.},
}
@article {pmid35686982,
year = {2022},
author = {Sanchez-Baltasar, R and Garcia-Torralba, A and Nieto-Romero, V and Page, A and Molinos-Vicente, A and López-Manzaneda, S and Ojeda-Pérez, I and Ramirez, A and Navarro, M and Segovia, JC and García-Bravo, M},
title = {Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {422-434},
doi = {10.1089/crispr.2022.0013},
pmid = {35686982},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; Mice, Knockout ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics ; *Zygote/metabolism ; },
abstract = {Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.},
}
@article {pmid35686981,
year = {2022},
author = {Lyons, LA},
title = {It's a Knockout for Cat Allergies?.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {356-357},
doi = {10.1089/crispr.2022.29148.lal},
pmid = {35686981},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; *Hypersensitivity/genetics ; },
}
@article {pmid35686980,
year = {2022},
author = {Shen, X and Lin, Q and Liang, Z and Wang, J and Yang, X and Liang, Y and Liang, H and Pan, H and Yang, J and Zhu, Y and Li, M and Xiang, W and Zhu, H},
title = {Reduction of Pre-Existing Adaptive Immune Responses Against SaCas9 in Humans Using Epitope Mapping and Identification.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {445-456},
doi = {10.1089/crispr.2021.0142},
pmid = {35686980},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Epitope Mapping ; *Gene Editing/methods ; HLA-A Antigens/genetics ; Humans ; Immunity ; Leukocytes, Mononuclear ; *Staphylococcus aureus/genetics ; },
abstract = {The CRISPR-Cas9 system is increasingly being used as a gene editing therapeutic technique in complex diseases but concerns remain regarding the clinical risks of Cas9 immunogenicity. In this study, we detected antibodies against Staphylococcus aureus Cas9 (SaCas9) and anti-SaCas9 T cells in 4.8% and 70% of Chinese donors, respectively. We predicted 135 SaCas9-derived B cell epitopes and 50 SaCas9-derived CD8+ T cell epitopes for HLA-A*24:02, HLA-A*11:01, and HLA-A*02:01. We identified R338 as an immunodominant SaCas9 B cell epitope and SaCas9_200-208 as an immunodominant CD8+ T cell epitope for the three human leukocyte antigen allotypes through immunological assays using sera positive for SaCas9-specific antibodies and peripheral blood mononuclear cells positive for SaCas9-reactive T cells, respectively. We also demonstrated that an SaCas9 variant bearing an R338G substitution reduces B cell immunogenicity and retains its gene-editing function. Our study highlights the immunological risks of the CRISPR-Cas9 system and provides a solution to mitigate pre-existing adaptive immune responses against Cas9 in humans.},
}
@article {pmid35686979,
year = {2022},
author = {Lo Presti, V and Cutilli, A and Dogariu, Y and Müskens, KF and Dünnebach, E and van den Beemt, DAMH and Cornel, AM and Plantinga, M and Nierkens, S},
title = {Gene Editing of Checkpoint Molecules in Cord Blood-Derived Dendritic Cells and CD8+ T Cells Using CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {435-444},
doi = {10.1089/crispr.2021.0133},
pmid = {35686979},
issn = {2573-1602},
mesh = {CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Dendritic Cells ; Fetal Blood ; *Gene Editing/methods ; Humans ; *Neoplasms/genetics ; },
abstract = {Immunotherapies targeting checkpoint inhibition and cell therapies are considered breakthroughs for cancer therapy. However, only a part of patients benefit from these treatments and resistance has been observed. Combining both approaches can potentially further enhance their efficacy. With the advent of gene editing techniques, such as clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), the elimination of checkpoint molecules became available as an option in good manufacturing practice conditions to improve persistence and efficacy. However, no data of CRISPR-Cas9 application have been reported in cord blood (CB)-derived immune cells, potentially usable for allogeneic cell therapy purposes. In this article, we describe the optimization of a protocol to deplete checkpoint molecules at the genomic level using CRISPR-Cas9 technology from CB-dendritic cells (DCs) and CB-CD8+ T cells. The protocol is based on the electroporation of a ribonucleoprotein complex, easily translatable to clinical settings. In both cell types, the knock-out (KO) was successful and did not affect cell viability. CB-DCs showed a decrease in expression of the targeted protein ranging from 50% to 95%, while CB-CD8+ T cells showed a reduction in the range of 25-45%. The procedure did not affect the stimulatory function of the CB-DCs or the response of CB-CD8+ T cells (proliferation or TNF-α production). In conclusion, we optimized a protocol to eliminate checkpoint molecules from CB-derived DCs and CD8+ T cells, with the aim to further implement allogeneic cell therapies for cancer.},
}
@article {pmid35686977,
year = {2022},
author = {Chen, S and Liu, Z and Xie, W and Yu, H and Lai, L and Li, Z},
title = {Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {472-486},
doi = {10.1089/crispr.2021.0143},
pmid = {35686977},
issn = {2573-1602},
mesh = {Adenine ; Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; *Proprotein Convertase 9/genetics ; },
abstract = {Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog-Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids-is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3-ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.},
}
@article {pmid35686976,
year = {2022},
author = {Bradford, J and Chappell, T and Perrin, D},
title = {Rapid Whole-Genome Identification of High Quality CRISPR Guide RNAs with the Crackling Method.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {410-421},
doi = {10.1089/crispr.2021.0102},
pmid = {35686976},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; *RNA, Guide/genetics ; Software ; },
abstract = {The design of CRISPR-Cas9 guide RNAs is not trivial and is a computationally demanding task. Design tools need to identify target sequences that will maximize the likelihood of obtaining the desired cut, while minimizing off-target risk. There is a need for a tool that can meet both objectives while remaining practical to use on large genomes. In this study, we present Crackling, a new method that is more suitable for meeting these objectives. We test its performance on 12 genomes and on data from validation studies. Crackling maximizes guide efficiency by combining multiple scoring approaches. On experimental data, the guides it selects are better than those selected by others. It also incorporates Inverted Signature Slice Lists (ISSL) for faster off-target scoring. ISSL provides a gain of an order of magnitude in speed compared with other popular tools, such as Cas-OFFinder, Crisflash, and FlashFry, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale. Crackling is available at https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.},
}
@article {pmid35686975,
year = {2022},
author = {},
title = {Special Issue on CRISPR Diagnostics.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {353},
doi = {10.1089/crispr.2022.29149.cfp},
pmid = {35686975},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35686974,
year = {2022},
author = {Barrangou, R},
title = {Celebrating a Decade of CRISPR-Fueled Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {354-355},
doi = {10.1089/crispr.2022.29150.rba},
pmid = {35686974},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35609450,
year = {2022},
author = {Hang, XM and Liu, PF and Tian, S and Wang, HY and Zhao, KR and Wang, L},
title = {Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114393},
doi = {10.1016/j.bios.2022.114393},
pmid = {35609450},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; *Hemorrhagic Fever, Ebola/diagnosis/genetics ; Humans ; RNA ; },
abstract = {A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/μL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.},
}
@article {pmid35580124,
year = {2022},
author = {Labude, MK and Xafis, V and Lai, PS and Mills, C},
title = {Vulnerability and the Ethics of Human Germline Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {358-363},
doi = {10.1089/crispr.2021.0053},
pmid = {35580124},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Ethics, Research ; *Gene Editing ; Genome, Human/genetics ; Germ Cells ; Humans ; },
abstract = {The concept of vulnerability has played an important role in theoretical bioethics as well as in numerous authoritative guidelines on research ethics. The concept helps to identify situations in which research participants and other individuals may be at a heightened risk of experiencing harm. However, existing guidance documents on the ethics of human germline gene editing largely fail to make any reference to considerations of vulnerability. In this article, we discuss this oversight and we highlight the role that vulnerability can play in ethical debates about human heritable genome editing. Future guidance documents on germline gene editing should pay attention to considerations of vulnerability and reference these appropriately.},
}
@article {pmid35506982,
year = {2022},
author = {Pulman, J and Sahel, JA and Dalkara, D},
title = {New Editing Tools for Gene Therapy in Inherited Retinal Dystrophies.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {377-388},
doi = {10.1089/crispr.2021.0141},
pmid = {35506982},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Humans ; *Retinal Dystrophies/genetics/therapy ; },
abstract = {Inherited retinal dystrophies (IRDs) are a heterogeneous group of diseases that affect more than 2 million people worldwide. Gene therapy (GT) has emerged as an exciting treatment modality with the potential to provide long-term benefit to patients. Today, gene addition is the most straightforward GT for autosomal recessive IRDs. However, there are three scenarios where this approach falls short. First, in autosomal dominant diseases caused by gain-of-function or dominant-negative mutations, the toxic mutated protein needs to be silenced. Second, a number of IRD genes exceed the limited carrying capacity of adeno-associated virus vectors. Third, there are still about 30% of patients with unknown mutations. In the first two contexts, precise editing tools, such as CRISPR-Cas9, base editors, or prime editors, are emerging as potential GT solutions for the treatment of IRDs. Here, we review gene editing tools based on CRISPR-Cas9 technology that have been used in vivo and the recent first-in-human application of CRISPR-Cas9 in an IRD.},
}
@article {pmid35398513,
year = {2022},
author = {Shin, S and Kim, SH and Park, JH and Lee, JS and Lee, GM},
title = {Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress.},
journal = {Metabolic engineering},
volume = {72},
number = {},
pages = {247-258},
doi = {10.1016/j.ymben.2022.03.017},
pmid = {35398513},
issn = {1096-7184},
mesh = {Animals ; Antibodies, Monoclonal ; *CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Kidney/metabolism ; Mammals/metabolism ; Recombinant Proteins/genetics ; *Recombinases/genetics ; },
abstract = {Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.},
}
@article {pmid35238624,
year = {2022},
author = {Rathbone, T and Ates, I and Fernando, L and Addlestone, E and Lee, CM and Richards, VP and Cottle, RN},
title = {Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {397-409},
doi = {10.1089/crispr.2021.0134},
pmid = {35238624},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Electroporation/methods ; *Gene Editing/methods ; Hepatocytes/metabolism ; Mice ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Adeno-associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models.},
}
@article {pmid35238619,
year = {2022},
author = {Chen, S and Liu, Z and Lai, L and Li, Z},
title = {Efficient C-to-G Base Editing with Improved Target Compatibility Using Engineered Deaminase-nCas9 Fusions.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {389-396},
doi = {10.1089/crispr.2021.0124},
pmid = {35238619},
issn = {2573-1602},
mesh = {Adenine ; Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing ; Genome ; Mice ; },
abstract = {CRISPR-guided DNA base editors (BEs) are potent genome editing tools in biotechnology and medicine. However, conventional cytosine and adenine BEs can only induce base transitions (C-to-T and A-to-G) and cannot induce base transversions. Recently, several C-to-G base editors (CGBEs) were generated and applied in human cells. By comparing them, we found that engineered deaminases rather than additional base excision repair proteins significantly improved the C-to-G efficiency. In addition, significant increase in C-to-G transversions in the GC context were determined by using rationally engineered eAID deaminase. The genome-targeting scope of CGBEs were further expanded by using SpRY Cas9 variant, which then successfully induced stop codon (TAC to TAG) to disrupt Tyr gene in mouse embryos. Taken together, these new CGBEs with engineered deaminase-nCas9 fusions broaden the BE toolsets for efficient gene modification and therapeutic applications.},
}
@article {pmid35051627,
year = {2022},
author = {Postma, ED and Hassing, EJ and Mangkusaputra, V and Geelhoed, J and de la Torre, P and van den Broek, M and Mooiman, C and Pabst, M and Daran, JM and Daran-Lapujade, P},
title = {Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.},
journal = {Metabolic engineering},
volume = {72},
number = {},
pages = {1-13},
doi = {10.1016/j.ymben.2021.12.013},
pmid = {35051627},
issn = {1096-7184},
mesh = {*Anthocyanins ; CRISPR-Cas Systems ; Chromosomes/genetics/metabolism ; *Metabolic Engineering ; Metabolic Networks and Pathways ; Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.},
}
@article {pmid35697070,
year = {2022},
author = {Ren, J and Wang, H and Yang, L and Li, F and Wu, Y and Luo, Z and Chen, Z and Zhang, Y and Feng, Y},
title = {Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {102124},
doi = {10.1016/j.jbc.2022.102124},
pmid = {35697070},
issn = {1083-351X},
abstract = {Prokaryotes evolved CRISPR and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements (MGEs) including harmful phages. To counteract this defense, many MGEs in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are also utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3, but does not hinder the recruitment of Cas2/3 to the CRISPR RNA (crRNA)-guided surveillance complex (the Csy complex). Also in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.},
}
@article {pmid35696906,
year = {2022},
author = {Li, Y and Gao, Z and Lu, J and Wei, X and Qi, M and Yin, Z and Li, T},
title = {SlSnRK2.3 interacts with SlSUI1 to modulate high temperature tolerance via Abscisic acid (ABA) controlling stomatal movement in tomato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {321},
number = {},
pages = {111305},
doi = {10.1016/j.plantsci.2022.111305},
pmid = {35696906},
issn = {1873-2259},
abstract = {Tomato is often exposed to high temperature stress during summer cultivation. Stomatal movement plays important roles in photosynthesis and transpiration which restricts the quality and yield of tomato under environmental stress. To elucidate the mechanism of stomatal movement in high temperature tolerance, SlSnRK2s (sucrose non-fermenting 1-related protein kinases) silenced plants were generated in tomato with CRISPR-Cas 9 gene editing techniques. Through the observation of stomatal parameters, SlSnRK2.3 regulated stomatal closure which was responded to ABA (abscisic acid) and activated signaling pathway of ROS (reactive oxygen species) in high temperature stress. Based on the positive functions of SlSnRK2.3, the cDNA library was generated to investigate interaction proteins of SlSnRK2s. The interaction between SlSnRK2.3 and SlSUI1 (protein translation factor SUI1 homolog) was employed by Yeast two hybrid assay (Y2H), Luciferase (LUC), and Bimolecular fluorescence complementation (BiFC). Finally, the specific interactive sites between SlSnRK2.3 and SlSUI1 were verified by site-directed mutagenesis. The consistent mechanism of SlSnRK2.3 and SlSUI1 in stomatal movement, indicating that SlSUI1 interacted with SlSnRK2.3 through ABA-dependent signaling pathway in high temperature stress. Our results provided evidence for improving the photosynthetic capacity of tomato under high temperature stress, and support the breeding and genetic engineering of tomato over summer facility cultivation.},
}
@article {pmid35696036,
year = {2022},
author = {Crispo, M and Chenouard, V and Dos Santos-Neto, P and Tesson, L and Souza-Neves, M and Heslan, JM and Cuadro, F and Anegón, I and Menchaca, A},
title = {Generation of a Human Deafness Sheep Model Using the CRISPR/Cas System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {233-244},
pmid = {35696036},
issn = {1940-6029},
abstract = {CRISPR/Cas9 system is a promising method for the generation of human disease models by genome editing in non-conventional experimental animals. Medium/large-sized animals like sheep have several advantages to study human diseases and medicine. Here, we present a protocol that describes the generation of an otoferlin edited sheep model via CRISPR-assisted single-stranded oligodinucleotide-mediated Homology-Directed Repair (HDR), through direct cytoplasmic microinjection in in vitro produced zygotes.Otoferlin is a protein expressed in the cochlear inner hair cells, with different mutations at the OTOF gene being the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. By using this protocol, we reported for the first time an OTOF KI model in sheep with 17.8% edited lambs showing indel mutations, and 61.5% of them bearing knock-in mutations by HDR . The reported method establishes the bases to produce a deafness model to test novel therapies in human disorders related to OTOF mutations.},
}
@article {pmid35696033,
year = {2022},
author = {Choo, A and Fung, E and Nguyen, TNM and Okada, A and Crisp, P},
title = {CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {151-171},
pmid = {35696033},
issn = {1940-6029},
abstract = {Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.},
}
@article {pmid35696026,
year = {2022},
author = {Shamshirgaran, Y and Liu, J and Sumer, H and Verma, PJ and Taheri-Ghahfarokhi, A},
title = {Tools for Efficient Genome Editing; ZFN, TALEN, and CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {29-46},
pmid = {35696026},
issn = {1940-6029},
abstract = {The last two decades have marked significant advancement in the genome editing field. Three generations of programmable nucleases (ZFNs, TALENs, and CRISPR-Cas system) have been adopted to introduce targeted DNA double-strand breaks (DSBs) in eukaryotic cells. DNA repair machinery of the cells has been exploited to introduce insertion and deletions (indels) at the targeted DSBs to study function of any gene-of-interest. The resulting indels were generally assumed to be "random" events produced by "error-prone" DNA repair pathways. However, recent advances in computational tools developed to study the Cas9-induced mutations have changed the consensus and implied the "non-randomness" nature of these mutations. Furthermore, CRISPR-centric tools are evolving at an unprecedented pace, for example, base- and prime-editors are the newest developments that have been added to the genome editing toolbox. Altogether, genome editing tools have revolutionized our way of conducting research in life sciences. Here, we present a concise overview of genome editing tools and describe the DNA repair pathways underlying the generation of genome editing outcome.},
}
@article {pmid35696025,
year = {2022},
author = {Montoliu, L},
title = {Historical DNA Manipulation Overview.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {3-28},
pmid = {35696025},
issn = {1940-6029},
abstract = {The history of DNA manipulation for the creation of genetically modified animals began in the 1970s, using viruses as the first DNA molecules microinjected into mouse embryos at different preimplantation stages. Subsequently, simple DNA plasmids were used to microinject into the pronuclei of fertilized mouse oocytes and that method became the reference for many years. The isolation of embryonic stem cells together with advances in genetics allowed the generation of gene-specific knockout mice, later on improved with conditional mutations. Cloning procedures expanded the gene inactivation to livestock and other non-model mammalian species. Lentiviruses, artificial chromosomes, and intracytoplasmic sperm injections expanded the toolbox for DNA manipulation. The last chapter of this short but intense history belongs to programmable nucleases, particularly CRISPR-Cas systems, triggering the development of genomic-editing techniques, the current revolution we are living in.},
}
@article {pmid35694537,
year = {2022},
author = {Maslennikova, A and Mazurov, D},
title = {Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {880030},
doi = {10.3389/fcimb.2022.880030},
pmid = {35694537},
issn = {2235-2988},
abstract = {Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.},
}
@article {pmid35694192,
year = {2022},
author = {Bexte, T and Alzubi, J and Reindl, LM and Wendel, P and Schubert, R and Salzmann-Manrique, E and von Metzler, I and Cathomen, T and Ullrich, E},
title = {CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma.},
journal = {Oncoimmunology},
volume = {11},
number = {1},
pages = {2081415},
doi = {10.1080/2162402X.2022.2081415},
pmid = {35694192},
issn = {2162-402X},
abstract = {Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.},
}
@article {pmid35693174,
year = {2022},
author = {Silva, FDA and Fontes, EPB},
title = {Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein System for Resistance Against Plant Viruses: Applications and Perspectives.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {904829},
doi = {10.3389/fpls.2022.904829},
pmid = {35693174},
issn = {1664-462X},
abstract = {Different genome editing approaches have been used to engineer resistance against plant viruses. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas; CRISPR/Cas) systems to create pinpoint genetic mutations have emerged as a powerful tool for molecular engineering of plant immunity and increasing resistance against plant viruses. This review presents (i) recent advances in engineering resistance against plant viruses by CRISPR/Cas and (ii) an overview of the potential host factors as targets for the CRISPR/Cas system-mediated broad-range resistance and immunity. Applications, challenges, and perspectives in enabling the CRISPR/Cas system for crop protection are also outlined.},
}
@article {pmid35693153,
year = {2022},
author = {Chen, Y and Hu, Y and Wang, X and Luo, S and Yang, N and Chen, Y and Li, Z and Zhou, Q and Li, W},
title = {Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing.},
journal = {Innovation (Cambridge (Mass.))},
volume = {3},
number = {4},
pages = {100264},
doi = {10.1016/j.xinn.2022.100264},
pmid = {35693153},
issn = {2666-6758},
abstract = {The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12i Max , exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12i Max (Cas12i HiFi) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.},
}
@article {pmid35692726,
year = {2022},
author = {Shim, H},
title = {Investigating the Genomic Background of CRISPR-Cas Genomes for CRISPR-Based Antimicrobials.},
journal = {Evolutionary bioinformatics online},
volume = {18},
number = {},
pages = {11769343221103887},
doi = {10.1177/11769343221103887},
pmid = {35692726},
issn = {1176-9343},
abstract = {CRISPR-Cas systems are an adaptive immunity that protects prokaryotes against foreign genetic elements. Genetic templates acquired during past infection events enable DNA-interacting enzymes to recognize foreign DNA for destruction. Due to the programmability and specificity of these genetic templates, CRISPR-Cas systems are potential alternative antibiotics that can be engineered to self-target antimicrobial resistance genes on the chromosome or plasmid. However, several fundamental questions remain to repurpose these tools against drug-resistant bacteria. For endogenous CRISPR-Cas self-targeting, antimicrobial resistance genes and functional CRISPR-Cas systems have to co-occur in the target cell. Furthermore, these tools have to outplay DNA repair pathways that respond to the nuclease activities of Cas proteins, even for exogenous CRISPR-Cas delivery. Here, we conduct a comprehensive survey of CRISPR-Cas genomes. First, we address the co-occurrence of CRISPR-Cas systems and antimicrobial resistance genes in the CRISPR-Cas genomes. We show that the average number of these genes varies greatly by the CRISPR-Cas type, and some CRISPR-Cas types (IE and IIIA) have over 20 genes per genome. Next, we investigate the DNA repair pathways of these CRISPR-Cas genomes, revealing that the diversity and frequency of these pathways differ by the CRISPR-Cas type. The interplay between CRISPR-Cas systems and DNA repair pathways is essential for the acquisition of new spacers in CRISPR arrays. We conduct simulation studies to demonstrate that the efficiency of these DNA repair pathways may be inferred from the time-series patterns in the RNA structure of CRISPR repeats. This bioinformatic survey of CRISPR-Cas genomes elucidates the necessity to consider multifaceted interactions between different genes and systems, to design effective CRISPR-based antimicrobials that can specifically target drug-resistant bacteria in natural microbial communities.},
}
@article {pmid35691167,
year = {2022},
author = {Wang, X and Qin, Y and Huang, Y and Hu, K and Zhao, S and Tian, J},
title = {A sensitive and facile microRNA detection based on CRISPR-Cas12a coupled with strand displacement amplification.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {279},
number = {},
pages = {121476},
doi = {10.1016/j.saa.2022.121476},
pmid = {35691167},
issn = {1873-3557},
abstract = {MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480-580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L-1 ∼ 40 pmol·L-1, the detection limit of 150 fmol·L-1 (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.},
}
@article {pmid35682838,
year = {2022},
author = {Happi Mbakam, C and Rousseau, J and Tremblay, G and Yameogo, P and Tremblay, JP},
title = {Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
doi = {10.3390/ijms23116160},
pmid = {35682838},
issn = {1422-0067},
support = {NA//Jesse's Journey - The Foundation for Gene and Cell Therapy/ ; NA//Centre Thématique de Recherche en Neurosciences/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Dystrophin/genetics/metabolism ; Gene Editing/methods ; HEK293 Cells ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Mutation ; },
abstract = {The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (DMD) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the DMD gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the DMD gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the DMD gene.},
}
@article {pmid35682671,
year = {2022},
author = {Smirnikhina, SA and Zaynitdinova, MI and Sergeeva, VA and Lavrov, AV},
title = {Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
doi = {10.3390/ijms23115992},
pmid = {35682671},
issn = {1422-0067},
support = {none//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Cell Division ; *Gene Editing ; Humans ; Mammals/genetics ; Recombinational DNA Repair ; },
abstract = {Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.},
}
@article {pmid35681543,
year = {2022},
author = {Liu, X and Wang, S and Ai, D},
title = {Predicting CRISPR/Cas9 Repair Outcomes by Attention-Based Deep Learning Framework.},
journal = {Cells},
volume = {11},
number = {11},
pages = {},
doi = {10.3390/cells11111847},
pmid = {35681543},
issn = {2073-4409},
support = {61873027//National Natural Science Foundation of China/ ; AQT-2020-YB6//the National Engineering Laboratory for Agri-product Quality Traceability/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Deep Learning ; Endonucleases/genetics ; Gene Editing/methods ; Mutation/genetics ; },
abstract = {As a simple and programmable nuclease-based genome editing tool, the CRISPR/Cas9 system has been widely used in target-gene repair and gene-expression regulation. The DNA mutation generated by CRISPR/Cas9-mediated double-strand breaks determines its biological and phenotypic effects. Experiments have demonstrated that CRISPR/Cas9-generated cellular-repair outcomes depend on local sequence features. Therefore, the repair outcomes after DNA break can be predicted by sequences near the cleavage sites. However, existing prediction methods rely on manually constructed features or insufficiently detailed prediction labels. They cannot satisfy clinical-level-prediction accuracy, which limit the performance of these models to existing knowledge about CRISPR/Cas9 editing. We predict 557 repair labels of DNA, covering the vast majority of Cas9-generated mutational outcomes, and build a deep learning model called Apindel, to predict CRISPR/Cas9 editing outcomes. Apindel, automatically, trains the sequence features of DNA with the GloVe model, introduces location information through Positional Encoding (PE), and embeds the trained-word vector matrixes into a deep learning model, containing BiLSTM and the Attention mechanism. Apindel has better performance and more detailed prediction categories than the most advanced DNA-mutation-predicting models. It, also, reveals that nucleotides at different positions relative to the cleavage sites have different influences on CRISPR/Cas9 editing outcomes.},
}
@article {pmid35679361,
year = {2022},
author = {Liu, N and Olson, EN},
title = {CRISPR Modeling and Correction of Cardiovascular Disease.},
journal = {Circulation research},
volume = {130},
number = {12},
pages = {1827-1850},
doi = {10.1161/CIRCRESAHA.122.320496},
pmid = {35679361},
issn = {1524-4571},
mesh = {Animals ; CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Mice ; },
abstract = {Cardiovascular disease remains the leading cause of morbidity and mortality in the developed world. In recent decades, extraordinary effort has been devoted to defining the molecular and pathophysiological characteristics of the diseased heart and vasculature. Mouse models have been especially powerful in illuminating the complex signaling pathways, genetic and epigenetic regulatory circuits, and multicellular interactions that underlie cardiovascular disease. The advent of CRISPR genome editing has ushered in a new era of cardiovascular research and possibilities for genetic correction of disease. Next-generation sequencing technologies have greatly accelerated the identification of disease-causing mutations, and advances in gene editing have enabled the rapid modeling of these mutations in mice and patient-derived induced pluripotent stem cells. The ability to correct the genetic drivers of cardiovascular disease through delivery of gene editing components in vivo, while still facing challenges, represents an exciting therapeutic frontier. In this review, we provide an overview of cardiovascular disease mechanisms and the potential applications of CRISPR genome editing for disease modeling and correction. We also discuss the extent to which mice can faithfully model cardiovascular disease and the opportunities and challenges that lie ahead.},
}
@article {pmid35679334,
year = {2022},
author = {Fan, S and Zhang, Z and Song, Y and Zhang, J and Wang, P},
title = {CRISPR/Cas9-mediated targeted mutagenesis of GmTCP19L increasing susceptibility to Phytophthora sojae in soybean.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0267502},
doi = {10.1371/journal.pone.0267502},
pmid = {35679334},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics ; Mutagenesis ; *Phytophthora/physiology ; Plant Breeding ; Plant Diseases/genetics ; Plant Proteins/metabolism ; Soybeans ; Transcription Factors/genetics/metabolism ; },
abstract = {The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors is one of the superfamilies of plant-specific transcription factors involved in plant growth, development, and biotic and abiotic stress. However, there is no report on the research of the TCP transcription factors in soybean response to Phytophthora sojae. In this study, Agrobacterium-mediated transformation was used to introduce the CRISPR/Cas9 expression vector into soybean cultivar "Williams 82" and generated targeted mutants of GmTCP19L gene, which was previously related to involve in soybean responses to P. sojae. We obtained the tcp19l mutants with 2-bp deletion at GmTCP19L coding region, and the frameshift mutations produced premature translation termination codons and truncated GmTCP19L proteins, increasing susceptibility to P. sojae in the T2-generation. These results suggest that GmTCP19L encodes a TCP transcription factor that affects plant defense in soybean. The new soybean germplasm with homozygous tcp19l mutations but the BAR and Cas9 sequences were undetectable using strip and PCR methods, respectively, suggesting directions for the breeding or genetic engineering of disease-resistant soybean plants.},
}
@article {pmid35382978,
year = {2022},
author = {Mou, Z and Zhao, D},
title = {Gene rational design: the dawn of crop breeding.},
journal = {Trends in plant science},
volume = {27},
number = {7},
pages = {633-636},
doi = {10.1016/j.tplants.2022.03.007},
pmid = {35382978},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; *Plant Breeding ; },
abstract = {Limited natural genetic diversity restricts the creation of excellent crops. Modeling-guided rational design represents a promising protein engineering technology to optimize existing genes for desired agronomic traits. Rational design coupled with other engineering approaches could also be applied in artificial gene improvement for the creation of economically valuable crops.},
}
@article {pmid35690249,
year = {2022},
author = {Santiago-Frangos, A and Nemudryi, A and Nemudraia, A and Wiegand, T and Nichols, JE and Krishna, P and Scherffius, AM and Zahl, TR and Wilkinson, RA and Wiedenheft, B},
title = {CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2022.06.002},
pmid = {35690249},
issn = {1095-9130},
abstract = {Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.},
}
@article {pmid35690065,
year = {2022},
author = {Vialetto, E and Yu, Y and Collins, SP and Wandera, KG and Barquist, L and Beisel, CL},
title = {A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2022.05.013},
pmid = {35690065},
issn = {1934-6069},
abstract = {CRISPR-Cas systems must enact robust immunity against foreign genetic material without inducing cytotoxic autoimmunity. For type VI systems that use Cas13 nucleases and recognize RNA targets, immune activation requires extensive CRISPR RNA (crRNA) guide-target complementarity and a target-flanking motif. Here, we report a third requirement shaping the immune response: the expression of the target transcript exceeding a threshold. We found that endogenous non-essential transcripts targeted by crRNAs rarely elicited autoimmunity. Instead, autoimmune induction required over-expressing the targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as a global determinant of cytotoxic autoimmunity and revealed that this threshold shifts with each guide-target pair. This threshold further ensured defense against a lytic bacteriophage yet allowed the tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as an additional criterion for immune defense by RNA-targeting CRISPR-Cas systems, preventing autoimmunity and distinguishing pathogenic and benign invaders.},
}
@article {pmid35689490,
year = {2022},
author = {Bhat, AI and Aman, R and Mahfouz, M},
title = {Onsite detection of plant viruses using isothermal amplification assays.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13871},
pmid = {35689490},
issn = {1467-7652},
abstract = {Plant diseases caused by viruses limit crop production and quality, resulting in significant losses. However, options for managing viruses are limited; for example, as systemic obligate parasites, they cannot be killed by chemicals. Sensitive, robust, affordable diagnostic assays are needed to detect the presence of viruses in plant materials such as seeds, vegetative parts, insect vectors, or alternative hosts, and then prevent or limit their introduction into the field by destroying infected plant materials or controlling insect hosts. Diagnostics based on biological and physical properties are not very sensitive and are time consuming, but assays based on viral proteins and nucleic acids are more specific, sensitive, and rapid. However, most such assays require laboratories with sophisticated equipment and technical skills. By contrast, isothermal-based assays such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are simple, easy to perform, reliable, specific, rapid, and do not require specialized equipment or skills. Isothermal amplification assays can be performed using lateral flow devices, making them suitable for onsite detection or testing in the field. To overcome non-specific amplification and cross-contamination issues, isothermal amplification assays can be coupled with CRISPR/Cas technology. Indeed, the collateral activity associated with some CRISPR/Cas systems has been successfully harnessed for visual detection of plant viruses. Here, we briefly describe traditional methods for detecting viruses, then examine the various isothermal assays that are being harnessed to detect viruses.},
}
@article {pmid35686978,
year = {2022},
author = {Torriano, S and Baulier, E and Garcia Diaz, A and Corneo, B and Farber, DB},
title = {CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {457-471},
doi = {10.1089/crispr.2021.0110},
pmid = {35686978},
issn = {2573-1602},
abstract = {Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.},
}
@article {pmid35686011,
year = {2022},
author = {Parsaeimehr, A and Ebirim, RI and Ozbay, G},
title = {CRISPR-Cas technology a new era in genomic engineering.},
journal = {Biotechnology reports (Amsterdam, Netherlands)},
volume = {34},
number = {},
pages = {e00731},
doi = {10.1016/j.btre.2022.e00731},
pmid = {35686011},
issn = {2215-017X},
abstract = {The CRISPR-Cas systems have offered a flexible, easy-to-use platform to precisely modify and control the genomes of organisms in various fields, ranging from agricultural biotechnology to therapeutics. This system is extensively used in the study of infectious, progressive, and life-threatening genetic diseases for the improvement of quality and quantity of major crops and in the development of sustainable methods for the generation of biofuels. As CRISPR-Cas technology continues to evolve, it is becoming more controllable and precise with the addition of molecular regulators, which will provide benefits for everyone and save many lives. Studies on the constant growth of CRISPR technology are important due to its rapid development. In this paper, we present the current applications and progress of CRISPR-Cas genome editing systems in several fields of research, we further highlight the applications of anti-CRISPR molecules to regulate CRISPR-Cas gene editing systems, and we discuss ethical considerations in CRISPR-Cas applications.},
}
@article {pmid35682737,
year = {2022},
author = {Bhardwaj, P and Kant, R and Behera, SP and Dwivedi, GR and Singh, R},
title = {Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
doi = {10.3390/ijms23116052},
pmid = {35682737},
issn = {1422-0067},
support = {Intramural//Indian Council of Medical Research/ ; },
abstract = {The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.},
}
@article {pmid35674887,
year = {2022},
author = {Andreyenkov, OV and Volkova, EI and Andreyenkova, NG and Demakov, SA},
title = {Using the CRISPR/Cas9 System for Dissection of Functional Sites of the Notch Gene in Drosophila melanogaster.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2472},
number = {},
pages = {1-14},
pmid = {35674887},
issn = {1940-6029},
mesh = {Animals ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Drosophila/metabolism ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics/metabolism ; },
abstract = {The Notch gene is a key factor in the signaling cascade that allows communication between neighboring cells in many organisms, from worms and insects to humans. The relative simplicity of the Notch pathway in Drosophila, combined with a powerful set of molecular and cytogenetic methods, makes this model attractive for studying the fundamental principles of Notch regulation and functioning. Here, using the CRISPR/Cas9 system in combination with homologous recombination, for the first time at the level of the whole organism, we obtained a directed deletion of the 5'-regulatory region and the first exon of the Notch gene, which were replaced by the attP integration site of the ΦC31 phage. Based on this approach, we obtained and characterized new Notch mutations. Thus, a new powerful tool is provided for studying the genetic regulation of the Notch gene and the organization of chromatin at this locus.},
}
@article {pmid35641106,
year = {2022},
author = {Zhai, H and Cui, L and Xiong, Z and Qi, Q and Hou, J},
title = {CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in Saccharomyces cerevisiae.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {5988-6000},
doi = {10.1093/nar/gkac463},
pmid = {35641106},
issn = {1362-4962},
support = {2021YFC2100500//National Key Research and Development Program of China/ ; 31970082//National Natural Science Foundation of China/ ; 2020CXGC010602//Key Research and Development Program of Shandong Province/ ; ZR2020YQ18//National Natural Science Foundation of Shandong Province/ ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/metabolism ; Gene Editing/methods ; Metabolic Engineering/methods ; *Saccharomyces cerevisiae/genetics/metabolism ; Transcriptional Activation ; },
abstract = {Saccharomyces cerevisiae is an important model eukaryotic microorganism and widely applied in fundamental research and the production of various chemicals. Its ability to efficiently and precisely control the expression of multiple genes is valuable for metabolic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)-mediated regulation enables complex gene expression programming; however, the regulation efficiency is often limited by the efficiency of pertinent regulators. Here, we developed CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression in S. cerevisiae. By introducing protein scaffolds (SPY and SunTag systems) to recruit multiple copies of regulators to different nuclease-deficient CRISPR proteins and design optimization, our system amplified gene regulation efficiency significantly. The gene activation and repression efficiencies reached as high as 34.9-fold and 95%, respectively, being 3.8- and 8.6-fold higher than those observed on the direct fusion of regulators with nuclease-deficient CRISPR proteins, respectively. We then applied the orthogonal bifunctional CRISPR-mediated transcriptional regulation system to regulate the expression of genes associated with 3-hydroxypropanoic acid production to deduce that CRISPR-associated regulator recruiting systems represent a robust method for simultaneously regulating multiple genes and rewiring metabolic pathways.},
}
@article {pmid35640600,
year = {2022},
author = {Shin, CH and Park, SC and Park, IG and Kim, H and An, B and Lee, C and Kim, SH and Lee, J and Lee, JM and Oh, SJ},
title = {Cytosolic microRNA-inducible nuclear translocation of Cas9 protein for disease-specific genome modification.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {5919-5933},
doi = {10.1093/nar/gkac431},
pmid = {35640600},
issn = {1362-4962},
support = {SRFC-MA2002-07//Samsung Research Funding &amp; Incubation Center of Samsung Electronics/ ; 2020R1C1C1009507//Korea government/ ; //KIST Institutional Program/ ; },
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Gene Editing ; *MicroRNAs/genetics ; RNA, Messenger/genetics ; },
abstract = {MicroRNA-dependent mRNA decay plays an important role in gene silencing by facilitating posttranscriptional and translational repression. Inspired by this intrinsic nature of microRNA-mediated mRNA cleavage, here, we describe a microRNA-targeting mRNA as a switch platform called mRNA bridge mimetics to regulate the translocation of proteins. We applied the mRNA bridge mimetics platform to Cas9 protein to confer it the ability to translocate into the nucleus via cleavage of the nuclear export signal. This system performed programmed gene editing in vitro and in vivo. Combinatorial treatment with cisplatin and miR-21-EZH2 axis-targeting CRISPR Self Check-In improved sensitivity to chemotherapeutic drugs in vivo. Using the endogenous microRNA-mediated mRNA decay mechanism, our platform is able to remodel a cell's natural biology to allow the entry of precise drugs into the nucleus, devoid of non-specific translocation. The mRNA bridge mimetics strategy is promising for applications in which the reaction must be controlled via intracellular stimuli and modulates Cas9 proteins to ensure safe genome modification in diseased conditions.},
}
@article {pmid35235944,
year = {2022},
author = {Economos, NG and Quijano, E and Carufe, KEW and Perera, JDR and Glazer, PM},
title = {Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {e59},
doi = {10.1093/nar/gkac095},
pmid = {35235944},
issn = {1362-4962},
support = {//NIH NHLBI/ ; U01 AI145965/AI/NIAID NIH HHS/United States ; F30 HL149185/HL/NHLBI NIH HHS/United States ; T32GM007205//MSTP/ ; R01 HL139756/HL/NHLBI NIH HHS/United States ; R01HL139756/NH/NIH HHS/United States ; R35 CA197574/CA/NCI NIH HHS/United States ; F30HL149185//National Research Service Award/ ; R01HL139756/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; *Peptide Nucleic Acids/pharmacology ; *RNA, Guide/genetics ; },
abstract = {Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.},
}
@article {pmid35217297,
year = {2022},
author = {Lu, L and Shen, X and Sun, X and Yan, Y and Wang, J and Yuan, Q},
title = {CRISPR-based metabolic engineering in non-model microorganisms.},
journal = {Current opinion in biotechnology},
volume = {75},
number = {},
pages = {102698},
doi = {10.1016/j.copbio.2022.102698},
pmid = {35217297},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems/genetics ; *Cyanobacteria/genetics ; Gene Editing ; *Metabolic Engineering ; Metabolic Networks and Pathways/genetics ; },
abstract = {Non-model microorganisms possess unique and versatile metabolic characteristics, offering great opportunities as cell factories for biosynthesis of target products. However, lack of efficient genetic tools for pathway engineering represents a big challenge to unlock the full production potential of these microbes. Over the past years, CRISPR systems have been extensively developed and applied to domesticate non-model microorganisms. In this paper, we summarize the current significant advances in designing and constructing CRISPR-mediated genetic modification systems in non-model microorganisms, such as bacteria, fungi and cyanobacteria. We particularly put emphasis on reviewing some successful implementations in metabolic pathway engineering via CRISPR-based genome editing tools. Moreover, the current barriers and future perspectives on improving the editing efficiency of CRISPR systems in non-model microorganisms are also discussed.},
}
@article {pmid35119139,
year = {2022},
author = {Xie, R and Wang, X and Wang, Y and Ye, M and Zhao, Y and Yandell, BS and Gong, S},
title = {pH-Responsive Polymer Nanoparticles for Efficient Delivery of Cas9 Ribonucleoprotein With or Without Donor DNA.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {34},
number = {23},
pages = {e2110618},
doi = {10.1002/adma.202110618},
pmid = {35119139},
issn = {1521-4095},
support = {UG3 NS111688/NS/NINDS NIH HHS/United States ; //University of Wisconsin-Madison, and the Wisconsin Institute for Discovery/ ; 4-UH3-NS111688/NH/NIH HHS/United States ; 4-UH3-NS111688/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/metabolism ; Hydrogen-Ion Concentration ; Mice ; *Nanoparticles ; Polymers ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) may offer new therapeutics for genetic diseases through gene disruption via nonhomologous end joining (NHEJ) or gene correction via homology-directed repair (HDR). However, clinical translation of CRISPR technology is limited by the lack of safe and efficient delivery systems. Here, facilely fabricated pH-responsive polymer nanoparticles capable of safely and efficiently delivering Cas9 ribonucleoprotein alone (termed NHEJ-NP, diameter = 29.4 nm), or together with donor DNA (termed HDR-NP, diameter = 33.3 nm) are reported. Moreover, intravenously, intratracheally, and intramuscularly injected NHEJ-NP induces efficient gene editing in mouse liver, lung, and skeletal muscle, respectively. Intramuscularly injected HDR-NP also leads to muscle strength recovery in a Duchenne muscular dystrophy mouse model. NHEJ-NP and HDR-NP possess many desirable properties including high payload loading content, small and uniform sizes, high editing efficiency, good biocompatibility, low immunogenicity, and ease of production, storage, and transport, making them great interest for various genome editing applications with clinical potentials.},
}
@article {pmid34217781,
year = {2021},
author = {Muto, Y and Humphreys, BD},
title = {Recent advances in lineage tracing for the kidney.},
journal = {Kidney international},
volume = {100},
number = {6},
pages = {1179-1184},
pmid = {34217781},
issn = {1523-1755},
support = {P30 DK020579/DK/NIDDK NIH HHS/United States ; R01 DK103740/DK/NIDDK NIH HHS/United States ; UC2 DK126024/DK/NIDDK NIH HHS/United States ; UH3 DK107374/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cell Lineage ; Gene Expression ; Genes, Reporter ; *Genomics ; Humans ; *Kidney ; },
abstract = {Lineage tracing was originally developed by developmental biologists to identify all progeny of a single cell during morphogenesis. More recently this approach has been applied to other fields, including organ homeostasis and recovery from injury. Modern lineage tracing techniques typically rely on reporter gene expression induced by cell-specific DNA recombination. There have been important scientific advances in the last 10 years that have impacted lineage tracing approaches, including intersectional genetics, optical clearing techniques, and the use of sequencing-based genomic lineage tracing. The latter combines CRISPR-Cas9-based genetic scarring with single-cell RNA-sequencing that, in theory, could allow comprehensive reconstruction of a lineage tree for an entire organism. This review summarizes recent advances in lineage tracing technologies and outlines potential applications for kidney research.},
}
@article {pmid35678575,
year = {2022},
author = {Sharma, N and Das, A and Raja, P and Marathe, SA},
title = {The CRISPR-Cas System Differentially Regulates Surface-Attached and Pellicle Biofilm in Salmonella enterica Serovar Typhimurium.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0020222},
doi = {10.1128/spectrum.00202-22},
pmid = {35678575},
issn = {2165-0497},
abstract = {The CRISPR-Cas mediated regulation of biofilm by Salmonella enterica serovar Typhimurium was investigated by deleting CRISPR-Cas components ΔcrisprI, ΔcrisprII, ΔΔcrisprI crisprII, and Δcas op. We determined that the system positively regulates surface biofilm while inhibiting pellicle biofilm formation. Results of real-time PCR suggest that the flagellar (fliC, flgK) and curli (csgA) genes were repressed in knockout strains, causing reduced surface biofilm. The mutants displayed altered pellicle biofilm architecture. They exhibited bacterial multilayers and a denser extracellular matrix with enhanced cellulose and less curli, ergo weaker pellicles than those of the wild type. The cellulose secretion was more in the knockout strains due to the upregulation of bcsC, which is necessary for cellulose export. We hypothesized that the secreted cellulose quickly integrates into the pellicle, leading to enhanced pellicular cellulose in the knockout strains. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD and, hence, also of csgA and bcsA. The conflicting upregulation of bcsC, the last gene of the bcsABZC operon, could be caused by independent regulation by the CRISPR-Cas system owing to a partial match between the CRISPR spacers and bcsC gene. The cAMP-regulated protein (CRP)-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of yddx governing the availability of the sigma factor σ28 that further regulates class 3 flagellar genes (fliC, fljB, and flgK). Additionally, the variations in the lipopolysaccharide (LPS) profile and expression of LPS-related genes (rfaC, rfbG, and rfbI) in knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle biofilm. IMPORTANCE In addition to being implicated in bacterial immunity and genome editing, the CRISPR-Cas system has recently been demonstrated to regulate endogenous gene expression and biofilm formation. While the function of individual cas genes in controlling Salmonella biofilm has been explored, the regulatory role of CRISPR arrays in biofilm is less studied. Moreover, studies have focused on the effects of the CRISPR-Cas system on surface-associated biofilms, and comprehensive studies on the impact of the system on pellicle biofilm remain an unexplored niche. We demonstrate that the CRISPR array and cas genes modulate the expression of various biofilm genes in Salmonella, whereby surface and pellicle biofilm formation is distinctively regulated.},
}
@article {pmid35671288,
year = {2022},
author = {Castle, AR and Wohlgemuth, S and Arce, L and Westaway, D},
title = {Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0269342},
doi = {10.1371/journal.pone.0269342},
pmid = {35671288},
issn = {1932-6203},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Mammals/genetics ; Mice ; *Prion Diseases/genetics ; Prion Proteins/genetics ; *Prions/genetics ; RNA, Guide ; *Wasting Disease, Chronic/genetics ; },
abstract = {Prion diseases are a group of fatal neurodegenerative disorders that includes chronic wasting disease, which affects cervids and is highly transmissible. Given that chronic wasting disease prevalence exceeds 30% in some endemic areas of North America, and that eventual transmission to other mammalian species, potentially including humans, cannot be ruled out, novel control strategies beyond population management via hunting and/or culling must be investigated. Prion diseases depend upon post-translational conversion of the cellular prion protein, encoded by the Prnp gene, into a disease-associated conformation; ablation of cellular prion protein expression, which is generally well-tolerated, eliminates prion disease susceptibility entirely. Inspired by demonstrations of gene drive in caged mosquito species, we aimed to test whether a CRISPR/Cas9-based gene drive mechanism could, in principle, promote the spread of a null Prnp allele among mammalian populations. First, we showed that transient co-expression of Cas9 and Prnp-directed guide RNAs in RK13 cells generates indels within the Prnp open-reading frame, indicating that repair of Cas9-induced double-strand breaks by non-homologous end-joining had taken place. Second, we integrated a ~1.2 kb donor DNA sequence into the Prnp open-reading frame in N2a cells by homology-directed repair following Cas9-induced cleavages and confirmed that integration occurred precisely in most cases. Third, we demonstrated that electroporation of Cas9/guide RNA ribonucleoprotein complexes into fertilised mouse oocytes resulted in pups with a variety of disruptions to the Prnp open reading frame, with a new coisogenic line of Prnp-null mice obtained as part of this work. However, a technical challenge in obtaining expression of Cas9 in the male germline prevented implementation of a complete gene drive mechanism in mice.},
}
@article {pmid35671066,
year = {2022},
author = {Klinnert, S and Chemnitzer, A and Rusert, P and Metzner, KJ},
title = {Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus.},
journal = {The Journal of general virology},
volume = {103},
number = {6},
pages = {},
doi = {10.1099/jgv.0.001754},
pmid = {35671066},
issn = {1465-2099},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *HIV Infections ; *HIV Seropositivity ; *HIV-1/genetics ; Humans ; Proviruses/genetics ; RNA, Guide/genetics ; Virus Activation/genetics ; Virus Latency/genetics ; },
abstract = {CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the 'shock and kill' cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5'LTR promoter, making them a promising 'shock' strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5'LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3' half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located -165 to -106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the 'shock and kill' cure approach.},
}
@article {pmid35446444,
year = {2022},
author = {Natsuga, K and Furuta, Y and Takashima, S and Nohara, T and Huang, HY and Shinkuma, S and Nakamura, H and Katsuda, Y and Higashi, H and Hsu, CK and Fukushima, S and Ujiie, H},
title = {Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.},
journal = {Human mutation},
volume = {43},
number = {7},
pages = {877-881},
doi = {10.1002/humu.24385},
pmid = {35446444},
issn = {1098-1004},
support = {//Japan Agency for Medical Research and Development/ ; //World-leading Innovative and Smart Education (WISE) Program/ ; },
mesh = {CRISPR-Cas Systems ; *Collagen Type VII/genetics ; *Epidermolysis Bullosa Dystrophica/genetics/pathology/therapy ; Genes, Recessive ; Haplotypes ; Humans ; Mutation ; },
abstract = {An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.},
}
@article {pmid35674844,
year = {2022},
author = {Bhattacharjee, R and Das Roy, L and Choudhury, A},
title = {Understanding on CRISPR/Cas9 mediated cutting-edge approaches for cancer therapeutics.},
journal = {Discover. Oncology},
volume = {13},
number = {1},
pages = {45},
pmid = {35674844},
issn = {2730-6011},
abstract = {The research focus on CRISPR/Cas9 has gained substantial concentration since the discovery of 'an unusual repeat sequence' reported by Ishino et al. (J Bacteriol 169:5429-5433, 1987) and the journey comprises the recent Nobel Prize award (2020), conferred to Emmanuelle Charpentier and Jennifer Doudna. Cumulatively, the CRISPR has a short, compact, and most discussed success of its application in becoming one of the most versatile and paradigm shifting technologies of Biological Research. Today, the CRISPR/Cas9 genome editing system is almost ubiquitously utilized in many facets of biological research where its tremendous gene manipulation capability has been harnessed to create miracles. From 2012, the CRISPR/Cas 9 system has been showcased in almost 15,000 research articles in the PubMed database, till date. Backed by some strong molecular evidence, the CRISPR system has been utilized in a few clinical trials targeted towards various pathologies. While the area covered by CRISPR is cosmic, this review will focus mostly on the utilization of CRISPR/Cas9 technology in the field of cancer therapy.},
}
@article {pmid35672603,
year = {2022},
author = {Negi, C and Vasistha, NK and Singh, D and Vyas, P and Dhaliwal, HS},
title = {Application of CRISPR-Mediated Gene Editing for Crop Improvement.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35672603},
issn = {1559-0305},
abstract = {Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology.},
}
@article {pmid35666449,
year = {2022},
author = {Guerra-Resendez, RS and Hilton, IB},
title = {Harnessing CRISPR-Cas9 for Epigenetic Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {237-251},
pmid = {35666449},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Epigenome ; Epigenomics ; Gene Editing/methods ; *RNA, Guide/genetics/metabolism ; },
abstract = {Epigenome editing has become more precise and effective by coupling epigenetic effectors to the dCas9 protein and targeting regulatory regions such as promoters and enhancers. Here, we describe a basic methodology for performing an epigenome editing experiment, starting from gRNA design and cloning to transiently transfecting the gRNA plasmid and the CRISPR/dCas9-based epigenetic effector and finalizing with chromatin immunoprecipitation (ChIP) to validate changes in epigenetic state at a targeted genomic region.},
}
@article {pmid35666448,
year = {2022},
author = {Jiao, C and Beisel, CL},
title = {Reprogramming TracrRNAs for Multiplexed RNA Detection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {217-235},
pmid = {35666448},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *RNA/genetics ; *RNA, Guide/genetics ; },
abstract = {CRISPR-based detection and recording technologies are gaining increasing attention in disease surveillance and prevention. In this chapter, we describe how our recent discovery of noncanonical crRNAs inspired the engineering of reprogrammed tracrRNAs and led to a powerful platform for multiplexed RNA detection. We provide detailed protocols regarding how to design reprogrammed tracrRNA and carry out assays in vitro and in vivo.},
}
@article {pmid35657952,
year = {2022},
author = {Spisak, S and O'Brien, B and Ostermeier, M},
title = {A bacterial dual positive and negative selection system for dCas9 activity.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0269270},
doi = {10.1371/journal.pone.0269270},
pmid = {35657952},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; },
abstract = {The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.},
}
@article {pmid35657788,
year = {2022},
author = {Lee, N and Park, J and Kim, JE and Shin, JY and Min, K and Son, H},
title = {Genome editing using preassembled CRISPR-Cas9 ribonucleoprotein complexes in Fusarium graminearum.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0268855},
doi = {10.1371/journal.pone.0268855},
pmid = {35657788},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; *Fusarium/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; },
abstract = {Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has greatly facilitated the genetic analysis of fungal pathogens. The head blight fungus, Fusarium graminearum, causes destructive losses of economically important cereal crops. The recent development of the CRISPR-Cas9 system for use with F. graminearum has enabled more efficient genome editing. In this study, we described a CRISPR-Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins (RNPs) into the protoplasts of F. graminearum. The use of RNPs significantly increased both the number of transformants and percentage of transformants in which the target gene was successfully replaced with a selectable marker. We showed that a single double-strand DNA break mediated by the Cas9 ribonucleoprotein was sufficient for gene deletion. In addition, short-homology recombination required only 50 base pair regions flanking the target gene. The high efficiency of Cas9 RNPs enables large-scale functional analysis, the identification of essential genes, and gene deletion that is difficult with conventional methods. We expect that our approach will accelerate genetic studies of F. graminearum.},
}
@article {pmid35657510,
year = {2022},
author = {Xu, S and Chen, T and Tian, M and Rahantaniaina, MS and Zhang, L and Wang, R and Xuan, W and Han, Y},
title = {Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2526},
number = {},
pages = {25-41},
pmid = {35657510},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant ; Homeostasis ; *Oryza/genetics ; Plants, Genetically Modified/genetics ; Reactive Oxygen Species ; },
abstract = {Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.},
}
@article {pmid35615910,
year = {2022},
author = {Ma, JY and Wang, SY and Du, YC and Wang, DX and Tang, AN and Wang, J and Kong, DM},
title = {"RESET" Effect: Random Extending Sequences Enhance the Trans-Cleavage Activity of CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {94},
number = {22},
pages = {8050-8057},
doi = {10.1021/acs.analchem.2c01401},
pmid = {35615910},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; },
abstract = {The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, a single-stranded DNA, which is too short to work as the activator, can efficiently activate CRISPR/Cas12a after being extended a random sequence from its 3'-end, even when the random sequence folds into secondary structures. The finding of the "RESET" effect enriches the CRISPR/Cas12a-based sensing strategies. Based on this effect, two CRISPR/Cas12a-based biosensors are designed for the sensitive and specific detection of two biologically important enzymes.},
}
@article {pmid35608169,
year = {2022},
author = {Huang, C and Zhang, L and Zhu, Y and Zhang, Z and Liu, Y and Liu, C and Ge, S and Yu, J},
title = {Dual-Engine Powered Paper Photoelectrochemical Platform Based on 3D DNA Nanomachine-Mediated CRISPR/Cas12a for Detection of Multiple miRNAs.},
journal = {Analytical chemistry},
volume = {94},
number = {22},
pages = {8075-8084},
doi = {10.1021/acs.analchem.2c01717},
pmid = {35608169},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrochemical Techniques/methods ; *MicroRNAs ; *Quantum Dots/chemistry ; },
abstract = {This work proposed a novel double-engine powered paper photoelectrochemical (PEC) biosensor based on an anode-cathode cooperative amplification strategy and various signal enhancement mechanisms, which realized the monitoring of multiple miRNAs (such as miRNA-141 and miRNA-21). Specifically, C3N4 quantum dots (QDs) sensitized ZnO nanostars and BiOI nanospheres simultaneously to construct a composite photoelectric layer that amplified the original photocurrent of the photoanode and photocathode, respectively. Through the independent design and partition of a flexible paper chip to functionalize injection holes and electrode areas, the bipolar combination completed the secondary upgrade of signals, which also provided biological reaction sites for multitarget detection. With the synergistic participation of a three-dimensional (3D) DNA nanomachine and programmable CRISPR/Cas12a shearing tool, C3N4 QDs lost their attachment away from the electrode surface to quench the signal. Moreover, electrode zoning significantly reduced the spatial cross talk of related substances for multitarget detection, while the universal trans-cleavage capability of CRISPR/Cas12a simplified the operation. The designed PEC biosensor revealed excellent linear ranges for detection of miRNA-141 and miRNA-21, for which the detection limits were 5.5 and 3.4 fM, respectively. With prominent selectivity and sensitivity, the platform established an effective approach for trace multitarget monitoring in clinical applications, and its numerous pioneering attempts owned favorable reference values.},
}
@article {pmid35598687,
year = {2022},
author = {Gonzalez-Salinas, F and Martinez-Amador, C and Trevino, V},
title = {Characterizing genes associated with cancer using the CRISPR/Cas9 system: A systematic review of genes and methodological approaches.},
journal = {Gene},
volume = {833},
number = {},
pages = {146595},
doi = {10.1016/j.gene.2022.146595},
pmid = {35598687},
issn = {1879-0038},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; Mutation ; *Neoplasms/genetics ; Oncogenes ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system enables a versatile set of genomes editing and genetic-based disease modeling tools due to its high specificity, efficiency, and accessible design and implementation. In cancer, the CRISPR/Cas9 system has been used to characterize genes and explore different mechanisms implicated in tumorigenesis. Different experimental strategies have been proposed in recent years, showing dependency on various intrinsic factors such as cancer type, gene function, mutation type, and technical approaches such as cell line, Cas9 expression, and transfection options. However, the successful methodological approaches, genes, and other experimental factors have not been analyzed. We, therefore, initially considered more than 1,300 research articles related to CRISPR/Cas9 in cancer to finally examine more than 400 full-text research publications. We summarize findings regarding target genes, RNA guide designs, cloning, Cas9 delivery systems, cell enrichment, and experimental validations. This analysis provides valuable information and guidance for future cancer gene validation experiments.},
}
@article {pmid35594500,
year = {2022},
author = {Kazemian, P and Yu, SY and Thomson, SB and Birkenshaw, A and Leavitt, BR and Ross, CJD},
title = {Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components.},
journal = {Molecular pharmaceutics},
volume = {19},
number = {6},
pages = {1669-1686},
doi = {10.1021/acs.molpharmaceut.1c00916},
pmid = {35594500},
issn = {1543-8392},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Lipids ; Liposomes ; *Nanoparticles ; },
abstract = {Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.},
}
@article {pmid35583489,
year = {2022},
author = {Cheng, MHY and Brimacombe, CA and Verbeke, R and Cullis, PR},
title = {Exciting Times for Lipid Nanoparticles: How Canadian Discoveries Are Enabling Gene Therapies.},
journal = {Molecular pharmaceutics},
volume = {19},
number = {6},
pages = {1663-1668},
doi = {10.1021/acs.molpharmaceut.2c00365},
pmid = {35583489},
issn = {1543-8392},
mesh = {CRISPR-Cas Systems ; Canada ; *Gene Editing ; Liposomes ; *Nanoparticles ; },
abstract = {In this brief perspective, we describe key events in the history of the lipid-based nanomedicine field, highlight Canadian contributions, and outline areas where lipid nanoparticle technology is poised to have a transformative effect on the future of medicine.},
}
@article {pmid35338712,
year = {2022},
author = {Tanouchi, M and Igawa, T and Suzuki, N and Suzuki, M and Hossain, N and Ochi, H and Ogino, H},
title = {Optimization of CRISPR/Cas9-mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique.},
journal = {Development, growth &amp; differentiation},
volume = {64},
number = {4},
pages = {219-225},
doi = {10.1111/dgd.12778},
pmid = {35338712},
issn = {1440-169X},
support = {//Frontier Development Program for Genome Editing in Hiroshima University/ ; 19K06689//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Phenotype ; RNA, Messenger/genetics ; Xenopus laevis/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi-quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10 nL each) of one-cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.},
}
@article {pmid34914197,
year = {2022},
author = {Riepe, C and Zelin, E and Frankino, PA and Meacham, ZA and Fernandez, SG and Ingolia, NT and Corn, JE},
title = {Double stranded DNA breaks and genome editing trigger loss of ribosomal protein RPS27A.},
journal = {The FEBS journal},
volume = {289},
number = {11},
pages = {3101-3114},
doi = {10.1111/febs.16321},
pmid = {34914197},
issn = {1742-4658},
support = {S10 OD018174/NH/NIH HHS/United States ; //Li Ka Shing Foundation/ ; //Shurl and Kay Curci Foundation/ ; //Heritage Medical Research Institute/ ; DGE 1106400//National Science Foundation/ ; DGE 1752814//National Science Foundation/ ; 4T32GM007232-40/NH/NIH HHS/United States ; DP2 CA195768/NH/NIH HHS/United States ; DP2 CA195768/CA/NCI NIH HHS/United States ; DP2 HL141006/NH/NIH HHS/United States ; 4T32GM007232-40/NH/NIH HHS/United States ; DP2 CA195768/NH/NIH HHS/United States ; DP2 HL141006/NH/NIH HHS/United States ; S10 OD018174/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Repair ; *Gene Editing/methods ; Ribosomal Proteins/genetics ; },
abstract = {DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.},
}
@article {pmid35671698,
year = {2022},
author = {Ke, X and Ou, Y and Lin, Y and Hu, T},
title = {Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a.},
journal = {Biosensors &amp; bioelectronics},
volume = {212},
number = {},
pages = {114428},
doi = {10.1016/j.bios.2022.114428},
pmid = {35671698},
issn = {1873-4235},
abstract = {CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.},
}
@article {pmid35670994,
year = {2022},
author = {Rahimi, H and Zaboli, KA and Thekkiniath, J and Mousavi, SH and Johari, B and Hashemi, MR and Nosrati, H and Goldschneider, D and Bernet, A and Danafar, H and Kaboli, S},
title = {BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35670994},
issn = {1559-0305},
support = {A-11-1294-3//Zanjan University of Medical Sciences/ ; },
abstract = {The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.},
}
@article {pmid35666447,
year = {2022},
author = {Villegas Kcam, MC and Chappell, J},
title = {Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {203-215},
pmid = {35666447},
issn = {1940-6029},
abstract = {CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.},
}
@article {pmid35666436,
year = {2022},
author = {Simmons, TR and Ellington, AD and Contreras, LM},
title = {RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {1-31},
pmid = {35666436},
issn = {1940-6029},
abstract = {Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.},
}
@article {pmid35665758,
year = {2022},
author = {Bessoltane, N and Charlot, F and Guyon-Debast, A and Charif, D and Mara, K and Collonnier, C and Perroud, PF and Tepfer, M and Nogué, F},
title = {Genome-wide specificity of plant genome editing by both CRISPR-Cas9 and TALEN.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {9330},
pmid = {35665758},
issn = {2045-2322},
support = {ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/ ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/ ; },
abstract = {CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.},
}
@article {pmid35663880,
year = {2022},
author = {Surachat, K and Kantachote, D and Wonglapsuwan, M and Chukamnerd, A and Deachamag, P and Mittraparp-Arthorn, P and Jeenkeawpiam, K},
title = {Complete Genome Sequence of Weissella cibaria NH9449 and Comprehensive Comparative-Genomic Analysis: Genomic Diversity and Versatility Trait Revealed.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {826683},
doi = {10.3389/fmicb.2022.826683},
pmid = {35663880},
issn = {1664-302X},
abstract = {Lactic acid bacteria (LAB) in the genus Weissella spp. contain traits in their genome that confer versatility. In particular, Weissella cibaria encodes several beneficial genes that are useful in biotechnological applications. The complete genome of W. cibaria NH9449 was sequenced and an in silico comparative analysis was performed to gain insight into the genomic diversity among members of the genus Weissella. A total of 219 Weissella genomes were used in a bioinformatics analysis of pan-genomes, phylogenetics, self-defense mechanisms, virulence factors, antimicrobial resistance, and carbohydrate-active enzymes. These investigations showed that the strain NH9449 encodes several restriction-modification-related genes and a CRISPR-Cas region in its genome. The identification of carbohydrate-active enzyme-encoding genes indicated that this strain could be beneficial in biotechnological applications. The comparative genomic analysis reveals the very high genomic diversity in this genus, and some marked differences in genetic variation and genes among Weissella species. The calculated average amino acid identity (AAI) and phylogenetic analysis of core and accessory genes shows the possible existence of three new species in this genus. These new genomic insights into Weissella species and their biological functions could be useful in the food industry and other applications.},
}
@article {pmid35659862,
year = {2022},
author = {Chen, H and Mayer, A and Balasubramanian, V},
title = {A scaling law in CRISPR repertoire sizes arises from the avoidance of autoimmunity.},
journal = {Current biology : CB},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cub.2022.05.021},
pmid = {35659862},
issn = {1879-0445},
abstract = {Some prokaryotes possess CRISPR-Cas systems that use DNA segments called spacers, which are acquired from invading phages, to guide immune defense. Here, we propose that cross-reactive CRISPR targeting can, however, lead to "heterologous autoimmunity," whereby foreign spacers guide self-targeting in a spacer-length-dependent fashion. Balancing antiviral defense against autoimmunity predicts a scaling relation between spacer length and CRISPR repertoire size. We find evidence for this scaling through a comparative analysis of sequenced prokaryotic genomes and show that this association also holds at the level of CRISPR types. By contrast, the scaling is absent in strains with nonfunctional CRISPR loci. Finally, we demonstrate that stochastic spacer loss can explain variations around the scaling relation, even between strains of the same species. Our results suggest that heterologous autoimmunity is a selective factor shaping the evolution of CRISPR-Cas systems, analogous to the trade-offs between immune specificity, breadth, and autoimmunity that constrain the diversity of adaptive immune systems in vertebrates.},
}
@article {pmid35659325,
year = {2022},
author = {Huang, CJ and Adler, BA and Doudna, JA},
title = {A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression.},
journal = {Molecular cell},
volume = {82},
number = {11},
pages = {2148-2160.e4},
doi = {10.1016/j.molcel.2022.04.020},
pmid = {35659325},
issn = {1097-4164},
abstract = {Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.},
}
@article {pmid35655130,
year = {2022},
author = {Rawashdeh, O and Rawashdeh, RY and Kebede, T and Kapp, D and Ralescu, A},
title = {Bio-informatic analysis of CRISPR protospacer adjacent motifs (PAMs) in T4 genome.},
journal = {BMC genomic data},
volume = {23},
number = {1},
pages = {40},
pmid = {35655130},
issn = {2730-6844},
mesh = {Amino Acids ; *Bacteriophages/genetics ; *Computational Biology ; },
abstract = {BACKGROUND: The existence of protospacer adjacent motifs (PAMs) sequences in bacteriophage genome is critical for the recognition and function of the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) machinery system. We further elucidate the significance of PAMs and their function, particularly as a part of transcriptional regulatory regions in T4 bacteriophages.<br><br>METHODS: A scripting language was used to analyze a sequence of T4 phage genome, and a list of few selected PAMs. Mann-Whitney Wilcoxon (MWW) test was used to compare the sequence hits for the PAMs versus the hits of all the possible sequences of equal lengths.<br><br>RESULTS: The results of MWW test show that certain PAMs such as: 'NGG' and 'TATA' are preferably located at the core of phage promoters: around -10 position, whereas the position around -35 appears to have no detectable count variation of any of the tested PAMs. Among all tested PAMs, the following three sequences: 5'-GCTV-3', 5'-TTGAAT-3' and 5'-TTGGGT-3' have higher prevalence in essential genes. By analyzing all the possible ways of reading PAM sequences as codons for the corresponding amino acids, it was found that deduced amino acids of some PAMs have a significant tendency to prefer the surface of proteins.<br><br>CONCLUSION: These results provide novel insights into the location and the subsequent identification of the role of PAMs as transcriptional regulatory elements. Also, CRISPR targeting certain PAM sequences is somehow likely to be connected to the hydrophilicity (water solubility) of amino acids translated from PAM's triplets. Therefore, these amino acids are found at the interacting unit at protein-protein interfaces.},
}
@article {pmid35654791,
year = {2022},
author = {Kobayashi, N and Okae, H and Hiura, H and Kubota, N and Kobayashi, EH and Shibata, S and Oike, A and Hori, T and Kikutake, C and Hamada, H and Kaji, H and Suyama, M and Bortolin-Cavaillé, ML and Cavaillé, J and Arima, T},
title = {The microRNA cluster C19MC confers differentiation potential into trophoblast lineages upon human pluripotent stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3071},
pmid = {35654791},
issn = {2041-1723},
support = {JP18bm0704021//Japan Agency for Medical Research and Development (AMED)/ ; JP17gm0510011//Japan Agency for Medical Research and Development (AMED)/ ; JP19gm1310001//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; Cell Differentiation/genetics ; Epigenesis, Genetic ; Humans ; Mammals ; *MicroRNAs/genetics ; *Pluripotent Stem Cells ; Trophoblasts ; },
abstract = {The first cell fate commitment during mammalian development is the specification of the inner cell mass and trophectoderm. This irreversible cell fate commitment should be epigenetically regulated, but the precise mechanism is largely unknown in humans. Here, we show that naïve human embryonic stem (hES) cells can transdifferentiate into trophoblast stem (hTS) cells, but primed hES cells cannot. Our transcriptome and methylome analyses reveal that a primate-specific miRNA cluster on chromosome 19 (C19MC) is active in naïve hES cells but epigenetically silenced in primed ones. Moreover, genome and epigenome editing using CRISPR/Cas systems demonstrate that C19MC is essential for hTS cell maintenance and C19MC-reactivated primed hES cells can give rise to hTS cells. Thus, we reveal that C19MC activation confers differentiation potential into trophoblast lineages on hES cells. Our findings are fundamental to understanding the epigenetic regulation of human early development and pluripotency.},
}
@article {pmid35658035,
year = {2022},
author = {Tran, NT and Danner, E and Li, X and Graf, R and Lebedin, M and de la Rosa, K and Kühn, R and Rajewsky, K and Chu, VT},
title = {Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells.},
journal = {Science advances},
volume = {8},
number = {22},
pages = {eabm9106},
doi = {10.1126/sciadv.abm9106},
pmid = {35658035},
issn = {2375-2548},
abstract = {While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the HBB, ELANE, IL7R, and PRF1 genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy.},
}
@article {pmid35653473,
year = {2022},
author = {Kaiser, J},
title = {A gentler way to tweak genes: epigenome editing.},
journal = {Science (New York, N.Y.)},
volume = {376},
number = {6597},
pages = {1034-1035},
doi = {10.1126/science.add2703},
pmid = {35653473},
issn = {1095-9203},
mesh = {Animals ; CRISPR-Cas Systems ; *Epigenome ; *Gene Editing ; Mice ; },
abstract = {Flipping genetic on-off switches can treat diseases in mice.},
}
@article {pmid35651625,
year = {2022},
author = {Yim, S and Hwang, W and Han, N and Lee, D},
title = {Computational Discovery of Cancer Immunotherapy Targets by Intercellular CRISPR Screens.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {884561},
doi = {10.3389/fimmu.2022.884561},
pmid = {35651625},
issn = {1664-3224},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Immunotherapy ; T-Lymphocytes, Cytotoxic ; *Triple Negative Breast Neoplasms/genetics ; },
abstract = {Cancer immunotherapy targets the interplay between immune and cancer cells. In particular, interactions between cytotoxic T lymphocytes (CTLs) and cancer cells, such as PD-1 (PDCD1) binding PD-L1 (CD274), are crucial for cancer cell clearance. However, immune checkpoint inhibitors targeting these interactions are effective only in a subset of patients, requiring the identification of novel immunotherapy targets. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening in either cancer or immune cells has been employed to discover regulators of immune cell function. However, CRISPR screens in a single cell type complicate the identification of essential intercellular interactions. Further, pooled screening is associated with high noise levels. Herein, we propose intercellular CRISPR screens, a computational approach for the analysis of genome-wide CRISPR screens in every interacting cell type for the discovery of intercellular interactions as immunotherapeutic targets. We used two publicly available genome-wide CRISPR screening datasets obtained while triple-negative breast cancer (TNBC) cells and CTLs were interacting. We analyzed 4825 interactions between 1391 ligands and receptors on TNBC cells and CTLs to evaluate their effects on CTL function. Intercellular CRISPR screens discovered targets of approved drugs, a few of which were not identifiable in single datasets. To evaluate the method's performance, we used data for cytokines and costimulatory molecules as they constitute the majority of immunotherapeutic targets. Combining both CRISPR datasets improved the recall of discovering these genes relative to using single CRISPR datasets over two-fold. Our results indicate that intercellular CRISPR screens can suggest novel immunotherapy targets that are not obtained through individual CRISPR screens. The pipeline can be extended to other cancer and immune cell types to discover important intercellular interactions as potential immunotherapeutic targets.},
}
@article {pmid35646725,
year = {2022},
author = {Wei, J and Li, Y and Cao, Y and Liu, Q and Yang, K and Song, X and Shao, Y and Qi, K and Tu, J},
title = {Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {879887},
doi = {10.3389/fcimb.2022.879887},
pmid = {35646725},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; *Parvovirus, Porcine/genetics ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Swine ; *Swine Diseases/genetics ; },
abstract = {Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 102 copies/μL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.},
}
@article {pmid35643551,
year = {2022},
author = {Baldanta, S and Guevara, G and Navarro-Llorens, JM},
title = {SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {103},
pmid = {35643551},
issn = {1475-2859},
support = {ALGATEC-CM (P2018/BAA-4532)//Comunidad de Madrid/ ; ALGATEC-CM (P2018/BAA-4532)//Comunidad de Madrid/ ; ALGATEC-CM (P2018/BAA-4532)//Comunidad de Madrid/ ; },
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; *Synechocystis/genetics/metabolism ; },
abstract = {BACKGROUND: Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology.<br><br>RESULTS: In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology.<br><br>CONCLUSIONS: The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.},
}
@article {pmid35643496,
year = {2022},
author = {Xin, Q and Chen, Y and Chen, Q and Wang, B and Pan, L},
title = {Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {99},
pmid = {35643496},
issn = {1475-2859},
support = {2018B020205002//Research and Development Plan in Key Areas of Guangdong Province/ ; 2020B020226007//Research and Development Plan in Key Areas of Guangdong Province/ ; 1920001000824//Core Technology Project of Foshan City/ ; 2021YFC2100200//National Key Research and Development Program of China/ ; },
mesh = {*Bacillus amyloliquefaciens/genetics ; *CRISPR-Cas Systems ; DNA Restriction-Modification Enzymes/genetics ; Gene Editing/methods ; alpha-Amylases/genetics ; },
abstract = {BACKGROUND: Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02.<br><br>RESULTS: In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain.<br><br>CONCLUSIONS: The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.},
}
@article {pmid35613622,
year = {2022},
author = {Prosser, BL and Helbig, I},
title = {Base editing the synapse: Modeling a complex neurological disorder in non-human primates.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {6},
pages = {2114-2116},
doi = {10.1016/j.ymthe.2022.05.009},
pmid = {35613622},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; *Neuromuscular Diseases ; *Primates/genetics ; RNA Editing ; Synapses ; },
}
@article {pmid35569695,
year = {2022},
author = {Zhang, X and Jin, X and Sun, R and Zhang, M and Lu, W and Zhao, M},
title = {Gene knockout in cellular immunotherapy: Application and limitations.},
journal = {Cancer letters},
volume = {540},
number = {},
pages = {215736},
doi = {10.1016/j.canlet.2022.215736},
pmid = {35569695},
issn = {1872-7980},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive ; Zinc Finger Nucleases/genetics ; },
abstract = {Cellular immunotherapy has achieved incremental success in recent years. Varieties of cell products are undergoing fundamental research and clinical trials, among which CAR-T cell therapy is approved for marketing. As research progresses, these cells need to be modified to promote their safety and efficacy. Gene-editing technologies have evolved from RNA interference (RNAi), including small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to new generations of zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and clusters of regularly spaced short palindromic repeats (CRISPR/Cas9), and delivery methods are widely used. Here, we summarize the ongoing clinical trials and fundamental research for genome editing therapy. Additionally, we highlight existing in vivo delivery systems and their limitations to find a better method to deliver genes.},
}
@article {pmid35483191,
year = {2022},
author = {Mitra, S and Sarker, J and Mojumder, A and Shibbir, TB and Das, R and Emran, TB and Tallei, TE and Nainu, F and Alshahrani, AM and Chidambaram, K and Simal-Gandara, J},
title = {Genome editing and cancer: How far has research moved forward on CRISPR/Cas9?.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {150},
number = {},
pages = {113011},
doi = {10.1016/j.biopha.2022.113011},
pmid = {35483191},
issn = {1950-6007},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer accounted for almost ten million deaths worldwide in 2020. Metastasis, characterized by cancer cell invasion to other parts of the body, is the main cause of cancer morbidity and mortality. Therefore, understanding the molecular mechanisms of tumor formation and discovery of potential drug targets are of great importance. Gene editing techniques can be used to find novel drug targets and study molecular mechanisms. In this review, we describe how popular gene-editing methods such as CRISPR/Cas9, TALEN and ZFNs work, and, by comparing them, we demonstrate that CRISPR/Cas9 has superior efficiency and precision. We further provide an overview of the recent applications of CRISPR/Cas9 to cancer research, focusing on the most common cancers such as breast cancer, lung cancer, colorectal cancer, and prostate cancer. We describe how these applications will shape future research and treatment of cancer, and propose new ways to overcome current challenges.},
}
@article {pmid35227602,
year = {2022},
author = {Lee, E and Shah, D and Porteus, M and Wright, JF and Bacchetta, R},
title = {Design of experiments as a decision tool for cell therapy manufacturing.},
journal = {Cytotherapy},
volume = {24},
number = {6},
pages = {590-596},
doi = {10.1016/j.jcyt.2022.01.009},
pmid = {35227602},
issn = {1477-2566},
mesh = {*CRISPR-Cas Systems/genetics ; Cell- and Tissue-Based Therapy ; Gene Editing ; Humans ; Prospective Studies ; *RNA, Guide/genetics ; },
abstract = {BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost.<br><br>METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.<br><br>RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 μg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 μg sgRNA.<br><br>CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.},
}
@article {pmid35221320,
year = {2022},
author = {Dorgalaleh, A and Kiani, J and Zaker, F and Safa, M},
title = {The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.},
journal = {Blood coagulation &amp; fibrinolysis : an international journal in haemostasis and thrombosis},
volume = {33},
number = {3},
pages = {153-158},
doi = {10.1097/MBC.0000000000001126},
pmid = {35221320},
issn = {1473-5733},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli ; Factor XIII/genetics ; *Factor XIII Deficiency/genetics/therapy ; *Gene Editing ; Humans ; Mutation ; },
abstract = {Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.},
}
@article {pmid35173102,
year = {2022},
author = {Tateno, M and Umeyama, T and Inukai, T and Takatsuka, S and Hoshino, Y and Yamagoe, S and Yamagata Murayama, S and Ishino, K and Miyazaki, Y},
title = {Examination of Cyp51A-Mediated Azole Resistance in Aspergillus lentulus Using CRISPR/Cas9 Genome Editing.},
journal = {Medical mycology journal},
volume = {63},
number = {2},
pages = {27-35},
doi = {10.3314/mmj.21-00024},
pmid = {35173102},
issn = {1882-0476},
mesh = {Antifungal Agents/pharmacology ; Aspergillus ; Aspergillus fumigatus/genetics ; *Azoles/pharmacology ; CRISPR-Cas Systems ; *Drug Resistance, Fungal/genetics ; Fungal Proteins/genetics/metabolism ; Gene Editing ; Humans ; Microbial Sensitivity Tests ; },
abstract = {Aspergillus lentulus was first reported in 2005 as a cryptic species of Aspergillus fumigatus, and since then, its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Although it has been reported that P450 14-α sterol demethylase (Cyp51) is involved in azole resistance in A. lentulus, the specific resistance mechanism has not been elucidated. In this study, we successfully introduced the entire A. fumigatus cyp51A gene into the cyp51A locus in A. lentulus using the CRISPR/Cas9 genome-editing system. The A. lentulus strains harboring A. fumigatus cyp51A showed reduced minimum inhibitory concentrations for itraconazole and voriconazole compared with those of the parent strain. This finding suggests that Cyp51A is involved in azole resistance in A. lentulus and may contribute to the elucidation of the mechanism of resistance to azole drugs via Cyp51A and to the development of new antifungal drugs. In addition, our successful application of the CRISPR/Cas9 system to A. lentulus opens the door to examination of other gene functions in this fungus.},
}
@article {pmid35143959,
year = {2022},
author = {Bengtsson, NE and Crudele, JM and Klaiman, JM and Halbert, CL and Hauschka, SD and Chamberlain, JS},
title = {Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {6},
pages = {2176-2185},
doi = {10.1016/j.ymthe.2022.02.003},
pmid = {35143959},
issn = {1525-0024},
mesh = {Aging ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Disease Progression ; Dogs ; *Dystrophin/genetics ; Gene Editing/methods ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; },
abstract = {Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.},
}
@article {pmid34880157,
year = {2022},
author = {Nakano, K and Shimizu, Y and Arai, T and Kaneko, T and Okamura, T},
title = {The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator.},
journal = {Experimental animals},
volume = {71},
number = {2},
pages = {214-223},
doi = {10.1538/expanim.21-0130},
pmid = {34880157},
issn = {1881-7122},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Mice, Knockout ; },
abstract = {Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.},
}
@article {pmid35654042,
year = {2022},
author = {Kaczmarska, Z and Czarnocki-Cieciura, M and Górecka-Minakowska, KM and Wingo, RJ and Jackiewicz, J and Zajko, W and Poznański, JT and Rawski, M and Grant, T and Peters, JE and Nowotny, M},
title = {Structural basis of transposon end recognition explains central features of Tn7 transposition systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2022.05.005},
pmid = {35654042},
issn = {1097-4164},
abstract = {Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.},
}
@article {pmid35652435,
year = {2022},
author = {Noureen, A and Zuhaib Khan, M and Amin, I and Zainab, T and Ahmad, N and Haider, S and Mansoor, S},
title = {Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop.},
journal = {GM crops &amp; food},
volume = {13},
number = {1},
pages = {97-111},
doi = {10.1080/21645698.2022.2080481},
pmid = {35652435},
issn = {2164-5701},
abstract = {Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.},
}
@article {pmid35641498,
year = {2022},
author = {Vos, PD and Rossetti, G and Mantegna, JL and Siira, SJ and Gandadireja, AP and Bruce, M and Raven, SA and Khersonsky, O and Fleishman, SJ and Filipovska, A and Rackham, O},
title = {Computationally designed hyperactive Cas9 enzymes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3023},
pmid = {35641498},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Engineering ; Genome ; Mammals ; },
abstract = {The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.},
}
@article {pmid35637227,
year = {2022},
author = {Corsi, GI and Qu, K and Alkan, F and Pan, X and Luo, Y and Gorodkin, J},
title = {CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3006},
pmid = {35637227},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Genome ; *RNA, Guide/genetics ; },
abstract = {A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 "sliding" on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design.},
}
@article {pmid35575140,
year = {2022},
author = {Liu, Q and Liu, M and Jin, Y and Li, B},
title = {Ratiometric fluorescent probe: a sensitive and reliable reporter for the CRISPR/Cas12a-based biosensing platform.},
journal = {The Analyst},
volume = {147},
number = {11},
pages = {2567-2574},
doi = {10.1039/d2an00613h},
pmid = {35575140},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; *Fluorescent Dyes ; },
abstract = {Due to the excellent activity of trans-cleavage and target recognition, the recently discovered CRISPR/Cas12a systems provide a promising opportunity for designing fluorescence biosensing. In the reported CRISPR/Cas12a-based biosensing platform, TaqMan probe is widely used as the reporter. However, the TaqMan probe provides single-wavelength fluorescence changes, which is easily influenced by various analyte-independent confounding factors to produce false-positive signals. In this study, a ratiometric fluorescent probe was designed to act as the reporter of a CRISPR/Cas12a-based system. As a proof-of-concept, fluorescein (FAM) and tetramethylrhodamine (TAMRA) were chosen as the two fluorescence dyes to label one short ssDNA at 5' and 3' ends, respectively, which was designed as one ratiometric fluorescent probe. When the ratiometric probe excites at 480 nm, duo to FRET effect, the probe emitted the 580 nm-fluorescence of TAMRA. The activated Cas12a can cleave the dual-labeled ssDNA, resulting in a decrease in the TAMRA's fluorescence and an increase in the FAM's fluorescence. This dual-response fluorescent probe can act as the reporter of the CRISPR/Cas12a-based biosensing platform. Compared with the classic TaqMan, CRISPR/Cas12a-based biosensing with the ratiometric probe as the reporter not only exhibited higher sensitivity but also could distinguish and avoid false positive signals.},
}
@article {pmid35536186,
year = {2022},
author = {Wagner, A and Seiler, J and Beye, M},
title = {Highly efficient site-specific integration of DNA fragments into the honeybee genome using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
doi = {10.1093/g3journal/jkac098},
pmid = {35536186},
issn = {2160-1836},
support = {//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Animals ; Base Sequence ; Bees/genetics ; *CRISPR-Cas Systems ; DNA ; Gene Editing/methods ; *Genome ; Mutation ; },
abstract = {Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal, and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9-mediated homology-directed repair. We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly, 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.},
}
@article {pmid35394026,
year = {2022},
author = {Yang, E and Metzloff, M and Langmüller, AM and Xu, X and Clark, AG and Messer, PW and Champer, J},
title = {A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
doi = {10.1093/g3journal/jkac081},
pmid = {35394026},
issn = {2160-1836},
support = {R21AI130635/NH/NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics ; Female ; *Gene Drive Technology/methods ; *RNA, Guide/genetics ; Translocation, Genetic ; },
abstract = {Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.},
}
@article {pmid35389492,
year = {2022},
author = {Carballar-Lejarazú, R and Tushar, T and Pham, TB and James, AA},
title = {Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae.},
journal = {Genetics},
volume = {221},
number = {2},
pages = {},
doi = {10.1093/genetics/iyac055},
pmid = {35389492},
issn = {1943-2631},
support = {//University of California Irvine Malaria Initiative/ ; //University of California/ ; },
mesh = {Alleles ; Animals ; *Anopheles/genetics ; CRISPR-Cas Systems ; Female ; *Malaria/prevention &amp; control ; Maternal Inheritance ; Mosquito Vectors/genetics ; },
abstract = {CRISPR/Cas9 technologies are important tools for the development of gene-drive systems to modify mosquito vector populations to control the transmission of pathogens that cause diseases such as malaria. However, one of the challenges for current Cas9-based drive systems is their ability to produce drive-resistant alleles resulting from insertions and deletions (indels) caused principally by nonhomologous end-joining following chromosome cleavage. Rapid increases in the frequency of such alleles may impair gene-drive dynamics. We explored the generation of indels in the germline and somatic cells in female gene-drive lineages using a series of selective crosses between a gene-drive line, AgNosCd-1, and wild-type mosquitoes. We find that potential drive-resistant mutant alleles are generated largely during embryonic development, most likely caused by deposition of the Cas9 endonuclease and guide RNAs in oocytes and resulting embryos by homozygous and hemizygous gene-drive mothers.},
}
@article {pmid35377421,
year = {2022},
author = {Luo, Z and Dai, W and Wang, C and Ye, Q and Zhou, Q and Wan, QL},
title = {Gene activation in Caenorhabditis elegans using the Campylobacter jejuni CRISPR-Cas9 feeding system.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
doi = {10.1093/g3journal/jkac068},
pmid = {35377421},
issn = {2160-1836},
support = {2018YFC2002000//National Key R&amp;D Program of China/ ; B16021//Program of Introducing Talents of Discipline to Universities (111 Project/ ; 2018A0303131003//Natural Science Foundation of Guangdong Province/ ; 202002030021//the Science and Technology Plan Project of Guangzhou, China/ ; 82001465//the National Natural Science Foundation of China/ ; 2020A1515111026//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Caenorhabditis elegans/genetics ; *Campylobacter jejuni/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Transcriptional Activation ; },
abstract = {Clustered regularly interspaced palindromic repeats-based activation system, a powerful genetic manipulation technology, can modulate endogenous gene transcription in various organisms through fusing nuclease-deficient Cas9 to transcriptional regulatory domains. At present, this clustered regularly interspaced palindromic repeats-based activation system has been applied to activate gene expression by microinjection manner in Caenorhabditis elegans. However, this complicated and time-consuming injection manner is not suitable for efficient and high-throughput gene regulation with clustered regularly interspaced palindromic repeats-Cas9 system. Here, we engineered a Campylobacter jejun clustered regularly interspaced palindromic repeats-Cas9-based gene activation system through bacteria feeding technique to delivering gene-specific sgRNA in C. elegans. It enables to activate various endogenous genes efficiently, as well as induce the corresponding phenotypes with a more efficient and labor-saving manner. Collectively, our results demonstrated that our novel dCjCas9-based activation feeding system holds great promise and potential in C. elegans.},
}
@article {pmid35191500,
year = {2022},
author = {Kumar, S and Liu, ZB and Sanyour-Doyel, N and Lenderts, B and Worden, A and Anand, A and Cho, HJ and Bolar, J and Harris, C and Huang, L and Xing, A and Richardson, A},
title = {Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.},
journal = {Plant physiology},
volume = {189},
number = {2},
pages = {585-594},
doi = {10.1093/plphys/kiac075},
pmid = {35191500},
issn = {1532-2548},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Targeting/methods ; *Ochrobactrum/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; *Soybeans/genetics ; },
abstract = {Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.},
}
@article {pmid35649413,
year = {2022},
author = {Wandera, KG and Alkhnbashi, OS and Bassett, HVI and Mitrofanov, A and Hauns, S and Migur, A and Backofen, R and Beisel, CL},
title = {Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2022.05.003},
pmid = {35649413},
issn = {1097-4164},
abstract = {As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.},
}
@article {pmid35647578,
year = {2022},
author = {Tripathi, L and Dhugga, KS and Ntui, VO and Runo, S and Syombua, ED and Muiruri, S and Wen, Z and Tripathi, JN},
title = {Genome Editing for Sustainable Agriculture in Africa.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {876697},
doi = {10.3389/fgeed.2022.876697},
pmid = {35647578},
issn = {2673-3439},
abstract = {Sustainable intensification of agriculture in Africa is essential for accomplishing food and nutritional security and addressing the rising concerns of climate change. There is an urgent need to close the yield gap in staple crops and enhance food production to feed the growing population. In order to meet the increasing demand for food, more efficient approaches to produce food are needed. All the tools available in the toolbox, including modern biotechnology and traditional, need to be applied for crop improvement. The full potential of new breeding tools such as genome editing needs to be exploited in addition to conventional technologies. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing has rapidly become the most prevalent genetic engineering approach for developing improved crop varieties because of its simplicity, efficiency, specificity, and easy to use. Genome editing improves crop variety by modifying its endogenous genome free of any foreign gene. Hence, genome-edited crops with no foreign gene integration are not regulated as genetically modified organisms (GMOs) in several countries. Researchers are using CRISPR/Cas-based genome editing for improving African staple crops for biotic and abiotic stress resistance and improved nutritional quality. Many products, such as disease-resistant banana, maize resistant to lethal necrosis, and sorghum resistant to the parasitic plant Striga and enhanced quality, are under development for African farmers. There is a need for creating an enabling environment in Africa with science-based regulatory guidelines for the release and adoption of the products developed using CRISPR/Cas9-mediated genome editing. Some progress has been made in this regard. Nigeria and Kenya have recently published the national biosafety guidelines for the regulation of gene editing. This article summarizes recent advances in developments of tools, potential applications of genome editing for improving staple crops, and regulatory policies in Africa.},
}
@article {pmid35646415,
year = {2022},
author = {Leibrock, NV and Santegoets, J and Mooijman, PJW and Yusuf, F and Zuijdgeest, XCL and Zutt, EA and Jacobs, JGM and Schaart, JG},
title = {The biological feasibility and social context of gene-edited, caffeine-free coffee.},
journal = {Food science and biotechnology},
volume = {31},
number = {6},
pages = {635-655},
doi = {10.1007/s10068-022-01082-3},
pmid = {35646415},
issn = {2092-6456},
abstract = {Coffee, especially the species Coffea arabica and Coffea canephora, is one of the world's most consumed beverages. The consumer demand for caffeine-free coffee is currently being met through chemical decaffeination processes. However, this method leads to loss of beverage quality. In this review, the feasibility of using gene editing to produce caffeine-free coffee plants is reviewed. The genes XMT (7-methylxanthosine methyltransferase) and DXMT (3,7-dimethylxanthine methyltransferase) were identified as candidate target genes for knocking out caffeine production in coffee plants. The possible effect of the knock-out of the candidate genes was assessed. Using Agrobacterium tumefaciens-mediated introduction of the CRISPR-Cas system to Knock out XMT or DXMT would lead to blocking caffeine biosynthesis. The use of CRISPR-Cas to genetically edit consumer products is not yet widely accepted, which may lead to societal hurdles for introducing gene-edited caffeine-free coffee cultivars onto the market. However, increased acceptance of CRISPR-Cas/gene editing on products with a clear benefit for consumers offers better prospects for gene editing efforts for caffeine-free coffee.},
}
@article {pmid35643959,
year = {2022},
author = {Yin, J and Hu, J},
title = {The origin of unwanted editing byproducts in gene editing.},
journal = {Acta biochimica et biophysica Sinica},
volume = {54},
number = {6},
pages = {1-15},
doi = {10.3724/abbs.2022056},
pmid = {35643959},
issn = {1745-7270},
abstract = {The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.},
}
@article {pmid35643083,
year = {2022},
author = {Kato, K and Zhou, W and Okazaki, S and Isayama, Y and Nishizawa, T and Gootenberg, JS and Abudayyeh, OO and Nishimasu, H},
title = {Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2022.05.003},
pmid = {35643083},
issn = {1097-4172},
abstract = {The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.},
}
@article {pmid35642647,
year = {2022},
author = {Verma, MK and Roychowdhury, S and Sahu, BD and Mishra, A and Sethi, KK},
title = {CRISPR-based point-of-care diagnostics incorporating Cas9, Cas12, and Cas13 enzymes advanced for SARS-CoV-2 detection.},
journal = {Journal of biochemical and molecular toxicology},
volume = {},
number = {},
pages = {e23113},
doi = {10.1002/jbt.23113},
pmid = {35642647},
issn = {1099-0461},
abstract = {An outbreak of the novel beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first came to light in December 2019, which has unfolded rapidly and turned out to be a global pandemic. Early prognosis of viral contamination involves speedy intervention, disorder control, and good-sized management of the spread of disease. Reverse transcription-polymerase chain reaction, considered the gold standard test for detecting nucleic acids and pathogen diagnosis, provides high sensitivity and specificity. However, reliance on high-priced equipped kits, associated reagents, and skilled personnel slow down sickness detection. Lately, the improvement of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated protein)-based diagnostic systems has reshaped molecular diagnosis due to their low cost, simplicity, speed, efficiency, high sensitivity, specificity, and versatility, which is vital for accomplishing point-of-care diagnostics. We reviewed and summarized CRISPR-Cas-based point-of-care diagnostic strategies and research in these paintings while highlighting their characteristics and challenges for identifying SARS-CoV-2.},
}
@article {pmid35642255,
year = {2022},
author = {de Bruijn, R and Wielstra, PCM and Calcines-Cruz, C and van Waveren, T and Hernandez-Garcia, A and van der Schoot, P},
title = {A kinetic model for the impact of packaging signal mimics on genome encapsulation.},
journal = {Biophysical journal},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bpj.2022.05.040},
pmid = {35642255},
issn = {1542-0086},
abstract = {Inspired by recent experiments on the spontaneous assembly of virus-like particles from a solution containing a synthetic coat protein and double-stranded DNA, we put forward a kinetic model that has as main ingredients a stochastic nucleation and a deterministic growth process. The efficiency and rate of DNA packaging strongly increase after tiling the DNA with CRISPR-Cas proteins at predesignated locations, mimicking assembly signals in viruses. Our model shows that treating these proteins as nucleation-inducing diffusion barriers is sufficient to explain the experimentally observed increase in encapsulation efficiency, but only if the nucleation rate is sufficiently high. We find an optimum in the encapsulation kinetics for conditions where the number of packaging signal mimics is equal to the number of nucleation events that can occur during the time required to fully encapsulate the DNA template, presuming that the nucleation events can only take place adjacent to a packaging signal. Our theory is in satisfactory agreement with the available experimental data.},
}
@article {pmid35641501,
year = {2022},
author = {Volke, DC and Martino, RA and Kozaeva, E and Smania, AM and Nikel, PI},
title = {Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3026},
pmid = {35641501},
issn = {2041-1723},
support = {NNF17CC0026768//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF20CC0035580//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF18OC0034818//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; PICT-2016-1545//Ministry of Science, Technology and Productive Innovation, Argentina | Agencia Nacional de Promoción Científica y Tecnológica (National Agency for Science and Technology, Argentina)/ ; PICT-2019-1590//Ministry of Science, Technology and Productive Innovation, Argentina | Agencia Nacional de Promoción Científica y Tecnológica (National Agency for Science and Technology, Argentina)/ ; 8021-00039B//Det Frie Forskningsråd (Danish Council for Independent Research)/ ; 814418//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
abstract = {CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.},
}
@article {pmid35475666,
year = {2022},
author = {Maeda, F and Kato, A and Takeshima, K and Shibazaki, M and Sato, R and Shibata, T and Miyake, K and Kozuka-Hata, H and Oyama, M and Shimizu, E and Imoto, S and Miyano, S and Adachi, S and Natsume, T and Takeuchi, K and Maruzuru, Y and Koyanagi, N and Jun, A and Yasushi, K},
title = {Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.},
journal = {Journal of virology},
volume = {96},
number = {10},
pages = {e0030622},
doi = {10.1128/jvi.00306-22},
pmid = {35475666},
issn = {1098-5514},
mesh = {Animals ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; *Herpesvirus 1, Human/genetics/physiology ; Humans ; *Membrane Transport Proteins/metabolism ; Nuclear Envelope/metabolism ; Nuclear Proteins ; Proteomics ; Vero Cells ; Viral Proteins/metabolism ; *Virus Release ; },
abstract = {This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.},
}
@article {pmid35298644,
year = {2022},
author = {Bewg, WP and Harding, SA and Engle, NL and Vaidya, BN and Zhou, R and Reeves, J and Horn, TW and Joshee, N and Jenkins, JW and Shu, S and Barry, KW and Yoshinaga, Y and Grimwood, J and Schmitz, RJ and Schmutz, J and Tschaplinski, TJ and Tsai, CJ},
title = {Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.},
journal = {Plant physiology},
volume = {189},
number = {2},
pages = {516-526},
doi = {10.1093/plphys/kiac128},
pmid = {35298644},
issn = {1532-2548},
support = {//The Center for Bioenergy Innovation/ ; //US Department of Energy Research Center/ ; //Office of Biological and Environmental Research in the DOE Office of Science/ ; IOS-1546867//Division of Integrative Organismal Systems/ ; //Community Science Program of the Joint Genome Institute/ ; //DOE Office of Science User Facility/ ; DE-AC02-05CH11231//US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy/ ; },
abstract = {As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.},
}
@article {pmid35637939,
year = {2021},
author = {Petraitytė, G and Preikšaitienė, E and Mikštienė, V},
title = {Genome Editing in Medicine: Tools and Challenges.},
journal = {Acta medica Lituanica},
volume = {28},
number = {2},
pages = {205-219},
doi = {10.15388/Amed.2021.28.2.8},
pmid = {35637939},
issn = {1392-0138},
abstract = {Studies which seek fundamental, thorough knowledge of biological processes, and continuous advancement in natural sciences and biotechnology enable the establishment of molecular strategies and tools to treat disorders caused by genetic mutations. Over the years biological therapy evolved from using stem cells and viral vectors to RNA therapy and testing different genome editing tools as promising gene therapy agents. These genome editing technologies (Zinc finger nucleases, TAL effector nucleases), specifically CRISPR-Cas system, revolutionized the field of genetic engineering and is widely applied to create cell and animal models for various hereditary, infectious human diseases and cancer, to analyze and understand the molecular and cellular base of pathogenesis, to find potential drug/treatment targets, to eliminate pathogenic DNA changes in various medical conditions and to create future "precise medication". Although different concerning factors, such as precise system delivery to the target cells, efficacy and accuracy of editing process, different approaches of making the DNA changes as well as worrying bioethical issues remain, the importance of genome editing technologies in medicine is undeniable. The future of innovative genome editing approach and strategies to treat diseases is complicated but interesting and exciting at once for all related parties - researchers, clinicians, and patients.},
}
@article {pmid35636828,
year = {2022},
author = {Rodino, KG and Smith, KP and Pettengill, MA},
title = {Novel Assays for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2.},
journal = {Clinics in laboratory medicine},
volume = {42},
number = {2},
pages = {299-307},
doi = {10.1016/j.cll.2022.02.004},
pmid = {35636828},
issn = {1557-9832},
abstract = {From the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 pandemic, there has been a major emphasis on molecular laboratory tests for the virus. Shortages in various testing supplies, the desire to increase testing capacity, and a push to make point-of-care or home-based testing available have fostered considerable innovation for SARS-CoV-2 molecular diagnostics, advancements likely to be applicable to other diagnostic uses. The authors attempt to cover some of the most compelling novel types of molecular assays or novel approaches in adapting established molecular methodologies for SARS-CoV-2 detection or characterization.},
}
@article {pmid35634923,
year = {2022},
author = {Ansai, S and Kitano, J},
title = {Speciation and adaptation research meets genome editing.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {377},
number = {1855},
pages = {20200516},
doi = {10.1098/rstb.2020.0516},
pmid = {35634923},
issn = {1471-2970},
abstract = {Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.},
}
@article {pmid35634921,
year = {2022},
author = {Kitano, J and Ishikawa, A and Ravinet, M and Courtier-Orgogozo, V},
title = {Genetic basis of speciation and adaptation: from loci to causative mutations.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {377},
number = {1855},
pages = {20200503},
doi = {10.1098/rstb.2020.0503},
pmid = {35634921},
issn = {1471-2970},
abstract = {Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.},
}
@article {pmid35633938,
year = {2022},
author = {Piskunen, P and Latham, R and West, CE and Castronovo, M and Linko, V},
title = {Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond.},
journal = {iScience},
volume = {25},
number = {6},
pages = {104389},
doi = {10.1016/j.isci.2022.104389},
pmid = {35633938},
issn = {2589-0042},
abstract = {Precise genome editing with CRISPR/Cas paves the way for many biochemical, biotechnological, and medical applications, and consequently, it may enable treatment of already known and still-to-be-found genetic diseases. Meanwhile, another rapidly emerging field-structural DNA nanotechnology-provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective.},
}
@article {pmid35628611,
year = {2022},
author = {Serrano, LJ and Garcia-Arranz, M and De Pablo-Moreno, JA and Segovia, JC and Olivera-Salazar, R and Garcia-Olmo, D and Liras, A},
title = {Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105802},
pmid = {35628611},
issn = {1422-0067},
support = {RETICS RD 16/0011/00011 and RD 16/0011/0013//European Regional Development/ ; AvanCell-CM; Ref S2017/BMD-3692//Directorate General for Research of the Community of Madrid/ ; PID2020-119637RB-I00//State Research Agency/ ; ASDEFAV 2021-23//Association for the Research and Cure of Factor V Deficiency (ASDEFAV)./ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Factor V/genetics ; *Factor V Deficiency/genetics ; Gene Editing ; Humans ; Mutation ; },
abstract = {Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.},
}
@article {pmid35628572,
year = {2022},
author = {Pavese, V and Moglia, A and Abbà, S and Milani, AM and Torello Marinoni, D and Corredoira, E and Martínez, MT and Botta, R},
title = {First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105762},
pmid = {35628572},
issn = {1422-0067},
support = {RF=2018.2549//Fondazione CRT/ ; PID2020-112627RB-C33//MICINN/ ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Plant Breeding ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.},
}
@article {pmid35628407,
year = {2022},
author = {Hu, H and Yu, F},
title = {A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105597},
pmid = {35628407},
issn = {1422-0067},
support = {90//Agriculture and Agriculture-Food Canada/ ; },
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in Arabidopsis thaliana (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the Arabidopsis background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.},
}
@article {pmid35627307,
year = {2022},
author = {Xiong, Y and Zhuang, R and Zhao, G and Liu, Y and Su, Y and Wang, W and Xi, X and Yang, Y and Han, X and Xie, S and Wang, H and Li, X and Zuo, B and Zhao, S and Feng, Z and Ruan, J},
title = {Identification of the CKM Gene as a Potential Muscle-Specific Safe Harbor Locus in Pig Genome.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
doi = {10.3390/genes13050921},
pmid = {35627307},
issn = {2073-4425},
support = {2021YFA0805903-3//National Key R&amp;D Program of China/ ; 2019B030301010//Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding/ ; No.2020ABA016//Major Science and Technology Projects in Hubei Province/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Genome/genetics ; Livestock/genetics ; Muscles ; Promoter Regions, Genetic ; Swine/genetics ; },
abstract = {Genetically modified pigs have shown considerable application potential in the fields of life science research and livestock breeding. Nevertheless, a barrier impedes the production of genetically modified pigs. There are too few safe harbor loci for the insertion of foreign genes into the pig genome. Only a few loci (pRosa26, pH11 and Pifs501) have been successfully identified to achieve the ectopic expression of foreign genes and produce gene-edited pigs. Here, we use CRISPR/Cas9-mediated homologous directed repair (HDR) to accurately knock the exogenous gene-of-interest fragments into an endogenous CKM gene in the porcine satellite cells. After porcine satellite cells are induced to differentiate, the CKM gene promoter simultaneously initiates the expression of the CKM gene and the exogenous gene. We infer preliminarily that the CKM gene can be identified as a potential muscle-specific safe harbor locus in pigs for the integration of exogenous gene-of-interest fragments.},
}
@article {pmid35627242,
year = {2022},
author = {Carrington, B and Bishop, K and Sood, R},
title = {A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
doi = {10.3390/genes13050857},
pmid = {35627242},
issn = {2073-4425},
support = {Intramural Research Program/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Transcription Activator-Like Effector Nucleases ; *Zebrafish/genetics ; },
abstract = {The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.},
}
@article {pmid35627121,
year = {2022},
author = {Jiang, C and Tao, D and Geng, Y and Yang, H and Xu, B and Chen, Y and Hu, C and Chen, H and Xie, S and Guo, A},
title = {Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
doi = {10.3390/genes13050734},
pmid = {35627121},
issn = {2073-4425},
support = {#2020BBA055//the Key Research and Development Program of Hubei Province/ ; CARS-37//China Agriculture Research System (beef/yaks) of MOF and MARA/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; DNA, Viral/genetics ; *Lumpy skin disease virus/genetics ; Lysergic Acid Diethylamide ; Nucleotidyltransferases/genetics ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; },
abstract = {Lumpy skin disease (LSD) is a severe and highly infectious pox disease of cattle caused by the lumpy skin disease virus (LSDV). To facilitate early control of LSD, this study aimed to develop a new rapid on-site LSDV detection method using an orf068 gene-based recombinase polymerase amplification assay (RPA) coupled with a CRISPR-Cas12a-based fluorescence assay (RPA-Cas12a-fluorescence assay). The results showed that the sensitivity of our RPA-Cas12a-fluorescence assay for detecting LSDV orf068 gene reached 5 copies/μL with plasmid as a template, and 102 TCID50/mL with viral genomic DNA as a template. No cross-reaction with other common bovine viruses was observed. Further, an on-site RPA-Cas12a-fluorescence assay of 40 clinical samples from cattle with or without LSD showed a diagnostic sensitivity of 96.3% (95% CI: 81.0-99.9%) and specificity of 92.31% (95% CI: 62.1-99.6%), which was close to those of the quantitative PCR assay. Therefore, our RPA-Cas12a-fluorescence assay has promising prospects in on-site rapid LSDV detection.},
}
@article {pmid35626649,
year = {2022},
author = {Abuhamad, AY and Mohamad Zamberi, NN and Sheen, L and Naes, SM and Mohd Yusuf, SNH and Ahmad Tajudin, A and Mohtar, MA and Amir Hamzah, AS and Syafruddin, SE},
title = {Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations.},
journal = {Cells},
volume = {11},
number = {10},
pages = {},
doi = {10.3390/cells11101612},
pmid = {35626649},
issn = {2073-4409},
support = {Geran Putra Berimpak, UPM/700-2/1/GBP/2017/9551200//Universiti Putra Malaysia/ ; Fundamental Research Grant Scheme FRGS/1/2020/SKK0/UKM/03/3//Malaysia Ministry of Higher Education/ ; Collaborative Research Programme - International Centre for Genetic Engineering and Biotech-nology Grant (CRP/MYS19-04_EC).//International Centre for Genetic Engineering and Biotechnology/ ; },
mesh = {*Breast Neoplasms/genetics ; *CRISPR-Cas Systems/genetics ; Female ; HEK293 Cells ; Humans ; Mutation/genetics ; Tumor Suppressor Protein p53/genetics ; Workflow ; },
abstract = {Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations' molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.},
}
@article {pmid35623786,
year = {2022},
author = {Zhou, S and Sun, H and Huo, D and Wang, X and Qi, N and Peng, L and Yang, M and Lu, P and Hou, C},
title = {A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation.},
journal = {Analytica chimica acta},
volume = {1212},
number = {},
pages = {339914},
doi = {10.1016/j.aca.2022.339914},
pmid = {35623786},
issn = {1873-4324},
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA/genetics ; *DNA Methylation ; *Deoxyribonuclease I ; Humans ; },
abstract = {Detection of methylation changes associated with oncogenic transformation is essential for early screening and treatment of cancer. Herein, we propose a novel DNA methylation detection assay based on the methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a (GlaI-DC-SDA-CRISPR/Cas12a). The GlaI enables highly specific recognition and digestion of methylated target site (dsDNA) but leaves unmethylated target intact. Therefore, only methylated DNA can be digested to produce two free 3'-OH terminus for triggering the next SDA-CRISPR/Cas12a. Compared with the fluorescence response under single amplification template, DC-SDA with double amplification templates shows higher sensitivity. Benefiting from the high specificity of GlaI and the cascaded amplification effect of DC-SDA combined with CRISPR/Cas12a, the proposed method shows excellent performance for DNA methylation detection with low LOD (1.28 × 10-13 M), ultra-low background interference and wide detection range (2 × 10-13 to 4 × 10-11, 4 × 10-11 to 1 × 10-8 M). 0.1% of DNA methylation can be discriminated from the mixture with a mass of unmethylated DNA. Most importantly, the proposed assay can be applied to the actual detection of human serum and genomic DNA, as well as to distinguish normal cells from cancer cells. It can also quantify DNA methylation in genomic DNA (HCT116) with a LOD of 37.95 ng, indicating its great potential in early clinical cancer screening.},
}
@article {pmid35430120,
year = {2022},
author = {Kleinboehl, E and Laoharawee, K and Moriarity, BS},
title = {Primary B cell engineering for therapeutic research.},
journal = {Trends in molecular medicine},
volume = {28},
number = {6},
pages = {528-529},
doi = {10.1016/j.molmed.2022.03.007},
pmid = {35430120},
issn = {1471-499X},
mesh = {B-Lymphocytes ; CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Genetic Engineering ; Humans ; *Therapeutic Human Experimentation ; },
}
@article {pmid35619054,
year = {2022},
author = {Arnan, C and Ullrich, S and Pulido-Quetglas, C and Nurtdinov, R and Esteban, A and Blanco-Fernandez, J and Aparicio-Prat, E and Johnson, R and Pérez-Lluch, S and Guigó, R},
title = {Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {402},
pmid = {35619054},
issn = {1471-2164},
support = {BIO2015-70777-P//Ministerio de Economía y Competitividad/ ; ERC-2011-AdG-294653-RNA-MAPS//European Union Seventh Framework Programme (FP7/2007-2013)/ ; },
mesh = {CRISPR-Cas Systems ; Cell Transdifferentiation ; Humans ; Macrophages ; *RNA, Guide/genetics ; *RNA, Long Noncoding/genetics ; },
abstract = {CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.},
}
@article {pmid35618895,
year = {2022},
author = {DeLuca, S and Bursac, N},
title = {CRISPR Library Screening in Cultured Cardiomyocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2485},
number = {},
pages = {1-13},
pmid = {35618895},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Gene Library ; Genome ; High-Throughput Nucleotide Sequencing ; *Myocytes, Cardiac ; },
abstract = {CRISPR-Cas9-based screening technologies enable precise, high-throughput genetic and epigenetic manipulation to study mechanisms of development and disease and identify new therapeutic targets. Here, we describe a general protocol for the generation of custom, pooled CRISPR sgRNA libraries for screening in cardiomyocyte cultures. This methodology can address a variety of lab-specific research questions in cardiomyocytes and other cell types, as the genes to be modified can be curated or whole genomes can be investigated. The use of lentiviral sgRNA delivery followed by high-throughput sequencing allows for rapid comparison and identification of candidate genes and epigenetic modifiers, which can be further validated individually or in sub-pooled libraries following screening.},
}
@article {pmid35617303,
year = {2022},
author = {Hernandez, VA and Carvajal-Moreno, J and Wang, X and Pietrzak, M and Yalowich, JC and Elton, TS},
title = {Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells.},
journal = {PloS one},
volume = {17},
number = {5},
pages = {e0265794},
doi = {10.1371/journal.pone.0265794},
pmid = {35617303},
issn = {1932-6203},
mesh = {Antigens, Neoplasm/genetics ; CRISPR-Cas Systems/genetics ; DNA Topoisomerases, Type II/genetics/metabolism ; Etoposide/pharmacology ; Humans ; Introns/genetics ; K562 Cells ; *Leukemia/genetics ; *RNA Splice Sites ; RNA, Messenger ; },
abstract = {DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5' splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5' splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.},
}
@article {pmid35616867,
year = {2022},
author = {Jansing, J and Bortesi, L},
title = {Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2480},
number = {},
pages = {241-284},
pmid = {35616867},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Glycosyltransferases/genetics/metabolism ; Humans ; Mammals/genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Polysaccharides/chemistry ; Recombinant Proteins/genetics/metabolism ; *Tobacco/genetics/metabolism ; Xylose/metabolism ; },
abstract = {Plants are excellent production hosts for the in vivo synthesis of complex glycosylated proteins such as antibodies. The plant N-glycosylation machinery is largely similar to that found in humans and other mammalian organisms, which is an advantage in comparison to microbial production systems in particular. However, there are some differences in the identity and chemical linkage of the sugars that plants and mammals use to build their N-glycans. These differences can affect important properties of glycosylated proteins produced recombinantly in plants. Here we describe the complete procedure of multiplex targeted gene knockout with CRISPR/Cas9 in Nicotiana benthamiana in order to eliminate the undesirable sugars α-1,3-fucose and β-1,2-xylose from the plant N-glycans. The workflow includes target gene identification, guide RNA design and testing, plant transformation, and the analysis of the regenerated transgenic plants by Sanger sequencing, immunoblot, and mass-spectrometric analysis of recombinant and endogenous proteins.},
}
@article {pmid35580804,
year = {2022},
author = {Petel Légaré, V and Rampal, CJ and Gurberg, TJN and Harji, ZA and Allard-Chamard, X and Rodríguez, EC and Armstrong, GAB},
title = {Development of an endogenously myc-tagged TARDBP (TDP-43) zebrafish model using the CRISPR/Cas9 system and homology directed repair.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {261},
number = {},
pages = {110756},
doi = {10.1016/j.cbpb.2022.110756},
pmid = {35580804},
issn = {1879-1107},
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Epitopes/metabolism ; *Neurodegenerative Diseases/genetics ; *Zebrafish/genetics/metabolism ; },
abstract = {Many of the modern advances in cellular biology have been made by the expression of engineered constructs with epitope tags for subsequent biochemical investigations. While the utility of epitope tags has permitted insights in cellular and animal models, these are often expressed using traditional transgenic approaches. Using the CRISPR/Cas9 system and homology directed repair we recombine a single myc epitope sequence following the start codon of the zebrafish ortholog of TARDBP (TDP-43). TDP-43 is an RNA binding protein that is involved in the neurodegenerative disease amyotrophic lateral sclerosis and frontotemporal dementia. We report that zebrafish expressing the myc-tardbp engendered allele produced a stable protein that was detected by both western blot and immunofluorescence. Furthermore, both heterozygous and homozygous carriers of the myc-tardbp allele developed to sexual maturity. We propose that the methodology used here will be useful for zebrafish researchers and other comparative animal biologists interested in developing animal models expressing endogenously tagged proteins.},
}
@article {pmid35527517,
year = {2022},
author = {Cao, G and Dong, J and Chen, X and Lu, P and Xiong, Y and Peng, L and Li, J and Huo, D and Hou, C},
title = {Simultaneous detection of CaMV35S and T-nos utilizing CRISPR/Cas12a and Cas13a with multiplex-PCR (MPT-Cas12a/13a).},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {43},
pages = {6328-6331},
doi = {10.1039/d2cc01300b},
pmid = {35527517},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural ; *Multiplex Polymerase Chain Reaction ; },
abstract = {Here, we established a strategy (MPT-Cas12a/13a) that combined CRISPR/Cas12a and Cas13a for simultaneously detecting CaMV35S and T-nos based on multiplex PCR (M-PCR) and transcription. It realized a simultaneous detection mode with different signals in the same space. The MPT-Cas12a/13a had excellent sensitivity with the limit of detection as low as 11 copies of T-nos and 13 copies of CaMV35S and it had outstanding specificity and anti-interference ability in actual sample analysis. Therefore, it is a potential candidate in the detection of GM crops.},
}
@article {pmid35526203,
year = {2022},
author = {Liu, L and Chen, Z and Tian, X and Chu, J},
title = {Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1.},
journal = {Biotechnology letters},
volume = {44},
number = {5-6},
pages = {755-766},
pmid = {35526203},
issn = {1573-6776},
support = {No. 2019YFA0904800//National Key Research Development Program of China/ ; },
mesh = {*Acremonium/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; Cephalosporins/metabolism ; GTP-Binding Proteins/genetics/metabolism ; Gene Editing ; Genes, Fungal ; Membrane Glycoproteins/genetics ; Saccharomyces cerevisiae/genetics ; *Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {OBJECTIVE: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum.<br><br>RESULT: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed.<br><br>CONCLUSION: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.},
}
@article {pmid35512461,
year = {2022},
author = {Song, G and Li, X and Wang, Z and Dong, C and Xie, X and Yan, X},
title = {Structure of AcrVIA2 and its binding mechanism to CRISPR-Cas13a.},
journal = {Biochemical and biophysical research communications},
volume = {612},
number = {},
pages = {84-90},
doi = {10.1016/j.bbrc.2022.04.091},
pmid = {35512461},
issn = {1090-2104},
mesh = {Bacteria/metabolism ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *RNA, Guide/genetics ; },
abstract = {Phages and non-phage derived bacteria have evolved many anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes. Thus Acrs can be applied as a regulatory tool for gene edition by CRISPR system. Recently, a non-phage derived AcrVIA2 has been identified as an inhibitor that blocks the editing activity of Cas13a in vitro by binding to Cas13a. Here, we solved the crystal structure of AcrVIA2 at a resolution of 2.59 Å and confirmed that AcrVIA2 can bind to Helical-I domain in LshCas13a. Structural analysis show that the V-shaped acidic groove formed by β3-β3 hairpin of AcrVIA2 dimer is the key region that mediates the interaction between AcrVIA2 and Helical-I domain. In addition, we also reveal that Asp37 of AcrVIA2 plays an essential role in the functioning of the V-shaped acidic groove, and the functional dimer conformation of AcrVIA2 is stabilized by hydrogen bonds formed between Tyr41 of one monomer with Glu35 and Asp37 of the other monomer. These data expand the current understanding of the diverse interaction mechanisms between Acrs and Cas proteins, and also provide new ideas for the development of CRISPR-Cas13a regulatory tool.},
}
@article {pmid35510603,
year = {2022},
author = {Li, Y and Zeng, R and Wang, W and Xu, J and Gong, H and Li, L and Li, M and Tang, D},
title = {Size-Controlled Engineering Photoelectrochemical Biosensor for Human Papillomavirus-16 Based on CRISPR-Cas12a-Induced Disassembly of Z-Scheme Heterojunctions.},
journal = {ACS sensors},
volume = {7},
number = {5},
pages = {1593-1601},
doi = {10.1021/acssensors.2c00691},
pmid = {35510603},
issn = {2379-3694},
mesh = {*Alphapapillomavirus ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/chemistry ; DNA, Single-Stranded ; Electrochemical Techniques/methods ; Human papillomavirus 16/genetics ; Humans ; },
abstract = {Photoelectrochemical (PEC) biosensors incorporating biomolecular recognition with photon-to-electron conversion capabilities of the photoactive species have been developed for molecular diagnosis, but most involve difficulty in adjusting band gap positions and are unsuitable for PEC biodetection. In this work, an innovative PEC biosensor combined with quantum size-controlled engineering based on quantum confinement by controlling the quantum size was designed for the detection of human papillomavirus-16 (HPV-16) through CRISPR-Cas12a (Cpf1)-induced disassembly of Z-scheme heterojunction. To the best of our knowledge, quantum size-controlled engineering that precisely tunes the properties of photoactive materials is first utilized in the PEC bioanalysis. Based on the quantum size effect, the light absorption efficiency and charge-transfer rate were tuned to suitable levels to obtain the best PEC performance. After incubation with target HPV-16, the binding of Cas12a-crRNA to the target double-stranded DNA (dsDNA) stimulated the activity of indiscriminate cleavage toward single-stranded DNA (ssDNA), resulting in a decrease in photocurrent due to the blocking of electron transfer through the heterojunction. By optimizing experimental conditions, the Z-scheme sensing system exhibited incredible photocurrent response to HPV-16 in the range from 3.0 pM to 600 nM with a detection limit of 1.0 pM. Impressively, the application of the quantum size effect could stimulate more interest in the precise design of band gap structure to improve PEC performance.},
}
@article {pmid35500431,
year = {2022},
author = {Navarro-Serna, S and Dehesa-Etxebeste, M and Piñeiro-Silva, C and Romar, R and Lopes, JS and López de Munaín, A and Gadea, J},
title = {Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination.},
journal = {Theriogenology},
volume = {186},
number = {},
pages = {175-184},
doi = {10.1016/j.theriogenology.2022.04.012},
pmid = {35500431},
issn = {1879-3231},
mesh = {Animals ; *CRISPR-Cas Systems ; *Calpain/genetics ; Electroporation/methods/veterinary ; Gene Editing/methods/veterinary ; Insemination ; Microinjections/veterinary ; Oocytes ; Swine/genetics ; },
abstract = {Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.},
}
@article {pmid35482449,
year = {2022},
author = {Li, YY and Li, HD and Fang, WK and Liu, D and Liu, MH and Zheng, MQ and Zhang, LL and Yu, H and Tang, HW},
title = {Amplification of the Fluorescence Signal with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a Based on Au Nanoparticle-DNAzyme Probe and On-Site Detection of Pb2+ Via the Photonic Crystal Chip.},
journal = {ACS sensors},
volume = {7},
number = {5},
pages = {1572-1580},
doi = {10.1021/acssensors.2c00516},
pmid = {35482449},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems ; *DNA, Catalytic ; Gold ; Lead ; *Metal Nanoparticles ; },
abstract = {Although great headway has been made in DNAzyme-based detection of Pb2+, its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb2+ and its suitability for precise detection of Pb2+ in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb2+ detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb2+ with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb2+ fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb2+ in complex biological or environmental samples, which is of great promise for widespread applications.},
}
@article {pmid35631721,
year = {2022},
author = {Hamdan, MF and Mohd Noor, SN and Abd-Aziz, N and Pua, TL and Tan, BC},
title = {Green Revolution to Gene Revolution: Technological Advances in Agriculture to Feed the World.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {10},
pages = {},
doi = {10.3390/plants11101297},
pmid = {35631721},
issn = {2223-7747},
support = {FRGS/1/2018/STG03/UM/02/2//Ministry of Higher Education/ ; NA170200; IF004-2018//Royal Society-Newton Advanced Fellowship/ ; ST003-2021//University of Malaya/ ; RU004A-2020//University of Malaya/ ; },
abstract = {Technological applications in agriculture have evolved substantially to increase crop yields and quality to meet global food demand. Conventional techniques, such as seed saving, selective breeding, and mutation breeding (variation breeding), have dramatically increased crop production, especially during the 'Green Revolution' in the 1990s. However, newer issues, such as limited arable lands, climate change, and ever-increasing food demand, pose challenges to agricultural production and threaten food security. In the following 'Gene Revolution' era, rapid innovations in the biotechnology field provide alternative strategies to further improve crop yield, quality, and resilience towards biotic and abiotic stresses. These innovations include the introduction of DNA recombinant technology and applications of genome editing techniques, such as transcription activator-like effector (TALEN), zinc-finger nucleases (ZFN), and clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas) systems. However, the acceptance and future of these modern tools rely on the regulatory frameworks governing their development and production in various countries. Herein, we examine the evolution of technological applications in agriculture, focusing on the motivations for their introduction, technical challenges, possible benefits and concerns, and regulatory frameworks governing genetically engineered product development and production.},
}
@article {pmid35631480,
year = {2022},
author = {He, J and Biswas, R and Bugde, P and Li, J and Liu, DX and Li, Y},
title = {Application of CRISPR-Cas9 System to Study Biological Barriers to Drug Delivery.},
journal = {Pharmaceutics},
volume = {14},
number = {5},
pages = {},
doi = {10.3390/pharmaceutics14050894},
pmid = {35631480},
issn = {1999-4923},
abstract = {In recent years, sequence-specific clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems have been widely used in genome editing of various cell types and organisms. The most developed and broadly used CRISPR-Cas system, CRISPR-Cas9, has benefited from the proof-of-principle studies for a better understanding of the function of genes associated with drug absorption and disposition. Genome-scale CRISPR-Cas9 knockout (KO) screen study also facilitates the identification of novel genes in which loss alters drug permeability across biological membranes and thus modulates the efficacy and safety of drugs. Compared with conventional heterogeneous expression models or other genome editing technologies, CRISPR-Cas9 gene manipulation techniques possess significant advantages, including ease of design, cost-effectiveness, greater on-target DNA cleavage activity and multiplexing capabilities, which makes it possible to study the interactions between membrane proteins and drugs more accurately and efficiently. However, many mechanistic questions and challenges regarding CRISPR-Cas9 gene editing are yet to be addressed, ranging from off-target effects to large-scale genetic alterations. In this review, an overview of the mechanisms of CRISPR-Cas9 in mammalian genome editing will be introduced, as well as the application of CRISPR-Cas9 in studying the barriers to drug delivery.},
}
@article {pmid35630321,
year = {2022},
author = {Layton, AM and Redding, KE},
title = {Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum.},
journal = {Microorganisms},
volume = {10},
number = {5},
pages = {},
doi = {10.3390/microorganisms10050876},
pmid = {35630321},
issn = {2076-2607},
support = {DE-SC0010575//United States Department of Energy/ ; },
abstract = {Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.},
}
@article {pmid35628596,
year = {2022},
author = {Clausing, M and William, D and Preussler, M and Biedermann, J and Grützmann, K and Richter, S and Buchholz, F and Temme, A and Schröck, E and Klink, B},
title = {Different Effects of RNAi-Mediated Downregulation or Chemical Inhibition of NAMPT in an Isogenic IDH Mutant and Wild-Type Glioma Cell Model.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105787},
pmid = {35628596},
issn = {1422-0067},
support = {Else Kröner-Promotionskolleg Dresden (M.C.)//Else Kröner-Fresenius-Stiftung/ ; #70112014//German Cancer Aid/ ; },
abstract = {The IDH1R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP+ were described, the consequences for the NAD+ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD+ in IDH1R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD+ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1R132H glioblastoma cells. Given this dependence of IDH1R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1R132H and IDH1wt cells. Altogether, our results indicate that targeting the NAD+ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.},
}
@article {pmid35628563,
year = {2022},
author = {Cai, Q and Guo, D and Cao, Y and Li, Y and Ma, R and Liu, W},
title = {Application of CRISPR/CasΦ2 System for Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105755},
pmid = {35628563},
issn = {1422-0067},
abstract = {CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.},
}
@article {pmid35628253,
year = {2022},
author = {Zhang, N and He, J and Muhammad, A and Shao, Y},
title = {CRISPR/Cas9-Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant-Associated Isolates.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105443},
pmid = {35628253},
issn = {1422-0067},
support = {32022081 and 31970483//National Natural Science Foundation of China/ ; LZ22C170001//Zhejiang Provincial Natural Science Foundation of China/ ; },
abstract = {As one of the most widespread groups of Gram-negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.},
}
@article {pmid35628210,
year = {2022},
author = {Kim, TH and Lee, SW},
title = {Therapeutic Application of Genome Editing Technologies in Viral Diseases.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
doi = {10.3390/ijms23105399},
pmid = {35628210},
issn = {1422-0067},
support = {2019M3E5D5065771, 2020M3A9C8017745//National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT/ ; },
abstract = {Viral infections can be fatal and consequently, they are a serious threat to human health. Therefore, the development of vaccines and appropriate antiviral therapeutic agents is essential. Depending on the virus, it can cause an acute or a chronic infection. The characteristics of viruses can act as inhibiting factors for the development of appropriate treatment methods. Genome editing technology, including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), is a technology that can directly target and modify genomic sequences in almost all eukaryotic cells. The development of this technology has greatly expanded its applicability in life science research and gene therapy development. Research on the use of this technology to develop therapeutics for viral diseases is being conducted for various purposes, such as eliminating latent infections or providing resistance to new infections. In this review, we will look at the current status of the development of viral therapeutic agents using genome editing technology and discuss how this technology can be used as a new treatment approach for viral diseases.},
}
@article {pmid35625953,
year = {2022},
author = {Coira, IF and Rincón, R and Cuendet, M},
title = {The Multiple Myeloma Landscape: Epigenetics and Non-Coding RNAs.},
journal = {Cancers},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/cancers14102348},
pmid = {35625953},
issn = {2072-6694},
support = {310030_184790/SNSF_/Swiss National Science Foundation/Switzerland ; },
abstract = {Despite advances in available treatments, multiple myeloma (MM) remains an incurable disease and represents a challenge in oncohematology. New insights into epigenetic factors contributing to MM development and progression have improved the knowledge surrounding its molecular basis. Beyond classical epigenetic factors, including methylation and acetylation, recent genome analyses have unveiled the importance of non-coding RNAs in MM pathogenesis. Non-coding RNAs have become of interest, as their dysregulation opens the door to new therapeutic approaches. The discovery, in the past years, of molecular techniques, such as CRISPR-Cas, has led to innovative therapies with potential benefits to achieve a better outcome for MM patients. This review summarizes the current knowledge on epigenetics and non-coding RNAs in MM pathogenesis.},
}
@article {pmid35623354,
year = {2022},
author = {Johnson, MC and Hille, LT and Kleinstiver, BP and Meeske, AJ and Bondy-Denomy, J},
title = {Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2022.05.002},
pmid = {35623354},
issn = {1097-4164},
abstract = {CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.},
}
@article {pmid35623245,
year = {2022},
author = {Li, K and Luo, S and Guan, S and Situ, B and Wu, Y and Ou, Z and Tao, M and Zheng, L and Cai, Z},
title = {Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection.},
journal = {Talanta},
volume = {247},
number = {},
pages = {123531},
doi = {10.1016/j.talanta.2022.123531},
pmid = {35623245},
issn = {1873-3573},
abstract = {The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a "one to more" amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL-1. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.},
}
@article {pmid35620343,
year = {2022},
author = {Akbari Kordkheyli, V and Rashidi, M and Shokri, Y and Fallahpour, S and Variji, A and Nabipour Ghara, E and Hosseini, SM},
title = {CRISPER/CAS System, a Novel Tool of Targeted Therapy of Drug-Resistant Lung Cancer.},
journal = {Advanced pharmaceutical bulletin},
volume = {12},
number = {2},
pages = {262-273},
doi = {10.34172/apb.2022.027},
pmid = {35620343},
issn = {2228-5881},
abstract = {Lung cancer (LC) is the most common cause of cancer-related death worldwide. Patients with LC are usually diagnosed at advanced phases. Five-year survival rate in LC patients is approximately 16%. Despite decades of research on LC treatments, clinical outcomes are still very poor, necessitating to develop novel technologies to manage the disease. Considering the role of genetic and epigenetic changes in oncogenes and tumor-suppressor genes in cancer progression, gene therapy provides a hot spot in cancer treatment research. Gene therapy offers less side effects compared to conventional methods such as chemotherapy. Unlike the traditional approaches of gene therapy that have temporary effects, using genetic modification tools can offer persistent cure. Over the past a few years, many studies have effectively used the CRISPR-Cas9 approach to modify gene expression in cells. This system is applied to induce site-specific mutagenesis and epigenetic modifications and regulate gene expression. In this review, we discuss recent applications of the CRISPR-Cas9 technology in treating LC.},
}
@article {pmid35613590,
year = {2022},
author = {Del Amo, VL and Juste, SS and Gantz, VM},
title = {A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila.},
journal = {Cell reports},
volume = {39},
number = {8},
pages = {110843},
doi = {10.1016/j.celrep.2022.110843},
pmid = {35613590},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA ; *Deoxyribonuclease I/metabolism ; Drosophila/metabolism ; *Gene Drive Technology ; Gene Editing ; },
abstract = {CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.},
}
@article {pmid35618430,
year = {2022},
author = {Woodside, WT and Vantsev, N and Catchpole, RJ and Garrett, SC and Olson, S and Graveley, BR and Terns, MP},
title = {Type III-A CRISPR-Cas systems as a versatile gene knockdown technology.},
journal = {RNA (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1261/rna.079206.122},
pmid = {35618430},
issn = {1469-9001},
abstract = {CRISPR-Cas systems are functionally diverse prokaryotic anti-viral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis and S. thermophilus) were each expressed on a single plasmid in E. coli and the efficiency and specificity of gene knockdown was assessed by Northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and non-coding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.},
}
@article {pmid35617958,
year = {2022},
author = {Omer-Javed, A and Pedrazzani, G and Albano, L and Ghaus, S and Latroche, C and Manzi, M and Ferrari, S and Fiumara, M and Jacob, A and Vavassori, V and Nonis, A and Canarutto, D and Naldini, L},
title = {Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2022.04.039},
pmid = {35617958},
issn = {1097-4172},
abstract = {Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.},
}
@article {pmid35616865,
year = {2022},
author = {González, B and Vazquez-Vilar, M and Sánchez-Vicente, J and Orzáez, D},
title = {Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2480},
number = {},
pages = {193-214},
pmid = {35616865},
issn = {1940-6029},
abstract = {New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.},
}
@article {pmid35611733,
year = {2022},
author = {Shang, L and Song, S and Zhang, T and Yan, K and Cai, H and Yuan, Y and Cheng, Y},
title = {[Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {5},
pages = {1847-1858},
doi = {10.13345/j.cjb.210541},
pmid = {35611733},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Muscle, Skeletal/metabolism ; Mutation ; *Myostatin/genetics/metabolism ; Phenotype ; Rabbits ; },
abstract = {Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.},
}
@article {pmid35610381,
year = {2022},
author = {Zhou, M and Cao, Y and Sui, M and Shu, X and Wan, F and Zhang, B},
title = {Dead Cas(t) light on new life: CRISPRa-mediated reprogramming of somatic cells into neurons.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {6},
pages = {315},
pmid = {35610381},
issn = {1420-9071},
support = {81471283//National Natural Science Foundation of China/ ; 82072795//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Gene Editing ; Neurons/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Overexpression of exogenous lineage-specific transcription factors could directly induce terminally differentiated somatic cells into target cell types. However, the low conversion efficiency and the concern about introducing exogenous genes limit the clinical application. With the rapid progress in genome editing, the application of CRISPR/dCas9 has been expanding rapidly, including converting somatic cells into other types of cells in vivo and in vitro. Using the CRISPR/dCas9 system, direct neuronal reprogramming could be achieved by activating endogenous genes. Here, we will discuss the latest progress, new insights, and future challenges of the application of the dCas9 system in direct neuronal reprogramming.},
}
@article {pmid35266285,
year = {2022},
author = {Nayeri, S and Baghban Kohnehrouz, B and Ahmadikhah, A and Mahna, N},
title = {CRISPR/Cas9-mediated P-CR domain-specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar.},
journal = {Plant biotechnology journal},
volume = {20},
number = {6},
pages = {1197-1212},
doi = {10.1111/pbi.13803},
pmid = {35266285},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Wall/genetics/metabolism ; Cellulose/metabolism ; Glucosyltransferases/genetics ; *Populus/genetics/metabolism ; },
abstract = {Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high-value bioproducts. The large transmembrane-localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para-crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant-conserved region (P-CR) of PalCESA4 subunit, which are involved in the CESA4-CESA8 heterodimerization. The CRISPR/Cas9-knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4-binding affinity resulting in normal roots, 1.5-2-fold higher plant growth and cell wall regeneration rates, 1.7-fold thinner cell wall, high hemicellulose content, 37%-67% decrease in cellulose content, high cellulose DP, 25%-37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2-fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9-mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose-based nanomaterial technologies.},
}
@article {pmid35610585,
year = {2022},
author = {Sun, L and Wang, J and Yan, F and Wang, G and Li, Y and Huang, J},
title = {CrisprVi: a software for visualizing and analyzing CRISPR sequences of prokaryotes.},
journal = {BMC bioinformatics},
volume = {23},
number = {Suppl 3},
pages = {172},
pmid = {35610585},
issn = {1471-2105},
support = {2018YFD0500500//National Key Research and Development Program of China/ ; R1805//Open Project Program of Jiangsu Key Laboratory of Zoonosis/ ; BK20190878//Natural Science Foundation of Jiangsu Province/ ; yzuxk202015//Cross-Disciplinary Project of the Animal Science Special Discipline of Yangzhou University/ ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi.<br><br>RESULTS: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes.<br><br>CONCLUSIONS: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.},
}
@article {pmid35609453,
year = {2022},
author = {Wu, H and Cao, X and Meng, Y and Richards, D and Wu, J and Ye, Z and deMello, AJ},
title = {DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114377},
doi = {10.1016/j.bios.2022.114377},
pmid = {35609453},
issn = {1873-4235},
abstract = {Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 102 cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.},
}
@article {pmid35608343,
year = {2022},
author = {Zhang, C and Li, N and Rao, L and Li, J and Liu, Q and Tian, C},
title = {Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0232121},
doi = {10.1128/spectrum.02321-21},
pmid = {35608343},
issn = {2165-0497},
abstract = {Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi. IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.},
}
@article {pmid35606745,
year = {2022},
author = {Metzloff, M and Yang, E and Dhole, S and Clark, AG and Messer, PW and Champer, J},
title = {Experimental demonstration of tethered gene drive systems for confined population modification or suppression.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {119},
pmid = {35606745},
issn = {1741-7007},
support = {R21AI130635/NH/NIH HHS/United States ; R01GM127418/NH/NIH HHS/United States ; F32AI138476/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila/genetics ; *Gene Drive Technology/methods ; },
abstract = {BACKGROUND: Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive.<br><br>RESULTS: Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency).<br><br>CONCLUSIONS: Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.},
}
@article {pmid35596077,
year = {2022},
author = {Pan, C and Li, G and Malzahn, AA and Cheng, Y and Leyson, B and Sretenovic, S and Gurel, F and Coleman, GD and Qi, Y},
title = {Boosting plant genome editing with a versatile CRISPR-Combo system.},
journal = {Nature plants},
volume = {8},
number = {5},
pages = {513-525},
pmid = {35596077},
issn = {2055-0278},
support = {IOS-1758745//National Science Foundation (NSF)/ ; },
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.},
}
@article {pmid35595842,
year = {2022},
author = {Flegler, A and Lipski, A},
title = {Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures.},
journal = {Current microbiology},
volume = {79},
number = {7},
pages = {199},
pmid = {35595842},
issn = {1432-0991},
mesh = {*Arthrobacter/genetics/metabolism ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Temperature ; },
abstract = {Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.},
}
@article {pmid35549163,
year = {2022},
author = {Zeng, R and Gong, H and Li, Y and Li, Y and Lin, W and Tang, D and Knopp, D},
title = {CRISPR-Cas12a-Derived Photoelectrochemical Biosensor for Point-Of-Care Diagnosis of Nucleic Acid.},
journal = {Analytical chemistry},
volume = {94},
number = {20},
pages = {7442-7448},
doi = {10.1021/acs.analchem.2c01373},
pmid = {35549163},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; Electrochemical Techniques ; Humans ; Hydrogen Peroxide ; *Nucleic Acids ; Point-of-Care Systems ; },
abstract = {This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) as the photoactive material. G-quadruplexes were capable of biocatalytic precipitation (H2O2-mediated 4-chloro-1-naphthol oxidation) on the In2O3-In2S3/SPE surface, resulting in a weakened photocurrent, but suffered from trans-cleavage when the CRISPR-Cas12a system specifically recognized the analyte. The photocurrent results could be directly observed with the card-sized electrochemical device via a smartphone, which displayed a high-value photocurrent for these positive samples, while a low-value photocurrent for the target-free samples. Such a system exhibited satisfying photocurrent responses toward HPV-16 within a wide working range from 5.0 to 5000 pM and allowed for detection of HPV-16 at a concentration as low as 1.2 pM. The proposed assay provided a smartphone signal readout to enable the rapid screening PEC determination of HPV-16 concentration without sophisticated instruments, thus meeting the requirements of remote areas and resource-limited settings. We envision that combining an efficient biometric PEC sensing platform with a wireless card-sized electrochemical device will enable high-throughput POC diagnostic analysis.},
}
@article {pmid35544322,
year = {2022},
author = {Liang, Y and Xie, J and Zhang, Q and Wang, X and Gou, S and Lin, L and Chen, T and Ge, W and Zhuang, Z and Lian, M and Chen, F and Li, N and Ouyang, Z and Lai, C and Liu, X and Li, L and Ye, Y and Wu, H and Wang, K and Lai, L},
title = {AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.},
journal = {Nucleic acids research},
volume = {50},
number = {9},
pages = {5384-5399},
pmid = {35544322},
issn = {1362-4962},
support = {81941004//National Natural Science Foundation of China/ ; 2021YFA0805903//National Key Research and Development Program of China/ ; ZDYF2021SHFZ052//Key Research &amp; Development Program of Hainan Province/ ; ZDKJ2021030//Major Science and Technology Project of Hainan Province/ ; KFJ-BRP-017-57//Chinese Academy of Sciences/ ; 2019347//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; 2018GZR110104004//Key Research &amp; Development Program of Bioland Laboratory/ ; 2020B1212060052//Science and Technology Planning Project of Guangdong Province/ ; 202007030003//Science and Technology Program of Guangzhou, China/ ; 202002011//2020 Research Program of Sanya Yazhou Bay Science and Technology City/ ; 2019-I2M-5-025//Chinese Academy of Medical Sciences/ ; YESS20200024//CAST/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; INDEL Mutation ; Mammals/genetics ; Mutation ; Uracil-DNA Glycosidase/genetics ; },
abstract = {Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.},
}
@article {pmid35483302,
year = {2022},
author = {Li, Y and Yang, F and Yuan, R and Zhong, X and Zhuo, Y},
title = {Electrochemiluminescence covalent organic framework coupling with CRISPR/Cas12a-mediated biosensor for pesticide residue detection.},
journal = {Food chemistry},
volume = {389},
number = {},
pages = {133049},
doi = {10.1016/j.foodchem.2022.133049},
pmid = {35483302},
issn = {1873-7072},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; *Metal-Organic Frameworks ; *Pesticide Residues ; },
abstract = {The trace detection of pesticide residue becomes particularly important since increasing attentions have been attached to food safety. Herein, we developed an electrochemiluminescence (ECL) covalent organic framework (COF) based-biosensor for trace pesticide detection coupling with CRISPR/Cas12a-mediated signal accumulation strategy. Firstly, the target conversion was carried out with an aptamer-assembled magnetic spherical nucleic acids, which can convert acetamiprid to activator DNA, triggering the CRISPR/Cas12a to make quenching probes far away from electrode for signal accumulation. The COF with stable and strong ECL was synthesized by a condensation reaction between the perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) and melamine (MA), due to the highly ordered arrangement of the PTCDA luminescence units among COF structure and the pore confinement effect. Moreover, the designed assay method was successfully employed to detect the residual level of acetamiprid in real sample and expected to be widely used in pesticide-related food safety.},
}
@article {pmid35418245,
year = {2022},
author = {Parashar, A and Bak, K and Murshed, M},
title = {Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {42},
number = {6},
pages = {e155-e167},
doi = {10.1161/ATVBAHA.122.317518},
pmid = {35418245},
issn = {1524-4636},
abstract = {BACKGROUND: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function.<br><br>METHODS: We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (MgpS3mut/S3mut) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging.<br><br>RESULTS: On a regular diet, the arterial walls in the Mgp-/-; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, MgpS3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp-/- mice, although with lesser severity.<br><br>CONCLUSIONS: For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.},
}
@article {pmid35378143,
year = {2022},
author = {Kovářová, J and Novotná, M and Faria, J and Rico, E and Wallace, C and Zoltner, M and Field, MC and Horn, D},
title = {CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.},
journal = {Molecular and biochemical parasitology},
volume = {249},
number = {},
pages = {111476},
doi = {10.1016/j.molbiopara.2022.111476},
pmid = {35378143},
issn = {1872-9428},
mesh = {CRISPR-Cas Systems ; Genes, Essential ; *Trypanosoma/genetics ; *Trypanosoma brucei brucei/genetics ; Untranslated Regions ; },
abstract = {Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.},
}
@article {pmid35304271,
year = {2022},
author = {Wen, W and Zhang, XB},
title = {CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells.},
journal = {Experimental hematology},
volume = {110},
number = {},
pages = {13-19},
doi = {10.1016/j.exphem.2022.03.002},
pmid = {35304271},
issn = {1873-2399},
mesh = {*CRISPR-Cas Systems ; DNA Repair ; *Gene Editing/methods ; Genetic Therapy ; Genome ; Humans ; },
abstract = {CRISPR-Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR-Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR-Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy.},
}
@article {pmid35247491,
year = {2022},
author = {Chae, SY and Jeong, E and Kang, S and Yim, Y and Kim, JS and Min, DH},
title = {Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {345},
number = {},
pages = {108-119},
doi = {10.1016/j.jconrel.2022.02.035},
pmid = {35247491},
issn = {1873-4995},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Nanoparticles ; Ribonucleoproteins/genetics ; },
abstract = {Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.},
}
@article {pmid34951457,
year = {2022},
author = {Paul, B and Chaubet, L and Verver, DE and Montoya, G},
title = {Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA.},
journal = {Nucleic acids research},
volume = {50},
number = {9},
pages = {5208-5225},
pmid = {34951457},
issn = {1362-4962},
support = {NNF14CC0001//Novo Nordisk Foundation/ ; NNF18OC0055061//Distinguished Investigator/ ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; DNA, Single-Stranded/genetics ; Endonucleases/metabolism ; Gene Editing ; RNA, Guide/genetics ; },
abstract = {Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage λ-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that λ-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.},
}
@article {pmid35605545,
year = {2022},
author = {Jia, HY and Zhao, HL and Wang, T and Chen, PR and Yin, BC and Ye, BC},
title = {A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114382},
doi = {10.1016/j.bios.2022.114382},
pmid = {35605545},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans-cleavage activity of Cas12a for further signal amplification. More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.},
}
@article {pmid35589728,
year = {2022},
author = {Yuan, Q and Gao, X},
title = {Multiplex base- and prime-editing with drive-and-process CRISPR arrays.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2771},
pmid = {35589728},
issn = {2041-1723},
support = {R01HL157714//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; RNA, Guide/genetics ; RNA, Transfer/genetics ; },
abstract = {Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.},
}
@article {pmid35545708,
year = {2022},
author = {Tang, L},
title = {Spatial CRISPR screens in tumors.},
journal = {Nature methods},
volume = {19},
number = {5},
pages = {517},
pmid = {35545708},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neoplasms/genetics ; RNA, Guide ; },
}
@article {pmid35481381,
year = {2022},
author = {Liang, Y and Zou, L and Lin, H and Li, B and Zhao, J and Wang, H and Sun, J and Chen, J and Mo, Y and Yang, X and Deng, X and Tang, S},
title = {Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1811-1823},
doi = {10.1021/acssynbio.1c00643},
pmid = {35481381},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Mutation ; *SARS-CoV-2/genetics ; },
abstract = {Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.},
}
@article {pmid35471824,
year = {2022},
author = {Lei, R and Li, L and Wu, P and Fei, X and Zhang, Y and Wang, J and Zhang, D and Zhang, Q and Yang, N and Wang, X},
title = {RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1772-1781},
doi = {10.1021/acssynbio.1c00620},
pmid = {35471824},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Toxoplasma/genetics/metabolism ; },
abstract = {Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/μL. The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.},
}
@article {pmid35471009,
year = {2022},
author = {Zhu, X and Wu, Y and Lv, X and Liu, Y and Du, G and Li, J and Liu, L},
title = {Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1897-1907},
doi = {10.1021/acssynbio.2c00041},
pmid = {35471009},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics ; *Gene Editing/methods ; Histidine/genetics ; Metabolic Engineering/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based gene-editing technology has been widely used in various microorganisms due to its advantages of low cost, high efficiency, easy operation, and multiple functions. In this study, an efficient and fast double-plasmid gene-editing system pEcCpf1/pcrEG was constructed in Escherichia coli based on CRISPR/Cpf1. First, gene knockout and integration efficiency were verified in eight different kinds of protospacer adjacent motif (PAM) regions. Then, the transformation method was optimized, and the efficiency of gene knockout or gene integration of this system increased to nearly 100%, and the large-length fragments could be integrated into the genome in E. coli BL21 (DE3). The system was also optimized by replacing the homologous recombination system in plasmid pEcCpf1, resulting in pEcCpf1H, which could perform precise single-point mutation, terminator insertion, short-sequence insertion, or gene knockout with high efficiency using a 90 nt (nucleotide) single-stranded primer. Further, multiple genes could be edited simultaneously. Next, these two systems were demonstrated in other E. coli strains. Finally, as an application, the system was used to engineer the synthesis pathway of l-histidine in the engineered strain. The titer of l-histidine in a shake flask reached 7.16 g/L, a value increased by 84.1% compared to the starting strain. Thus, this study provided an effective tool for metabolic engineering of E. coli.},
}
@article {pmid35412812,
year = {2022},
author = {Scott, H and Sun, D and Beal, J and Kiani, S},
title = {Simulation-Based Engineering of Time-Delayed Safety Switches for Safer Gene Therapies.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1782-1789},
doi = {10.1021/acssynbio.1c00621},
pmid = {35412812},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Humans ; },
abstract = {CRISPR-based gene editing is a powerful tool with great potential for applications in the treatment of many inherited and acquired diseases. The longer that CRISPR gene therapy is maintained within a patient, however, the higher the likelihood that it will result in problematic side effects such as off-target editing or immune response. One approach to mitigating these issues is to link the operation of the therapeutic system to a safety switch that autonomously disables its operation and removes the delivered therapeutics after some amount of time. We present here a simulation-based analysis of the potential for regulating the time delay of such a safety switch using one or two transcriptional regulators and/or recombinases. Combinatorial circuit generation identifies 30 potential architectures for such circuits, which we evaluate in simulation with respect to tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation. This modeling predicts one of these circuit architectures to have the desired dynamics and robustness, which can be further tested and applied in the context of CRISPR therapeutics.},
}
@article {pmid35338523,
year = {2022},
author = {Chen, Y and Jong, TT and Chen, C and Sidransky, E},
title = {CRISPR/Cas9-Based Functional Genomics in Human Induced Pluripotent Stem Cell-Derived Models: Can "the Stars Align" for Neurodegenerative Diseases?.},
journal = {Movement disorders : official journal of the Movement Disorder Society},
volume = {37},
number = {5},
pages = {886-890},
doi = {10.1002/mds.28998},
pmid = {35338523},
issn = {1531-8257},
support = {/HG/NHGRI NIH HHS/United States ; //Intramural Research Program/ ; /NH/NIH HHS/United States ; //The Michael J. Fox Foundation for Parkinson's Research (MJFF)/ ; //Aligning Science Across Parkinson's (ASAP)/ ; /HG/NHGRI NIH HHS/United States ; /NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genomics ; Humans ; *Induced Pluripotent Stem Cells ; *Neurodegenerative Diseases/genetics ; },
}
@article {pmid35598011,
year = {2022},
author = {Herrera-Uribe, J and Zaldívar-López, S and Aguilar, C and Entrenas-García, C and Bautista, R and Claros, MG and Garrido, JJ},
title = {Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.},
journal = {Veterinary research},
volume = {53},
number = {1},
pages = {35},
pmid = {35598011},
issn = {1297-9716},
support = {AGL2014-54089-R//Ministerio de Economía y Competitividad/ ; FPDI-2013-15619//Ministerio de Economía y Competitividad/ ; BES-2012-058642//Ministerio de Economía y Competitividad/ ; AGL2017-87415-R//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; IJCI-2017-31382//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; },
abstract = {Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.},
}
@article {pmid35597144,
year = {2022},
author = {Wei, Y and Tao, Z and Wan, L and Zong, C and Wu, J and Tan, X and Wang, B and Guo, Z and Zhang, L and Yuan, H and Wang, P and Yang, Z and Wan, Y},
title = {Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114282},
doi = {10.1016/j.bios.2022.114282},
pmid = {35597144},
issn = {1873-4235},
abstract = {CRISPR-Cas systems have been employed to detect a large variety of pathogenic microorganisms by simply changing the guide RNA sequence. However, these platforms usually rely on nucleic acid extraction and amplification to achieve good sensitivity. Herein, we developed a new platform for the highly specific and sensitive detection of live staphylococcus aureus (S. aureus) based on an Aptamer-based Cas14a1 Biosensor (ACasB), without the need for nucleic acid extraction or amplification. First, the S. aureus specific aptamer was hybrid with a blocker DNA. After the live S. aureus was added, the blocker can be released upon bacteria-aptamer binding. Finally, the released blocker can activate Cas14a1 protein by binding with the sgRNA to generate a change of fluorescent intensity. The ACasB indicates high specificity and sensitivity: it can directly distinguish 400 CFU/ml live S. aureus cells. Comparable to qPCR, the Cas14a1-aptamer biosensor can detect S. aureus with 100% accuracy in complex samples. Therefore, this ACasB for the on-site detection of live S. aureus can broaden its applications in food safety and environmental monitoring.},
}
@article {pmid35595757,
year = {2022},
author = {McGaw, C and Garrity, AJ and Munoz, GZ and Haswell, JR and Sengupta, S and Keston-Smith, E and Hunnewell, P and Ornstein, A and Bose, M and Wessells, Q and Jakimo, N and Yan, P and Zhang, H and Alfonse, LE and Ziblat, R and Carte, JM and Lu, WC and Cerchione, D and Hilbert, B and Sothiselvam, S and Yan, WX and Cheng, DR and Scott, DA and DiTommaso, T and Chong, S},
title = {Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2833},
pmid = {35595757},
issn = {2041-1723},
abstract = {The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.},
}
@article {pmid35595728,
year = {2022},
author = {Schwartz, EA and McBride, TM and Bravo, JPK and Wrapp, D and Fineran, PC and Fagerlund, RD and Taylor, DW},
title = {Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2829},
pmid = {35595728},
issn = {2041-1723},
support = {R35GM138348//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.},
}
@article {pmid35595574,
year = {2022},
author = {Li, L and Shen, G and Wu, M and Jiang, J and Xia, Q and Lin, P},
title = {CRISPR-Cas-mediated diagnostics.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.04.006},
pmid = {35595574},
issn = {1879-3096},
abstract = {An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.},
}
@article {pmid35594718,
year = {2022},
author = {Nath, A and Bhattacharjee, R and Nandi, A and Sinha, A and Kar, S and Manoharan, N and Mitra, S and Mojumdar, A and Panda, PK and Patro, S and Dutt, A and Ahuja, R and Verma, SK and Suar, M},
title = {Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {151},
number = {},
pages = {113122},
doi = {10.1016/j.biopha.2022.113122},
pmid = {35594718},
issn = {1950-6007},
abstract = {The Host-microbiome interactions that exist inside the gut microbiota operate in a synergistic and abnormal manner. Additionally, the normal homeostasis and functioning of gut microbiota are frequently disrupted by the intervention of Multi-Drug Resistant (MDR) pathogens. CRISPR-Cas (CRISPR-associated protein with clustered regularly interspersed short palindromic repeats) recognized as a prokaryotic immune system has emerged as an effective genome-editing tool to edit and delete specific microbial genes for the expulsion of bacteria through bactericidal action. In this review, we demonstrate many functioning CRISPR-Cas systems against the anti-microbial resistance of multiple pathogens, which infiltrate the gastrointestinal tract. Moreover, we discuss the advancement in the development of a phage-delivered CRISPR-Cas system for killing a gut MDR pathogen. We also discuss a combinatorial approach to use bacteriophage as a delivery system for the CRISPR-Cas gene for targeting a pathogenic community in the gut microbiome to resensitize the drug sensitivity. Finally, we discuss engineered phage as a plausible potential option for the CRISPR-Cas system for pathogenic killing and improvement of the efficacy of the system.},
}
@article {pmid35592579,
year = {2022},
author = {Chen, H and Neubauer, M and Wang, JP},
title = {Enhancing HR Frequency for Precise Genome Editing in Plants.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {883421},
pmid = {35592579},
issn = {1664-462X},
abstract = {Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.},
}
@article {pmid35589712,
year = {2022},
author = {Philippe, C and Morency, C and Plante, PL and Zufferey, E and Achigar, R and Tremblay, DM and Rousseau, GM and Goulet, A and Moineau, S},
title = {A truncated anti-CRISPR protein prevents spacer acquisition but not interference.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2802},
pmid = {35589712},
issn = {2041-1723},
abstract = {CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.},
}
@article {pmid35585651,
year = {2022},
author = {Osteikoetxea, X and Silva, A and Lázaro-Ibáñez, E and Salmond, N and Shatnyeva, O and Stein, J and Schick, J and Wren, S and Lindgren, J and Firth, M and Madsen, A and Mayr, LM and Overman, R and Davies, R and Dekker, N},
title = {Engineered Cas9 extracellular vesicles as a novel gene editing tool.},
journal = {Journal of extracellular vesicles},
volume = {11},
number = {5},
pages = {e12225},
doi = {10.1002/jev2.12225},
pmid = {35585651},
issn = {2001-3078},
support = {739593//European Union's Horizon 2020 Research and Innovation Programme/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; *Gene Editing ; HEK293 Cells ; Humans ; Proprotein Convertase 9/genetics ; },
abstract = {Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.},
}
@article {pmid35584220,
year = {2022},
author = {Fan, N and Bian, X and Li, M and Chen, J and Wu, H and Peng, Q and Bai, H and Cheng, W and Kong, L and Ding, S and Li, S and Cheng, W},
title = {Hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons for spatial-controlled genome editing and precise cancer therapy.},
journal = {Science advances},
volume = {8},
number = {20},
pages = {eabn7382},
doi = {10.1126/sciadv.abn7382},
pmid = {35584220},
issn = {2375-2548},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Neoplasms/genetics/therapy ; RNA ; RNA, Messenger/genetics ; },
abstract = {CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.},
}
@article {pmid35583740,
year = {2022},
author = {Wen, Z and Qian, F and Zhang, J and Jiang, Y and Yang, S},
title = {Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {189-206},
pmid = {35583740},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics ; *Gene Editing/methods ; },
abstract = {Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.},
}
@article {pmid35583739,
year = {2022},
author = {Hong, W and Zhang, J and Cui, G and Zhou, Q and Wang, P and Wang, Y},
title = {Highly Efficient Genome Editing in Clostridium difficile Using the CRISPR-Cpf1 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {175-187},
pmid = {35583739},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Clostridioides difficile/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.},
}
@article {pmid35583738,
year = {2022},
author = {Wozniak, KJ and Simmons, LA},
title = {Genome Editing Methods for Bacillus subtilis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {159-174},
pmid = {35583738},
issn = {1940-6029},
mesh = {*Bacillus subtilis/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering ; Humans ; },
abstract = {Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.},
}
@article {pmid35583737,
year = {2022},
author = {Penewit, K and Salipante, SJ},
title = {Recombineering in Staphylococcus aureus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {135-157},
pmid = {35583737},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; Recombinases/genetics ; *Staphylococcal Infections/genetics ; *Staphylococcus aureus/genetics ; },
abstract = {Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.},
}
@article {pmid35583736,
year = {2022},
author = {Ellington, AJ and Reisch, CR},
title = {Generating Single Nucleotide Point Mutations in E. coli with the No-SCAR System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {119-133},
pmid = {35583736},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing/methods ; Nucleotides ; Point Mutation ; },
abstract = {Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is one of the most widely used gene-editing methods, enabling site-specific insertions, deletions, and point mutations of any genomic locus. The no-SCAR system combines λ-Red recombineering with CRISPR/Cas9 for programmable selection of recombinant cells. Recombineering results in the transient production of heteroduplex DNA, as only one strand of DNA is initially altered, leaving the mismatched bases susceptible to repair by the host methyl-directed mismatch repair (MMR) system and reduces the efficiency of generating single nucleotide point mutations. Here we describe a method, where expression of cas9 and the MMR-inhibiting mutLE32K variant are independently controlled by anhydrotetracycline- and cumate-inducible promoters from the pCas9CyMutL plasmid. Thus, MMR is selectively inhibited until recombinant cells have undergone replication and the desired mutation is permanently incorporated. By transiently inhibiting MMR, the accumulation of off-target mutations typically associated with MMR-deficient cell types is minimized. Methods for designing the editing template and sgRNA, cloning of the sgRNA, induction of λ-Red and MutLE32K, the transformation of editing oligo, and induction of Cas9 for mutant selection are detailed within.},
}
@article {pmid35583735,
year = {2022},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {105-117},
pmid = {35583735},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; *Gene Editing/methods ; *Klebsiella pneumoniae/genetics/metabolism ; Technology ; },
abstract = {CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.},
}
@article {pmid35395152,
year = {2022},
author = {Hobbs, SJ and Wein, T and Lu, A and Morehouse, BR and Schnabel, J and Leavitt, A and Yirmiya, E and Sorek, R and Kranzusch, PJ},
title = {Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity.},
journal = {Nature},
volume = {605},
number = {7910},
pages = {522-526},
pmid = {35395152},
issn = {1476-4687},
mesh = {Bacteria/metabolism ; Bacterial Proteins/metabolism ; Bacteriophage T4/metabolism ; *Bacteriophages/physiology ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Escherichia coli/metabolism ; Nucleotides, Cyclic/metabolism ; Oligonucleotides ; Pyrimidines/metabolism ; },
abstract = {The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.},
}
@article {pmid35587321,
year = {2022},
author = {Kumar, G and Jagadeeshwari, U and Sreya, P and Shabbir, A and Sasikala, C and Ramana, CV},
title = {A genomic overview including polyphasic taxonomy of Thalassoroseus pseudoceratinae gen. nov., sp. nov. isolated from a marine sponge, Pseudoceratina sp.},
journal = {Antonie van Leeuwenhoek},
volume = {},
number = {},
pages = {},
pmid = {35587321},
issn = {1572-9699},
abstract = {A pink-coloured, salt- and alkali-tolerant planctomycetal strain (JC658T) with oval to pear-shaped, motile, aerobic, Gram-negative stained cells was isolated from a marine sponge, Pseudoceratina sp. Strain JC658T shares the highest 16S rRNA gene sequence identity with Maioricimonas rarisocia Mal4T (< 89.2%) in the family Planctomycetaceae. The genomic analysis of the new strain indicates its biotechnological potential for the production of various industrially important enzymes, notably sulfatases and carbohydrate-active enzymes (CAZymes), and also potential antimicrobial compounds. Several genes encoding restriction-modification (RM) and CRISPR-CAS systems are also present. NaCl is obligate for growth, of which strain JC658T can tolerate a concentration up to 6% (w/v). Optimum pH and temperature for growth are 8.0 (range 7.0-9.0) and 25 ºC (range 10-40 °C), respectively. The major respiratory quinone of strain JC658T is MK6. Major fatty acids are C16:1ω7c/C16:1ω6c, C18:0 and C16:0. Major polar lipids are phosphatidylcholine, phosphatidyl-dimethylethanolamine and phosphatidyl-monomethylethanolamine. The genomic size of strain JC658T is 7.36 Mb with a DNA G + C content of 54.6 mol%. Based on phylogenetic, genomic (ANI, AAI, POCP, dDDH), chemotaxonomic, physiological and biochemical characteristics, we conclude that strain JC658T belongs to a novel genus and constitutes a novel species within the family Planctomycetaceae, for which we propose the name Thalassoroseus pseudoceratinae gen. nov., sp. nov. The novel species is represented by the type strain JC658T (= KCTC 72881 T = NBRC 114371 T).},
}
@article {pmid35587292,
year = {2022},
author = {Van Vu, T and Das, S and Hensel, G and Kim, JY},
title = {Genome editing and beyond: what does it mean for the future of plant breeding?.},
journal = {Planta},
volume = {255},
number = {6},
pages = {130},
pmid = {35587292},
issn = {1432-2048},
support = {2020R1I1A1A01072130//National Research Foundation of Korea/ ; 2020M3A9I4038352//National Research Foundation of Korea/ ; 2020R1A6A1A03044344//National Research Foundation of Korea/ ; Germany's Excellence Strategy - EXC-2048/1 - project ID 390686111//Deutsche Forschungsgemeinschaft/ ; 426557363//Deutsche Forschungsgemeinschaft/ ; 458717903//Deutsche Forschungsgemeinschaft/ ; ZS/2018/06/93171//European Regional Development Fund/ ; CZ.02.1.01./0.0/0.0/16_019/0000827//Czech Science Foundation/ ; SPP 813103381//Czech Science Foundation/ ; },
abstract = {MAIN CONCLUSION: Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.},
}
@article {pmid35586709,
year = {2022},
author = {Yin, T and Luo, J and Huang, D and Li, H},
title = {Current Progress of Mitochondrial Genome Editing by CRISPR.},
journal = {Frontiers in physiology},
volume = {13},
number = {},
pages = {883459},
doi = {10.3389/fphys.2022.883459},
pmid = {35586709},
issn = {1664-042X},
}
@article {pmid35581343,
year = {2022},
author = {Perdigoto, CN},
title = {TALEDs complete the toolkit for editing human mitochondrial DNA.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {5},
pages = {415},
doi = {10.1038/s41594-022-00781-z},
pmid = {35581343},
issn = {1545-9985},
mesh = {CRISPR-Cas Systems/genetics ; *DNA, Mitochondrial/genetics ; *Gene Editing ; Humans ; Mitochondria/genetics ; },
}
@article {pmid35573771,
year = {2022},
author = {Wu, X and Wang, S and Li, C and Shi, J and Peng, Z and Liu, C and Han, H and Ma, Y and Zheng, L and Xu, S and Du, W and Li, J and Zhang, F},
title = {CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {865744},
pmid = {35573771},
issn = {2235-2988},
mesh = {Animals ; Autophagy ; *CRISPR-Cas Systems ; DNA Viruses ; Female ; *Picornaviridae/genetics ; RNA Interference ; Swine ; Virus Replication ; },
abstract = {RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-DicerΔ- and BHK-Ago2Δ-. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.},
}
@article {pmid35559673,
year = {2022},
author = {Feng, X and Tang, M and Dede, M and Su, D and Pei, G and Jiang, D and Wang, C and Chen, Z and Li, M and Nie, L and Xiong, Y and Li, S and Park, JM and Zhang, H and Huang, M and Szymonowicz, K and Zhao, Z and Hart, T and Chen, J},
title = {Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.},
journal = {Science advances},
volume = {8},
number = {19},
pages = {eabm6638},
doi = {10.1126/sciadv.abm6638},
pmid = {35559673},
issn = {2375-2548},
mesh = {*Antineoplastic Agents ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Tumor Suppressor ; Humans ; *Neoplasms/genetics ; Synthetic Lethal Mutations ; },
abstract = {Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.},
}
@article {pmid35534557,
year = {2022},
author = {Eisenstein, M},
title = {Base editing marches on the clinic.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {623-625},
doi = {10.1038/s41587-022-01326-x},
pmid = {35534557},
issn = {1546-1696},
mesh = {Ambulatory Care Facilities ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid35436106,
year = {2022},
author = {Park, H and Osman, EA and Cromwell, CR and St Laurent, CD and Liu, Y and Kitova, EN and Klassen, JS and Hubbard, BP and Macauley, MS and Gibbs, JM},
title = {CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.},
journal = {Bioconjugate chemistry},
volume = {33},
number = {5},
pages = {858-868},
doi = {10.1021/acs.bioconjchem.2c00106},
pmid = {35436106},
issn = {1520-4812},
mesh = {Alkynes ; Azides/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide/genetics/metabolism ; },
abstract = {Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.},
}
@article {pmid35427721,
year = {2022},
author = {Avci-Adali, M and A Santos, H},
title = {Current trends in delivery of non-viral nucleic acid-based therapeutics for improved efficacy.},
journal = {Advanced drug delivery reviews},
volume = {185},
number = {},
pages = {114297},
doi = {10.1016/j.addr.2022.114297},
pmid = {35427721},
issn = {1872-8294},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Nanomedicine ; *Nucleic Acids ; },
}
@article {pmid35422459,
year = {2022},
author = {},
title = {Forum: CRISPR screening roundtable with Stegmaier and Doench.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {655},
doi = {10.1038/s41587-022-01303-4},
pmid = {35422459},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Mass Screening ; Research ; },
}
@article {pmid35350865,
year = {2022},
author = {Wasala, NB and Million, ED and Watkins, TB and Wasala, LP and Han, J and Yue, Y and Lu, B and Chen, SJ and Hakim, CH and Duan, D},
title = {The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.},
journal = {Human gene therapy},
volume = {33},
number = {9-10},
pages = {518-528},
doi = {10.1089/hum.2021.130},
pmid = {35350865},
issn = {1557-7422},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics/metabolism ; *Dystrophin/genetics/metabolism ; Gene Editing/methods ; Genetic Therapy/methods ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; RNA, Guide/genetics/metabolism ; },
abstract = {Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.},
}
@article {pmid34989166,
year = {2022},
author = {Zhang, C and Ren, H and Liu, G and Li, J and Wang, X and Zhang, Y},
title = {Effective Genome Editing Using CRISPR-Cas9 Nanoflowers.},
journal = {Advanced healthcare materials},
volume = {11},
number = {10},
pages = {e2102365},
doi = {10.1002/adhm.202102365},
pmid = {34989166},
issn = {2192-2659},
support = {2021YFC2102300//National Key Research and Development Program/ ; 32071384//National Natural Science Foundation of China/ ; //One-thousand Young Talent Program of China/ ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Micelles ; *Nanoparticles ; Polymers/metabolism ; },
abstract = {CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.},
}
@article {pmid34887556,
year = {2022},
author = {Anzalone, AV and Gao, XD and Podracky, CJ and Nelson, AT and Koblan, LW and Raguram, A and Levy, JM and Mercer, JAM and Liu, DR},
title = {Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {731-740},
pmid = {34887556},
issn = {1546-1696},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; RM1 HG00949//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Chromosome Inversion ; DNA/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.},
}
@article {pmid34719304,
year = {2022},
author = {Zocca, VFB and Corrêa, GG and Lins, MRDCR and de Jesus, VN and Tavares, LF and Amorim, LADS and Kundlatsch, GE and Pedrolli, DB},
title = {The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {6},
pages = {813-826},
doi = {10.1080/07388551.2021.1983516},
pmid = {34719304},
issn = {1549-7801},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {CRISPR has revolutionized the way we engineer genomes. Its simplicity and modularity have enabled the development of a great number of tools to edit genomes and to control gene expression. This powerful technology was first adapted to Bacillus subtilis in 2016 and has been intensely upgraded since then. Many tools have been successfully developed to build a CRISPR toolbox for this Gram-positive model and important industrial chassis. The toolbox includes tools, such as double-strand and single-strand cutting CRISPR for point mutation, gene insertion, and gene deletion up to 38 kb. Moreover, catalytic dead Cas proteins have been used for base editing, as well as for the control of gene expression (CRISPRi and CRISPRa). Many of these tools have been used for multiplex CRISPR with the most successful one targeting up to six loci simultaneously for point mutation. However, tools for efficient multiplex CRISPR for other functionalities are still missing in the toolbox. CRISPR engineering has already resulted in efficient protein and metabolite-producing strains, demonstrating its great potential. In this review, we cover all the important additions made to the B. subtilis CRISPR toolbox since 2016, and strain developments fomented by the technology.},
}
@article {pmid34349239,
year = {2022},
author = {Gao, C and Wu, P and Yu, L and Liu, L and Liu, H and Tan, X and Wang, L and Huang, X and Wang, H},
title = {The application of CRISPR/Cas9 system in cervical carcinogenesis.},
journal = {Cancer gene therapy},
volume = {29},
number = {5},
pages = {466-474},
pmid = {34349239},
issn = {1476-5500},
support = {81830074//National Natural Science Foundation of China (National Science Foundation of China)/ ; 81902667//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Female ; Human papillomavirus 16/genetics/metabolism ; Humans ; Mice ; Mice, Transgenic ; *Oncogene Proteins, Viral/genetics ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections/complications/genetics/therapy ; *Precancerous Conditions/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; *Uterine Cervical Neoplasms/genetics/pathology/therapy ; },
abstract = {Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.},
}
@article {pmid33864024,
year = {2022},
author = {Marayati, R and Stafman, LL and Williams, AP and Bownes, LV and Quinn, CH and Markert, HR and Easlick, JL and Stewart, JE and Crossman, DK and Mroczek-Musulman, E and Beierle, EA},
title = {CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.},
journal = {Cancer gene therapy},
volume = {29},
number = {5},
pages = {558-572},
pmid = {33864024},
issn = {1476-5500},
support = {T32 CA183926/CA/NCI NIH HHS/United States ; P30 CA013148/CA/NCI NIH HHS/United States ; CA013148//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 AR048311/AR/NIAMS NIH HHS/United States ; T32 CA091078/CA/NCI NIH HHS/United States ; 5T32GM008361//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32 GM008361/GM/NIGMS NIH HHS/United States ; P30 AI027767/AI/NIAID NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; T32 CA091078/CA/NCI NIH HHS/United States ; T32 CA183926/CA/NCI NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Transformation, Neoplastic/genetics ; Child ; *Hepatoblastoma/genetics/metabolism/pathology ; Humans ; *Liver Neoplasms/metabolism ; Mice ; Protein Serine-Threonine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; },
abstract = {Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.},
}
@article {pmid35584409,
year = {2022},
author = {Lee, HJ and Kim, HJ and Park, YJ and Lee, SJ},
title = {Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00054},
pmid = {35584409},
issn = {2161-5063},
abstract = {Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3'-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3'-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3'-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3'-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.},
}
@article {pmid35583743,
year = {2022},
author = {Vento, JM and Beisel, CL},
title = {Genome Editing with Cas9 in Lactobacilli.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {245-261},
pmid = {35583743},
issn = {1940-6029},
abstract = {The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.},
}
@article {pmid35583742,
year = {2022},
author = {Xu, T and Tao, X and Kempher, ML and Zhou, J},
title = {Cas9 Nickase-Based Genome Editing in Clostridium cellulolyticum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {227-243},
pmid = {35583742},
issn = {1940-6029},
abstract = {Clostridium cellulolyticum is a model mesophilic, cellulolytic bacterium, with the potential to produce biofuels from lignocellulose. However, the natural cellulose utilization efficiency is quite low and, therefore, metabolically engineered strains with increased efficiency can decrease both the overall cost and time required for biofuel production. Traditional genetic tools are inefficient, expensive, and time-consuming, but recent developments in the use of CRISPR-Cas genetic editing systems have greatly expanded our ability to reprogram cells. Here we describe an established protocol enabling one-step versatile genome editing in C. cellulolyticum. It integrates Cas9 nickase (Cas9n) which introduces a single nick that triggers repair via homologous recombination (SNHR) to edit genomic loci with high efficiency and accuracy. This one-step editing is achieved by transforming an all-in-one vector to coexpress Cas9n and a single guide RNA (gRNA) and carries a user-defined homologous donor template to promote SNHR at a desired target site. Additionally, this system has high specificity and allows for various types of genomic editing, including markerless insertions, deletions, substitutions, and even multiplex editing.},
}
@article {pmid35583741,
year = {2022},
author = {Tan, LL and Heng, E and Zulkarnain, N and Hsiao, WC and Wong, FT and Zhang, MM},
title = {CRISPR/Cas-Mediated Genome Editing of Streptomyces.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {207-225},
pmid = {35583741},
issn = {1940-6029},
abstract = {Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.},
}
@article {pmid35583202,
year = {2022},
author = {Luo, G and Najafi, J and Correia, PMP and Trinh, MDL and Chapman, EA and Østerberg, JT and Thomsen, HC and Pedas, PR and Larson, S and Gao, C and Poland, J and Knudsen, S and DeHaan, L and Palmgren, M},
title = {Accelerated Domestication of New Crops: Yield is Key.},
journal = {Plant &amp; cell physiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/pcp/pcac065},
pmid = {35583202},
issn = {1471-9053},
support = {DEEPROOTS//Innovationsfonden/ ; LESSISMORE//Innovationsfonden/ ; CF18-1113//Carlsbergfondet/ ; 2019OC53580//Novo Nordisk Fonden/ ; },
abstract = {Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients, and can be cultivated with a minimal carbon footprint. Wild plants that fulfil these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity, and increases our carbon footprint. Sustainable intensification can be achieved by increasing yield in underutilized or wild plant species that are already resilient but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yield. With new precise molecular techniques it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.},
}
@article {pmid35581233,
year = {2022},
author = {Sansbury, BM and Hewes, AM and Tharp, OM and Masciarelli, SB and Kaouser, S and Kmiec, EB},
title = {Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {8132},
pmid = {35581233},
issn = {2045-2322},
abstract = {Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.},
}
@article {pmid35579071,
year = {2022},
author = {Zhang, L and Zhao, X and Hu, X and Zhang, Y and Liu, R and Peng, H and Chen, Y and Zhang, H and Luo, Y},
title = {Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2an00170e},
pmid = {35579071},
issn = {1364-5528},
abstract = {Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation from clinical samples remains a challenge. Herein, we propose an easy-to-operate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system Assisted Methylation (CAM) approach for the sensitive detection of DNA methylation through the integration of rolling circle amplification and CRISPR-Cas12a-assisted cascade amplification. Briefly, bisulfite was employed to prepare the clinical samples so that the methylated DNA sequences trigger the subsequent triple signal amplifications, whilst the normal counterparts do not. The triple signal amplification procedure consists of methylated DNA sequence-based rolling circle amplification for a preliminary signal enhancement, a nicking enzyme-initiated target cleavage for a secondary amplification, and CRISPR-Cas12a enzyme-mediated trans-cleavage for a tertiary signal enhancement. This proposed approach reveals high sensitivity, which can even distinguish as low as 0.01% methylation levels from mixtures, paving the way towards the acceleration of methylation-based cancer diagnostics and management.},
}
@article {pmid35577099,
year = {2022},
author = {Panda, G and Ray, A},
title = {Decrypting the mechanistic basis of CRISPR/Cas9 protein.},
journal = {Progress in biophysics and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.pbiomolbio.2022.05.001},
pmid = {35577099},
issn = {1873-1732},
abstract = {CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.},
}
@article {pmid35573787,
year = {2022},
author = {Shankar, C and Vasudevan, K and Jacob, JJ and Baker, S and Isaac, BJ and Neeravi, AR and Sethuvel, DPM and George, B and Veeraraghavan, B},
title = {Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {875116},
doi = {10.3389/fcimb.2022.875116},
pmid = {35573787},
issn = {2235-2988},
mesh = {Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Humans ; *Klebsiella Infections ; *Klebsiella pneumoniae ; Plasmids/genetics ; Virulence/genetics ; beta-Lactamases/genetics ; },
abstract = {Background: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.<br><br>Methods: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.<br><br>Results: Among the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, bla OXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.<br><br>Conclusion: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.},
}
@article {pmid35573047,
year = {2022},
author = {Kath, J and Du, W and Pruene, A and Braun, T and Thommandru, B and Turk, R and Sturgeon, ML and Kurgan, GL and Amini, L and Stein, M and Zittel, T and Martini, S and Ostendorf, L and Wilhelm, A and Akyüz, L and Rehm, A and Höpken, UE and Pruß, A and Künkele, A and Jacobi, AM and Volk, HD and Schmueck-Henneresse, M and Stripecke, R and Reinke, P and Wagner, DL},
title = {Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {25},
number = {},
pages = {311-330},
doi = {10.1016/j.omtm.2022.03.018},
pmid = {35573047},
issn = {2329-0501},
abstract = {Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.},
}
@article {pmid35572739,
year = {2022},
author = {Abdullah, M and Okemo, P and Furtado, A and Henry, R},
title = {Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {875243},
doi = {10.3389/fgeed.2022.875243},
pmid = {35572739},
issn = {2673-3439},
abstract = {Rice, a staple food worldwide and a model crop, could benefit from the introduction of novel genetics from wild relatives. Wild rice in the AA genome group closely related to domesticated rice is found across the tropical world. Due to their locality outside the range of domesticated rice, Australian wild rice populations are a potential source of unique traits for rice breeding. These rice species provide a diverse gene pool for improvement that could be utilized for desirable traits such as stress resistance, disease tolerance, and nutritional qualities. However, they remain poorly characterized. The CRISPR/Cas system has revolutionized gene editing and has improved our understanding of gene functions. Coupled with the increasing availability of genomic information on the species, genes in Australian wild rice could be modified through genome editing technologies to produce new domesticates. Alternatively, beneficial alleles from these rice species could be incorporated into cultivated rice to improve critical traits. Here, we summarize the beneficial traits in Australian wild rice, the available genomic information and the potential of gene editing to discover and understand the functions of novel alleles. Moreover, we discuss the potential domestication of these wild rice species for health and economic benefits to rice production globally.},
}
@article {pmid35572197,
year = {2022},
author = {Nieland, L and van Solinge, TS and Cheah, PS and Morsett, LM and El Khoury, J and Rissman, JI and Kleinstiver, BP and Broekman, MLD and Breakefield, XO and Abels, ER},
title = {CRISPR-Cas knockout of miR21 reduces glioma growth.},
journal = {Molecular therapy oncolytics},
volume = {25},
number = {},
pages = {121-136},
doi = {10.1016/j.omto.2022.04.001},
pmid = {35572197},
issn = {2372-7705},
abstract = {Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.},
}
@article {pmid35572153,
year = {2022},
author = {Christian, A},
title = {Addressing Conflicts of Interest and Conflicts of Commitment in Public Advocacy and Policy Making on CRISPR/Cas-Based Human Genome Editing.},
journal = {Frontiers in research metrics and analytics},
volume = {7},
number = {},
pages = {775336},
doi = {10.3389/frma.2022.775336},
pmid = {35572153},
issn = {2504-0537},
abstract = {Leading experts on CRISPR/Cas-based genome editing-such as 2020 Nobel laureates Jennifer Doudna and Emmanuelle Charpentier-are not only renowned specialists in their fields, but also public advocates for upcoming regulatory frameworks on CRISPR/Cas. These frameworks will affect large portions of biomedical research on human genome editing. In advocating for particular ways of handling the risks and prospects of this technology, high-profile scientists not only serve as scientific experts, but also as moral advisers. The majority of them currently intend to bring about a "responsible pathway" toward human genome interventions in clinical therapy. Engaging in advocacy for such a pathway, they issue moral judgments on the risks and benefits of this new technology. They declare that there actually is a responsible pathway, they draft resolutions on temporary moratoria, they make judgments on which groups and individuals are credible and should participate in public and semi-public debates, so they also set the standards for deciding who counts as well-informed, as well as the standards of evidence for adopting or rejecting research policies. This degree of influence on public debates and policy making is, at the very least, noteworthy. This contribution sounds a note of caution with regard to the endeavor of a responsible pathway to human genome editing and in particular scrutinizes the legitimacy of expert-driven research policies given commercial conflicts of interest and conflicts of commitment among first-rank scholars.},
}
@article {pmid35569864,
year = {2022},
author = {Islam, MM and Koirala, D},
title = {Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses.},
journal = {Analytica chimica acta},
volume = {1209},
number = {},
pages = {339338},
doi = {10.1016/j.aca.2021.339338},
pmid = {35569864},
issn = {1873-4324},
abstract = {As the COVID-19 pandemic continues to affect human health across the globe rapid, simple, point-of-care (POC) diagnosis of infectious viruses such as SARS-CoV-2 remains challenging. Polymerase chain reaction (PCR)-based diagnosis has risen to meet these demands and despite its high-throughput and accuracy, it has failed to gain traction in the rapid, low-cost, point-of-test settings. In contrast, different emerging isothermal amplification-based detection methods show promise in the rapid point-of-test market. In this comprehensive study of the literature, several promising isothermal amplification methods for the detection of SARS-CoV-2 are critically reviewed that can also be applied to other infectious viruses detection. Starting with a brief discussion on the SARS-CoV-2 structure, its genomic features, and the epidemiology of the current pandemic, this review focuses on different emerging isothermal methods and their advancement. The potential of isothermal amplification combined with the revolutionary CRISPR/Cas system for a more powerful detection tool is also critically reviewed. Additionally, the commercial success of several isothermal methods in the pandemic are highlighted. Different variants of SARS-CoV-2 and their implication on isothermal amplifications are also discussed. Furthermore, three most crucial aspects in achieving a simple, fast, and multiplexable platform are addressed.},
}
@article {pmid35568950,
year = {2022},
author = {Cai, P and Han, M and Zhang, R and Ding, S and Zhang, D and Liu, D and Liu, S and Hu, QN},
title = {SynBioStrainFinder: A microbial strain database of manually curated CRISPR/Cas genetic manipulation system information for biomanufacturing.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {87},
pmid = {35568950},
issn = {1475-2859},
support = {2019YFA0904300//National Key Research and Development Program of China/ ; 31700081//National Natural Science Foundation of China/ ; 31570092//National Natural Science Foundation of China/ ; QYZDB-SSW-SMC012//CAS STS program/ ; 153D31KYSB20170121//International Partnership Program of Chinese Academy of Sciences of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {BACKGROUND: Microbial strain information databases provide valuable data for microbial basic research and applications. However, they rarely contain information on the genetic operating system of microbial strains.<br><br>RESULTS: We established a comprehensive microbial strain database, SynBioStrainFinder, by integrating CRISPR/Cas gene-editing system information with cultivation methods, genome sequence data, and compound-related information. It is presented through three modules, Strain2Gms/PredStrain2Gms, Strain2BasicInfo, and Strain2Compd, which combine to form a rapid strain information query system conveniently curated, integrated, and accessible on a single platform. To date, 1426 CRISPR/Cas gene-editing records of 157 microbial strains have been manually extracted from the literature in the Strain2Gms module. For strains without established CRISPR/Cas systems, the PredStrain2Gms module recommends the system of the most closely related strain as a reference to facilitate the construction of a new CRISPR/Cas gene-editing system. The database contains 139,499 records of strain cultivation and genome sequences, and 773,298 records of strain-related compounds. To facilitate simple and intuitive data application, all microbial strains are also labeled with stars based on the order and availability of strain information. SynBioStrainFinder provides a user-friendly interface for querying, browsing, and visualizing detailed information on microbial strains, and it is publicly available at http://design.rxnfinder.org/biosynstrain/ .<br><br>CONCLUSION: SynBioStrainFinder is the first microbial strain database with manually curated information on the strain CRISPR/Cas system as well as other microbial strain information. It also provides reference information for the construction of new CRISPR/Cas systems. SynBioStrainFinder will serve as a useful resource to extend microbial strain research and application for biomanufacturing.},
}
@article {pmid35568789,
year = {2022},
author = {Chaudhary, M and Mukherjee, TK and Singh, R and Gupta, M and Goyal, S and Singhal, P and Kumar, R and Bhusal, N and Sharma, P},
title = {CRISPR/Cas technology for improving nutritional values in the agricultural sector: an update.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {35568789},
issn = {1573-4978},
abstract = {BACKGROUND: The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system was initially identified in bacteria and archaea as a defense mechanism to confer immunity against phages. Later on, it was developed as a gene editing tool for both prokaryotic and eukaryotic cells including plant cells.<br><br>METHODS AND RESULTS: CRISPR/Cas9 approach has wider applications in reverse genetics as well as in crop improvement. Various characters involved in enhancing economic value and crop sustainability against biotic/abiotic stresses can be targeted through this tool. Currently, CRISPR/Cas9 gene editing mechanism has been applied on around 20 crop species for improvement in several traits including yield enhancement and resistance against biotic and abiotic stresses. In the last five years, maximum genome editing research has been validated in rice, wheat, maize and soybean. Genes targeted in these plants has been involved in causing male sterility, conferring resistance against pathogens or having certain nutritional value.<br><br>CONCLUSIONS: Current review summarizes various applications of CRISPR/Cas system and its future prospects in plant biotechnology targeting crop improvement with higher yield, disease tolerance and enhanced nutritional value.},
}
@article {pmid35565854,
year = {2022},
author = {Quéré, M and Alberto, JM and Broly, F and Hergalant, S and Christov, C and Gauchotte, G and Guéant, JL and Namour, F and Battaglia-Hsu, SF},
title = {ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.},
journal = {Nutrients},
volume = {14},
number = {9},
pages = {},
pmid = {35565854},
issn = {2072-6643},
support = {NEGERE 2019//La Ligue Contre Cancer/ ; BMS Incitatif 2020//Université de Lorraine/ ; },
mesh = {Cell Line, Tumor ; *Glioblastoma/metabolism ; Humans ; Methionine/metabolism ; Neoplastic Stem Cells/metabolism ; Oxidative Stress ; Reactive Oxygen Species/metabolism ; },
abstract = {Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.},
}
@article {pmid35563876,
year = {2022},
author = {Gómez-García, F and Martínez-Pulleiro, R and Carrera, N and Allegue, C and Garcia-Gonzalez, MA},
title = {Genetic Kidney Diseases (GKDs) Modeling Using Genome Editing Technologies.},
journal = {Cells},
volume = {11},
number = {9},
pages = {},
pmid = {35563876},
issn = {2073-4409},
support = {PI18/00378//Instituto de Salud Carlos III/ ; IN607B-2016/020//Axencia Galega de Innovacion/ ; ED431G 2019/02//Xunta de Galicia/ ; RD21/0005/0020//Redes de Investigación Cooperativa Orientadas a Resultados en Salud/ ; RD16/0009/0024//Red de Investigación Renal/ ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Female ; *Gene Editing/methods ; Humans ; *Kidney Diseases/genetics/therapy ; Male ; Zinc Finger Nucleases ; },
abstract = {Genetic kidney diseases (GKDs) are a group of rare diseases, affecting approximately about 60 to 80 per 100,000 individuals, for which there is currently no treatment that can cure them (in many cases). GKDs usually leads to early-onset chronic kidney disease, which results in patients having to undergo dialysis or kidney transplant. Here, we briefly describe genetic causes and phenotypic effects of six GKDs representative of different ranges of prevalence and renal involvement (ciliopathy, glomerulopathy, and tubulopathy). One of the shared characteristics of GKDs is that most of them are monogenic. This characteristic makes it possible to use site-specific nuclease systems to edit the genes that cause GKDs and generate in vitro and in vivo models that reflect the genetic abnormalities of GKDs. We describe and compare these site-specific nuclease systems (zinc finger nucleases (ZFNs), transcription activator-like effect nucleases (TALENs) and regularly clustered short palindromic repeat-associated protein (CRISPR-Cas9)) and review how these systems have allowed the generation of cellular and animal GKDs models and how they have contributed to shed light on many still unknown fields in GKDs. We also indicate the main obstacles limiting the application of these systems in a more efficient way. The information provided here will be useful to gain an accurate understanding of the technological advances in the field of genome editing for GKDs, as well as to serve as a guide for the selection of both the genome editing tool and the gene delivery method most suitable for the successful development of GKDs models.},
}
@article {pmid35563479,
year = {2022},
author = {Feser, CJ and Lees, CJ and Lammers, DT and Riddle, MJ and Bingham, JR and Eckert, MJ and Tolar, J and Osborn, MJ},
title = {Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563479},
issn = {1422-0067},
support = {W81XWH-20-C-0052//US Special Operations Command/ ; },
mesh = {*Blood Coagulation Factors/genetics ; *CRISPR-Cas Systems ; Fibrinogen/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Transcriptional Activation ; },
abstract = {Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120-4700-fold and 60-680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700-92,000-fold increases and 80-5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.},
}
@article {pmid35563453,
year = {2022},
author = {Shin, NR and Shin, YH and Kim, HS and Park, YD},
title = {Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563453},
issn = {1422-0067},
support = {NRF-2021M3E5E6025387//National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT/ ; },
mesh = {*Brassica/genetics ; *CRISPR-Cas Systems ; China ; Gene Editing ; Mutagenesis ; Plant Breeding ; },
abstract = {Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.},
}
@article {pmid35563435,
year = {2022},
author = {Fischer, B and Schmidt, V and Ly, TD and Kleine, A and Knabbe, C and Faust-Hinse, I},
title = {First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563435},
issn = {1422-0067},
support = {FA 1381/1-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; *Fibroblasts/metabolism ; Gene Editing ; Humans ; Infant, Newborn ; Pentosyltransferases/genetics/metabolism ; Skin/metabolism ; },
abstract = {BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue.<br><br>METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis.<br><br>RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization.<br><br>CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.},
}
@article {pmid35563412,
year = {2022},
author = {Yao, M and Ren, T and Pan, Y and Xue, X and Li, R and Zhang, L and Li, Y and Huang, K},
title = {A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563412},
issn = {1422-0067},
support = {32070654//National Natural Science Foundation of China/ ; 201901D211193//Shanxi Province Science Foundation for Outstanding Youths, China/ ; 32071454//National Natural Science Foundation of China/ ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage/genetics ; Gene Editing ; *Single-Cell Analysis ; *Transcriptome ; },
abstract = {Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts.},
}
@article {pmid35563297,
year = {2022},
author = {Moniruzzaman, M and Zhong, Y and Huang, Z and Zhong, G},
title = {Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563297},
issn = {1422-0067},
support = {2019B030316005//Guangdong Provincial Science and Technology Program/ ; 2018B020202009//Guangdong Provincial Science and Technology Program/ ; 32072535//National Natural Science Foundation of China/ ; 32002016//National Natural Science Foundation of China/ ; BZ201902//Dean's Foundation of Guangdong Academy of Agricultural Sciences/ ; 2020KJ108//Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Special Teams/ ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Gene Editing ; Mutagenesis ; *RNA, Guide/genetics ; },
abstract = {Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.},
}
@article {pmid35563243,
year = {2022},
author = {Tian, J and Xing, B and Li, M and Xu, C and Huo, YX and Guo, S},
title = {Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563243},
issn = {1422-0067},
support = {2019YFA0904104//National Key R&amp;D Program of China/ ; 2021YFC2100500//National Key R&amp;D Program of China/ ; },
mesh = {*Bacillus subtilis/genetics ; Biofuels ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial ; Metabolic Engineering ; },
abstract = {Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.},
}
@article {pmid35563030,
year = {2022},
author = {Toinga-Villafuerte, S and Vales, MI and Awika, JM and Rathore, KS},
title = {CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563030},
issn = {1422-0067},
mesh = {Amylopectin/metabolism ; Amylose/metabolism ; CRISPR-Cas Systems/genetics ; Gold/metabolism ; Mutagenesis ; *Solanum tuberosum/genetics/metabolism ; Starch/metabolism ; *Starch Synthase/genetics ; Yukon Territory ; },
abstract = {Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.},
}
@article {pmid35562887,
year = {2022},
author = {Wang, YY and Hsu, SH and Tsai, HY and Cheng, FY and Cheng, MC},
title = {Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35562887},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carrier Proteins ; Chromatography, Liquid ; HEK293 Cells ; Humans ; Microfilament Proteins ; Mitochondrial Proteins ; *Proteomics ; Tandem Mass Spectrometry ; *Transcriptome ; },
abstract = {Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.},
}
@article {pmid35562427,
year = {2022},
author = {Wang, JY and Pausch, P and Doudna, JA},
title = {Structural biology of CRISPR-Cas immunity and genome editing enzymes.},
journal = {Nature reviews. Microbiology},
volume = {},
number = {},
pages = {},
pmid = {35562427},
issn = {1740-1534},
abstract = {CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.},
}
@article {pmid35560111,
year = {2022},
author = {Worthington, AK and Forsberg, EC},
title = {A CRISPR View of Hematopoietic Stem Cells: Moving Innovative Bioengineering into the Clinic.},
journal = {American journal of hematology},
volume = {},
number = {},
pages = {},
doi = {10.1002/ajh.26588},
pmid = {35560111},
issn = {1096-8652},
abstract = {CRISPR/Cas genome engineering has emerged as a powerful tool to modify precise genomic sequences with unparalleled accuracy and efficiency. Major advances in CRISPR technologies over the last five years have fueled the development of novel techniques in hematopoiesis research to interrogate the complexities of hematopoietic stem cell (HSC) biology. In particular, high throughput CRISPR based screens using various "flavors" of Cas coupled with sequencing and/or functional outputs are becoming increasingly efficient and accessible. In this review, we discuss recent achievements in CRISPR-mediated genomic engineering and how these new tools have advanced the understanding of HSC heterogeneity and function throughout life. Additionally, we highlight how these techniques can be used to answer previously inaccessible questions and the challenges to implement them. Finally, we focus on their translational potential to both model and treat hematological diseases in the clinic. This article is protected by copyright. All rights reserved.},
}
@article {pmid35559022,
year = {2022},
author = {Mandal, S and Ghorai, M and Anand, U and Roy, D and Kant, N and Mishra, T and Mane, AB and Jha, NK and Lal, MK and Tiwari, RK and Kumar, M and Radha, and Ghosh, A and Bhattacharjee, R and Proćków, J and Dey, A},
title = {Cytokinins: A Genetic Target for Increasing Yield Potential in the CRISPR Era.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {883930},
pmid = {35559022},
issn = {1664-8021},
abstract = {Over the last decade, remarkable progress has been made in our understanding the phytohormones, cytokinin's (CKs) biosynthesis, perception, and signalling pathways. Additionally, it became apparent that interfering with any of these steps has a significant effect on all stages of plant growth and development. As a result of their complex regulatory and cross-talk interactions with other hormones and signalling networks, they influence and control a wide range of biological activities, from cellular to organismal levels. In agriculture, CKs are extensively used for yield improvement and management because of their wide-ranging effects on plant growth, development and physiology. One of the primary targets in this regard is cytokinin oxidase/dehydrogenase (CKO/CKX), which is encoded by CKX gene, which catalyses the irreversible degradation of cytokinin. The previous studies on various agronomically important crops indicated that plant breeders have targeted CKX directly. In recent years, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been increasingly used in editing the CKO/CKX gene and phenomenal results have been achieved. This review provides an updated information on the applications of CRISPR-based gene-editing tools in manipulating cytokinin metabolism at the genetic level for yield improvement. Furthermore, we summarized the current developments of RNP-mediated DNA/transgene-free genomic editing of plants which would broaden the application of this technology. The current review will advance our understanding of cytokinins and their role in sustainably increase crop production through CRISPR/Cas genome editing tool.},
}
@article {pmid35558825,
year = {2022},
author = {Rahman, F and Mishra, A and Gupta, A and Sharma, R},
title = {Spatiotemporal Regulation of CRISPR/Cas9 Enables Efficient, Precise, and Heritable Edits in Plant Genomes.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {870108},
pmid = {35558825},
issn = {2673-3439},
abstract = {CRISPR/Cas-mediated editing has revolutionized crop engineering. Due to the broad scope and potential of this technology, many studies have been carried out in the past decade towards optimizing genome editing constructs. Clearly, the choice of the promoter used to drive gRNA and Cas9 expression is critical to achieving high editing efficiency, precision, and heritability. While some important considerations for choosing a promoter include the number and nature of targets, host organism, mode of transformation and goal of the experiment, spatiotemporal regulation of Cas9 expression using tissue-specific or inducible promoters enables higher heritability and efficiency of targeted mutagenesis with reduced off-target effects. In this review, we discuss specific studies that highlight the prospects and trade-offs associated with the choice of promoters on genome editing and emphasize the need for inductive exploration and discovery to further advance this area of research in crop plants.},
}
@article {pmid35557039,
year = {2022},
author = {Ye, J and Xi, H and Chen, Y and Chen, Q and Lu, X and Lv, J and Chen, Y and Gu, F and Zhao, J},
title = {Can SpRY recognize any PAM in human cells?.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {5},
pages = {382-391},
doi = {10.1631/jzus.B2100710},
pmid = {35557039},
issn = {1862-1783},
support = {18331105//Lin HE's Academician Workstation of New Medicine and Clinical Translation/ ; H22010011//Program for Basic Science and Technology Cooperation Projects of Wenzhou City/ ; },
abstract = {The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.},
}
@article {pmid35552388,
year = {2022},
author = {Vicencio, J and Sánchez-Bolaños, C and Moreno-Sánchez, I and Brena, D and Vejnar, CE and Kukhtar, D and Ruiz-López, M and Cots-Ponjoan, M and Rubio, A and Melero, NR and Crespo-Cuadrado, J and Carolis, C and Pérez-Pulido, AJ and Giráldez, AJ and Kleinstiver, BP and Cerón, J and Moreno-Mateos, MA},
title = {Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2601},
pmid = {35552388},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics/metabolism ; *Gene Editing/methods ; RNA, Guide/genetics ; RNA, Messenger ; Zebrafish/genetics/metabolism ; },
abstract = {The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.},
}
@article {pmid35551240,
year = {2022},
author = {Zhang, D and Wang, G and Yu, X and Wei, T and Farbiak, L and Johnson, LT and Taylor, AM and Xu, J and Hong, Y and Zhu, H and Siegwart, DJ},
title = {Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy.},
journal = {Nature nanotechnology},
volume = {},
number = {},
pages = {},
pmid = {35551240},
issn = {1748-3395},
support = {RP160157//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; RP190251//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; Predoctoral Fellowship in Drug Delivery//Pharmaceutical Research and Manufacturers of America Foundation (PhRMA Foundation)/ ; R01 CA251928/CA/NCI NIH HHS/United States ; 5P30CA142543//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA269787-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; RSG-17-012-01//American Cancer Society (American Cancer Society, Inc.)/ ; I-1855//Welch Foundation/ ; SIEGWA18XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; },
abstract = {Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.},
}
@article {pmid35550915,
year = {2022},
author = {Li, Y and Mensah, EO and Fordjour, E and Bai, J and Yang, Y and Bai, Z},
title = {Recent advances in high-throughput metabolic engineering: Generation of oligonucleotide-mediated genetic libraries.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {107970},
doi = {10.1016/j.biotechadv.2022.107970},
pmid = {35550915},
issn = {1873-1899},
abstract = {The preparation of genetic libraries is an essential step to evolve microorganisms and study genotype-phenotype relationships by high-throughput screening/selection. As the large-scale synthesis of oligonucleotides becomes easy, cheap, and high-throughput, numerous novel strategies have been developed in recent years to construct high-quality oligo-mediated libraries, leveraging state-of-art molecular biology tools for genome editing and gene regulation. This review presents an overview of recent advances in creating and characterizing in vitro and in vivo genetic libraries, based on CRISPR/Cas, regulatory RNAs, and recombineering, primarily for Escherichia coli and Saccharomyces cerevisiae. These libraries' applications in high-throughput metabolic engineering, strain evolution and protein engineering are also discussed.},
}
@article {pmid35550024,
year = {2022},
author = {Mesa, V and Monot, M and Ferraris, L and Popoff, M and Mazuet, C and Barbut, F and Delannoy, J and Dupuy, B and Butel, MJ and Aires, J},
title = {Core-, pan- and accessory genome analyses of Clostridium neonatale: insights into genetic diversity.},
journal = {Microbial genomics},
volume = {8},
number = {5},
pages = {},
doi = {10.1099/mgen.0.000813},
pmid = {35550024},
issn = {2057-5858},
mesh = {*Clostridium/genetics ; Genetic Variation ; *Genome, Bacterial ; Humans ; Infant, Newborn ; Phylogeny ; },
abstract = {Clostridium neonatale is a potential opportunistic pathogen recovered from faecal samples in cases of necrotizing enterocolitis (NEC), a gastrointestinal disease affecting preterm neonates. Although the C. neonatale species description and name validation were published in 2018, comparative genomics are lacking. In the present study, we provide the closed genome assembly of the C. neonatale ATCC BAA-265T (=250.09) reference strain with a manually curated functional annotation of the coding sequences. Pan-, core- and accessory genome analyses were performed using the complete 250.09 genome (4.7 Mb), three new assemblies (4.6-5.6 Mb), and five publicly available draft genome assemblies (4.6-4.7 Mb). The C. neonatale pan-genome contains 6840 genes, while the core-genome has 3387 genes. Pan-genome analysis revealed an 'open' state and genomic diversity. The strain-specific gene families ranged from five to 742 genes. Multiple mobile genetic elements were predicted, including a total of 201 genomic islands, 13 insertion sequence families, one CRISPR-Cas type I-B system and 15 predicted intact prophage signatures. Primary virulence classes including offensive, defensive, regulation of virulence-associated genes and non-specific virulence factors were identified. The presence of a tet(W/N/W) gene encoding a tetracycline resistance ribosomal protection protein and a 23S rRNA methyltransferase ermQ gene were identified in two different strains. Together, our results revealed a genetic diversity and plasticity of C. neonatale genomes and provide a comprehensive view of this species genomic features, paving the way for the characterization of its biological capabilities.},
}
@article {pmid35549895,
year = {2022},
author = {Bernas, G and Ouellet, M and Barrios, A and Jamann, H and Larochelle, C and Lévy, É and Schmouth, JF},
title = {Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.},
journal = {BMC biotechnology},
volume = {22},
number = {1},
pages = {14},
pmid = {35549895},
issn = {1472-6750},
support = {EGID 3322//Multiple Sclerosis Society of Canada/ ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Electroporation/methods ; Embryo, Mammalian ; Exons ; Mammals/genetics ; Mice ; *Zygote ; },
abstract = {BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project.<br><br>RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%.<br><br>CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.},
}
@article {pmid35549133,
year = {2021},
author = {Kanduri, V and LaVigne, D and Larsen, J},
title = {Current Advances Toward the Encapsulation of Cas9.},
journal = {ACS macro letters},
volume = {10},
number = {12},
pages = {1576-1589},
doi = {10.1021/acsmacrolett.1c00538},
pmid = {35549133},
issn = {2161-1653},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Lipids ; Liposomes ; Nanoparticles ; Polymers/metabolism ; Quality of Life ; },
abstract = {Genetic diseases present formidable hurdles in maintaining a good quality of life for those suffering from these ailments. Often, patients look to inadequate treatments to manage symptoms, which can result in harmful effects on the body. Through genetic engineering, scientists utilize the clustered regularly short palindromic repeat (CRISPR)-associated protein, known as Cas9, to treat the root of the problem. The Cas9 protein is often codelivered with guide RNAs or in ribonucleoprotein complexes (RNP) to ensure targeted delivery of the genetic tool as well as to limit off-target effects. This paper provides an overview of the current advances made toward the encapsulation and delivery of Cas9 to desired locations in the body through encapsulating nanoparticles. Several factors must be considered when employing the Cas9 system to allow gene editing to occur. Material selection is crucial to protect the payload of the delivery vector. Current literature indicates that lipid- and polymer-based nanoparticles show the most potential as delivery vessels for Cas9. Lipid nanoparticles greatly outpace polymer-based nanoparticles in the clinic, despite the benefits that polymers may introduce. When developing translatable systems, there are factors that have not yet been considered that are relevant to Cas9 delivery that are highlighted in this Viewpoint. The proper functioning of Cas9 is dependent on maintaining a proper internal environment; however, there are gaps in the literature regarding these optimal conditions. Interactions between charges of the Cas9 protein, codelivered molecules, and delivery vehicles could impact the effectiveness of the gene editing taking place. While the internal charges of nanoparticles and their effects on Cas9 are presently undetermined, nanoparticles currently offer the ideal delivery method for the Cas9 protein due to their adequate size, modifiable external charge, and ability to be modified. Overall, a cationic lipid-/polymer-based nanoparticle system was found to have the most prospects in Cas9 delivery thus far. By understanding the successes of other systems, translatable, polymer-based delivery vehicles may be developed.},
}
@article {pmid35548699,
year = {2022},
author = {Sturme, MHJ and van der Berg, JP and Bouwman, LMS and De Schrijver, A and de Maagd, RA and Kleter, GA and Battaglia-de Wilde, E},
title = {Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants.},
journal = {ACS agricultural science &amp; technology},
volume = {2},
number = {2},
pages = {192-201},
pmid = {35548699},
issn = {2692-1952},
abstract = {CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants. Most observed off-target changes were small insertions or deletions (1-22 bp) or nucleotide substitutions, and large deletions (>100 bp) were rare. One study detected the insertion of vector-derived DNA sequences, which is important considering the risk assessment of gene-edited plants. Off-target sites had few mismatches (1-3 nt) with the target sequence and were mainly located in protein-coding regions, often in target gene homologues. Off-targets edits were predominantly detected via biased analysis of predicted off-target sites instead of unbiased genome-wide analysis. CRISPR-Cas-edited plants showed lower off-target mutation frequencies than conventionally bred plants. This Review can aid discussions on the relevance of evaluating off-target modifications for risk assessment of CRISPR-Cas-edited plants.},
}
@article {pmid35547744,
year = {2022},
author = {Chan, YT and Lu, Y and Wu, J and Zhang, C and Tan, HY and Bian, ZX and Wang, N and Feng, Y},
title = {CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives.},
journal = {Theranostics},
volume = {12},
number = {7},
pages = {3329-3344},
pmid = {35547744},
issn = {1838-7640},
mesh = {*Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; *Neoplasms/drug therapy/genetics ; },
abstract = {CRISPR-Cas9 is a Nobel Prize-winning robust gene-editing tool developed in the last decade. This technique enables a stable genetic engineering method with high precision on the genomes of all organisms. The latest advances in the technology include a genome library screening approach, which can detect survival-essential and drug resistance genes via gain or loss of function. The versatile machinery allows genomic screening for gene activation or inhibition, and targets non-coding sequences, such as promoters, miRNAs, and lncRNAs. In this review, we introduce the emerging high-throughput CRISPR-Cas9 library genome screening technology and its working principles to detect survival and drug resistance genes through positive and negative selection. The technology is compared with other existing approaches while focusing on the advantages of its variable applications in anti-cancer drug discovery, including functions and target identification, non-coding RNA information, actions of small molecules, and drug target discoveries. The combination of the CRISPR-Cas9 system with multi-omic platforms represents a dynamic field expected to advance anti-cancer drug discovery and precision medicine in the clinic.},
}
@article {pmid35544771,
year = {2022},
author = {Liu, W and An, C and Shu, X and Meng, X and Yao, Y and Zhang, J and Chen, F and Xiang, H and Yang, S and Gao, X and Gao, SS},
title = {Correction to "A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi".},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00228},
pmid = {35544771},
issn = {2161-5063},
}
@article {pmid35543560,
year = {2022},
author = {Li, Y and Zhang, L and Yang, H and Xia, Y and Liu, L and Chen, X and Shen, W},
title = {Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0005922},
doi = {10.1128/spectrum.00059-22},
pmid = {35543560},
issn = {2165-0497},
abstract = {Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.},
}
@article {pmid35538629,
year = {2022},
author = {Bellingrath, JS and McClements, ME and Shanks, M and Clouston, P and Fischer, MD and MacLaren, RE},
title = {Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.},
journal = {Ophthalmic genetics},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/13816810.2022.2073599},
pmid = {35538629},
issn = {1744-5094},
abstract = {BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms.<br><br>PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing.<br><br>METHODS: Case report.<br><br>RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window.<br><br>CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.},
}
@article {pmid35538076,
year = {2022},
author = {Dong, C and Fu, S and Karvas, RM and Chew, B and Fischer, LA and Xing, X and Harrison, JK and Popli, P and Kommagani, R and Wang, T and Zhang, B and Theunissen, TW},
title = {A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2548},
pmid = {35538076},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; DNA-Binding Proteins/genetics/metabolism ; Female ; Humans ; *Placenta/metabolism ; Pregnancy ; Protein Tyrosine Phosphatases, Non-Receptor/genetics ; Stem Cells/metabolism ; Transcription Factors/genetics/metabolism ; *Trophoblasts/metabolism ; },
abstract = {The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.},
}
@article {pmid35536747,
year = {2022},
author = {Gotoh, Y and Atsuta, Y and Taniguchi, T and Nishida, R and Nakamura, K and Ogura, Y and Misawa, N and Hayashi, T},
title = {Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex.},
journal = {Microbial genomics},
volume = {8},
number = {5},
pages = {},
doi = {10.1099/mgen.0.000830},
pmid = {35536747},
issn = {2057-5858},
mesh = {Animals ; *Bacteremia ; Cricetinae ; Dogs ; *Helicobacter/genetics ; *Helicobacter Infections ; Humans ; Rats ; },
abstract = {Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.},
}
@article {pmid35534475,
year = {2022},
author = {Bishop, AL and López Del Amo, V and Okamoto, EM and Bodai, Z and Komor, AC and Gantz, VM},
title = {Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2595},
pmid = {35534475},
issn = {2041-1723},
support = {MCB-2048207//National Science Foundation (NSF)/ ; DP5OD023098//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; R01AI162911//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Germ Cells ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; },
abstract = {Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.},
}
@article {pmid35534455,
year = {2022},
author = {Bodai, Z and Bishop, AL and Gantz, VM and Komor, AC},
title = {Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2351},
pmid = {35534455},
issn = {2041-1723},
support = {DP5OD023098//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; MCB-2048207//National Science Foundation (NSF)/ ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; *Gene Editing/methods ; Mammals/genetics ; *RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.},
}
@article {pmid35532260,
year = {2022},
author = {Chambers, C and Quan, L and Yi, G and Esquela-Kerscher, A},
title = {CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {182},
pages = {},
doi = {10.3791/63704},
pmid = {35532260},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing/methods ; Humans ; *MicroRNAs/genetics ; RNA, Guide/genetics ; RNA, Untranslated ; },
abstract = {MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30% of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic. The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment. The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.},
}
@article {pmid35531207,
year = {2022},
author = {Raza, SHA and Hassanin, AA and Pant, SD and Bing, S and Sitohy, MZ and Abdelnour, SA and Alotaibi, MA and Al-Hazani, TM and Abd El-Aziz, AH and Cheng, G and Zan, L},
title = {Potentials, prospects and applications of genome editing technologies in livestock production.},
journal = {Saudi journal of biological sciences},
volume = {29},
number = {4},
pages = {1928-1935},
pmid = {35531207},
issn = {1319-562X},
abstract = {In recent years, significant progress has been achieved in genome editing applications using new programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs) and the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9). These genome editing tools are capable of nicking DNA precisely by targeting specific sequences, and enable the addition, removal or substitution of nucleotides via double-stranded breakage at specific genomic loci. CRISPR/Cas system, one of the most recent genome editing tools, affords the ability to efficiently generate multiple genomic nicks in single experiment. Moreover, CRISPR/Cas systems are relatively easy and cost effective when compared to other genome editing technologies. This is in part because CRISPR/Cas systems rely on RNA-DNA binding, unlike other genome editing tools that rely on protein-DNA interactions, which affords CRISPR/Cas systems higher flexibility and more fidelity. Genome editing tools have significantly contributed to different aspects of livestock production such as disease resistance, improved performance, alterations of milk composition, animal welfare and biomedicine. However, despite these contributions and future potential, genome editing technologies also have inherent risks, and therefore, ethics and social acceptance are crucial factors associated with implementation of these technologies. This review emphasizes the impact of genome editing technologies in development of livestock breeding and production in numerous species such as cattle, pigs, sheep and goats. This review also discusses the mechanisms behind genome editing technologies, their potential applications, risks and associated ethics that should be considered in the context of livestock.},
}
@article {pmid35525561,
year = {2022},
author = {Cassidy, AM and Kuliyev, E and Thomas, DB and Chen, H and Pelletier, S},
title = {Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology.},
journal = {Methods in enzymology},
volume = {667},
number = {},
pages = {775-812},
doi = {10.1016/bs.mie.2022.03.053},
pmid = {35525561},
issn = {1557-7988},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Targeting ; Mice ; Mutagenesis ; Technology ; },
abstract = {Allelic series are extremely valuable genetic tools to study gene function and identify essential structural features of gene products. In mice, allelic series have been engineered using conventional gene targeting in embryonic stem cells or chemical mutagenesis. While these approaches have provided valuable information about the function of genes, they remain cumbersome. Modern approaches such as CRISPR-Cas9 technologies now allow for the precise and cost-effective generation of mouse models with specific mutations, facilitating the development of allelic series. Here, we describe procedures for the generation of three types of mutations used to dissect protein function in vivo using CRISPR-Cas9 technology. This step-by-step protocol describes the generation of missense mutations, large in-frame deletions, and insertions of genetic material using SCY1-like 1 (Scyl1) as a model gene.},
}
@article {pmid35525543,
year = {2022},
author = {Jacobsen, AV and Murphy, JM},
title = {CRISPR deletions in cell lines for reconstitution studies of pseudokinase function.},
journal = {Methods in enzymology},
volume = {667},
number = {},
pages = {229-273},
doi = {10.1016/bs.mie.2022.03.054},
pmid = {35525543},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing/methods ; Humans ; *Protein Kinases/genetics ; },
abstract = {The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.},
}
@article {pmid35524183,
year = {2022},
author = {Zhou, S and Kalds, P and Luo, Q and Sun, K and Zhao, X and Gao, Y and Cai, B and Huang, S and Kou, Q and Petersen, B and Chen, Y and Ma, B and Wang, X},
title = {Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {348},
pmid = {35524183},
issn = {1471-2164},
support = {31872332//National Natural Science Foundation of China/ ; 31972526//National Natural Science Foundation of China/ ; NXTS2021-001//Local Grant/ ; 2021YFF1000700//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Goats/genetics ; Meat ; *Myostatin/genetics ; RNA, Guide/genetics ; RNA, Messenger ; Sheep/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner.<br><br>RESULTS: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs.<br><br>CONCLUSIONS: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.},
}
@article {pmid35524126,
year = {2022},
author = {Després, PC and Dubé, AK and Yachie, N and Landry, CR},
title = {High-Throughput Gene Mutagenesis Screening Using Base Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2477},
number = {},
pages = {331-348},
pmid = {35524126},
issn = {1940-6029},
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutagenesis/genetics ; RNA, Guide/genetics ; },
abstract = {Base editing is a CRISPR-Cas9 genome engineering tool that allows programmable mutagenesis without the creation of double-stranded breaks. Here, we describe the design and execution of large-scale base editing screens using the Target-AID base editor in yeast. Using this approach, thousands of sites can be mutated simultaneously. The effects of these mutations on fitness can be measured using a pooled growth competition assay followed by DNA sequencing of gRNAs as barcodes.},
}
@article {pmid35524121,
year = {2022},
author = {Dubé, AK and Dandage, R and Dibyachintan, S and Dionne, U and Després, PC and Landry, CR},
title = {Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2477},
number = {},
pages = {237-259},
pmid = {35524121},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Point Mutation ; },
abstract = {Deep mutational scanning (DMS) generates mutants of a protein of interest in a comprehensive manner. CRISPR-Cas9 technology enables large-scale genome editing with high efficiency. Using both DMS and CRISPR-Cas9 therefore allows us to investigate the effects of thousands of mutations inserted directly in the genome. Combined with protein-fragment complementation assay (PCA), which enables the quantitative measurement of protein-protein interactions (PPIs) in vivo, these methods allow for the systematic assessment of the effects of mutations on PPIs in living cells. Here, we describe a method leveraging DMS, CRISPR-Cas9, and PCA to study the effect of point mutations on PPIs mediated by protein domains in yeast.},
}
@article {pmid35524052,
year = {2022},
author = {Chhun, A and Alberti, F},
title = {CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2489},
number = {},
pages = {201-222},
pmid = {35524052},
issn = {1940-6029},
mesh = {*Actinobacteria/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Streptomyces/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.},
}
@article {pmid35524051,
year = {2022},
author = {Massicard, JM and Su, L and Jacob, C and Weissman, KJ},
title = {Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2489},
number = {},
pages = {173-200},
pmid = {35524051},
issn = {1940-6029},
mesh = {Animals ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Polyketides/metabolism ; RNA, Guide/genetics/metabolism ; *Streptomyces/genetics/metabolism ; },
abstract = {The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.},
}
@article {pmid35522691,
year = {2022},
author = {Matozel, EK and Parziale, S and Price, AC},
title = {A programmable DNA roadblock system using dCas9 and multivalent target sites.},
journal = {PloS one},
volume = {17},
number = {5},
pages = {e0268099},
pmid = {35522691},
issn = {1932-6203},
mesh = {Binding Sites ; CRISPR-Cas Systems ; *DNA/metabolism ; DNA-Binding Proteins/genetics ; *Endonucleases/metabolism ; RNA, Guide/genetics ; },
abstract = {A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.},
}
@article {pmid35521548,
year = {2022},
author = {Shor, O and Rabinowitz, R and Offen, D and Benninger, F},
title = {Computational normal mode analysis accurately replicates the activity and specificity profiles of CRISPR-Cas9 and high-fidelity variants.},
journal = {Computational and structural biotechnology journal},
volume = {20},
number = {},
pages = {2013-2019},
pmid = {35521548},
issn = {2001-0370},
abstract = {The CRISPR-Cas system has transformed the field of gene-editing and created opportunities for novel genome engineering therapeutics. The field has significantly progressed, and recently, CRISPR-Cas9 was utilized in clinical trials to target disease-causing mutations. Existing tools aim to predict the on-target efficacy and potential genome-wide off-targets by scoring a particular gRNA according to an array of gRNA design principles or machine learning algorithms based on empirical results of large numbers of gRNAs. However, such tools are unable to predict the editing outcome by variant Cas enzymes and can only assess potential off-targets related to reference genomes. Here, we employ normal mode analysis (NMA) to investigate the structure of the Cas9 protein complexed with its gRNA and target DNA and explore the function of the protein. Our results demonstrate the feasibility and validity of NMA to predict the activity and specificity of SpyCas9 in the presence of mismatches by comparison to empirical data. Furthermore, despite the absence of their exact structures, this method accurately predicts the enzymatic activity of known high-fidelity engineered Cas9 variants.},
}
@article {pmid35513429,
year = {2022},
author = {Li, R and Klingbeil, O and Monducci, D and Young, MJ and Rodriguez, DJ and Bayyat, Z and Dempster, JM and Kesar, D and Yang, X and Zamanighomi, M and Vakoc, CR and Ito, T and Sellers, WR},
title = {Comparative optimization of combinatorial CRISPR screens.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2469},
pmid = {35513429},
issn = {2041-1723},
support = {W81XWH-19-1-0271//U.S. Department of Defense (United States Department of Defense)/ ; 500506//Ludwig Institute for Cancer Research (Ludwig Cancer Research)/ ; },
mesh = {*Acidaminococcus/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.},
}
@article {pmid35512092,
year = {2022},
author = {Taylor, JH and Walton, JC and McCann, KE and Norvelle, A and Liu, Q and Vander Velden, JW and Borland, JM and Hart, M and Jin, C and Huhman, KL and Cox, DN and Albers, HE},
title = {CRISPR-Cas9 editing of the arginine-vasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {19},
pages = {e2121037119},
doi = {10.1073/pnas.2121037119},
pmid = {35512092},
issn = {1091-6490},
support = {IOS-1035960//National Science Foundation (NSF)/ ; MH109302/MH/NIMH NIH HHS/United States ; MH122622/MH/NIMH NIH HHS/United States ; },
mesh = {Aggression/physiology ; Animals ; Arginine/metabolism ; Arginine Vasopressin/genetics ; *CRISPR-Cas Systems ; Cricetinae ; Mesocricetus ; *Receptors, Vasopressin/genetics/metabolism ; Social Behavior ; },
abstract = {SignificanceArginine-vasopressin (AVP) acting on V1a receptors (Avpr1as) represents a key signaling mechanism in a brain circuit that increases the expression of social communication and aggression. We produced Syrian hamsters that completely lack Avpr1as (Avpr1a knockout [KO] hamsters) using the CRISPR-Cas9 system to more fully examine the role of Avpr1a in the expression of social behaviors. We confirmed the absence of Avpr1as in these hamsters by demonstrating 1) a complete lack of Avpr1a-specific receptor binding throughout the brain, 2) a behavioral insensitivity to centrally administered AVP, and 3) an absence of the well-known blood-pressure response produced by activating Avpr1as. Unexpectedly, however, Avpr1a KO hamsters displayed more social communication behavior and aggression toward same-sex conspecifics than did their wild-type (WT) littermates.},
}
@article {pmid35509467,
year = {2022},
author = {Wang, DX and Wang, YX and Wang, J and Ma, JY and Liu, B and Tang, AN and Kong, DM},
title = {MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a.},
journal = {Chemical science},
volume = {13},
number = {15},
pages = {4364-4371},
pmid = {35509467},
issn = {2041-6520},
abstract = {Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO2 nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn2+ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO2 nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO2 nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.},
}
@article {pmid35509363,
year = {2022},
author = {Ebrahimi, S and Khanbabaei, H and Abbasi, S and Fani, M and Soltani, S and Zandi, M and Najafimemar, Z},
title = {CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19.},
journal = {Avicenna journal of medical biotechnology},
volume = {14},
number = {1},
pages = {3-9},
pmid = {35509363},
issn = {2008-2835},
abstract = {More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.},
}
@article {pmid35508982,
year = {2022},
author = {Shojaei Baghini, S and Gardanova, ZR and Abadi, SAH and Zaman, BA and İlhan, A and Shomali, N and Adili, A and Moghaddar, R and Yaseri, AF},
title = {CRISPR/Cas9 application in cancer therapy: a pioneering genome editing tool.},
journal = {Cellular &amp; molecular biology letters},
volume = {27},
number = {1},
pages = {35},
pmid = {35508982},
issn = {1689-1392},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {The progress of genetic engineering in the 1970s brought about a paradigm shift in genome editing technology. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a flexible means to target and modify particular DNA sequences in the genome. Several applications of CRISPR/Cas9 are presently being studied in cancer biology and oncology to provide vigorous site-specific gene editing to enhance its biological and clinical uses. CRISPR's flexibility and ease of use have enabled the prompt achievement of almost any preferred alteration with greater efficiency and lower cost than preceding modalities. Also, CRISPR/Cas9 technology has recently been applied to improve the safety and efficacy of chimeric antigen receptor (CAR)-T cell therapies and defeat tumor cell resistance to conventional treatments such as chemotherapy and radiotherapy. The current review summarizes the application of CRISPR/Cas9 in cancer therapy. We also discuss the present obstacles and contemplate future possibilities in this context.},
}
@article {pmid35508977,
year = {2022},
author = {Trung, NT and Son, LHP and Hien, TX and Quyen, DT and Bang, MH and Song, LH},
title = {CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis.},
journal = {BMC infectious diseases},
volume = {22},
number = {1},
pages = {429},
pmid = {35508977},
issn = {1471-2334},
support = {108.06-2017.21//Vietnam National Foundation for Science and Technology Development (NAFOSTED)/ ; 364/2020/HD-NCKHCN//The Vietnamese Ministry of National Defence/ ; },
mesh = {*CRISPR-Cas Systems ; DNA ; Humans ; Molecular Diagnostic Techniques ; *Neisseria meningitidis/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA ; },
abstract = {BACKGROUND: Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen's DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.<br><br>METHODS: The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).<br><br>RESULTS: In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.<br><br>CONCLUSIONS: In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.},
}
@article {pmid35508460,
year = {2022},
author = {Mac Kain, A and Maarifi, G and Aicher, SM and Arhel, N and Baidaliuk, A and Munier, S and Donati, F and Vallet, T and Tran, QD and Hardy, A and Chazal, M and Porrot, F and OhAinle, M and Carlson-Stevermer, J and Oki, J and Holden, K and Zimmer, G and Simon-Lorière, E and Bruel, T and Schwartz, O and van der Werf, S and Jouvenet, N and Nisole, S and Vignuzzi, M and Roesch, F},
title = {Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2442},
pmid = {35508460},
issn = {2041-1723},
support = {ANR-20-COVI-000//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Co-Repressor Proteins/genetics/metabolism ; Humans ; Interferons/metabolism ; Molecular Chaperones/genetics/metabolism ; Proteasome Endopeptidase Complex/metabolism ; *SARS-CoV-2 ; },
abstract = {Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.},
}
@article {pmid35507186,
year = {2022},
author = {Desjardins, J and Cowan, M and Yamanaka, Y},
title = {Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {515-531},
pmid = {35507186},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome/genetics ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; Zygote ; },
abstract = {Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.},
}
@article {pmid35507174,
year = {2022},
author = {Ford, MJ and Yamanaka, Y},
title = {Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {367-377},
pmid = {35507174},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Electroporation/methods ; Epithelial Cells ; Fallopian Tubes ; Female ; *Gene Editing/methods ; Humans ; Mice ; },
abstract = {Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.},
}
@article {pmid35507170,
year = {2022},
author = {Devaraju, N and Rajendiran, V and Ravi, NS and Mohankumar, KM},
title = {Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {307-331},
pmid = {35507170},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Mice ; Transplantation, Autologous ; },
abstract = {Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.},
}
@article {pmid35507169,
year = {2022},
author = {Park, SH and Lee, CM and Bao, G},
title = {Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {281-306},
pmid = {35507169},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; High-Throughput Nucleotide Sequencing ; *RNA, Guide/genetics/metabolism ; },
abstract = {Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.},
}
@article {pmid35506667,
year = {2022},
author = {Kever, L and Hardy, A and Luthe, T and Hünnefeld, M and Gätgens, C and Milke, L and Wiechert, J and Wittmann, J and Moraru, C and Marienhagen, J and Frunzke, J},
title = {Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.},
journal = {mBio},
volume = {},
number = {},
pages = {e0078322},
doi = {10.1128/mbio.00783-22},
pmid = {35506667},
issn = {2150-7511},
abstract = {In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.},
}
@article {pmid35506451,
year = {2022},
author = {Kalafati, E and Papanikolaou, E and Marinos, E and Anagnou, NP and Pappa, KI},
title = {Mimiviruses: Giant viruses with novel and intriguing features (Review).},
journal = {Molecular medicine reports},
volume = {25},
number = {6},
pages = {},
doi = {10.3892/mmr.2022.12723},
pmid = {35506451},
issn = {1791-3004},
mesh = {*Amoeba ; CRISPR-Cas Systems ; Capsid ; *Giant Viruses/genetics ; Humans ; *Mimiviridae/genetics ; },
abstract = {The Mimivirus is a giant virus that infects amoebae and was long considered to be a bacterium due to its size. The viral particles are composed of a protein capsid of ~500 nm in diameter, which is enclosed in a polysaccharide layer in which ~120‑140 nm long fibers are embedded, resulting in an overall diameter of 700 nm. The virus has a genome size of 1.2 Mb DNA, and surprisingly, replicates only in the cytoplasm of the infected cells without entering the nucleus, which is a unique characteristic among DNA viruses. Their existence is undeniable; however, as with any novel discovery, there is still uncertainty concerning their pathogenicity mechanisms in humans and the nature of the Mimivirus virophage resistance element system (MIMIVIRE), a term given to describe the immune network of the Mimivirus, which closely resembles the CRISPR‑Cas system. The scope of the present review is to discuss the recent developments derived from structural and functional studies performed on the distinctive characteristics of the Mimivirus, and from studies concerning their putative clinical relevance in humans.},
}
@article {pmid35504953,
year = {2022},
author = {Warmt, C and Yaslanmaz, C and Henkel, J},
title = {Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {7137},
pmid = {35504953},
issn = {2045-2322},
mesh = {DNA Primers ; Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques/methods ; *Nucleotides ; Spectrometry, Fluorescence ; },
abstract = {Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.},
}
@article {pmid35504940,
year = {2022},
author = {Sommerkamp, P and Sommerkamp, AC and Zeisberger, P and Eiben, PL and Narr, A and Korkmaz, A and Przybylla, A and Sohn, M and van der Hoeven, F and Schönig, K and Trumpp, A},
title = {CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {7181},
pmid = {35504940},
issn = {2045-2322},
support = {FOR2033//Deutsche Forschungsgemeinschaft/ ; RiskY-AML//Deutschen Konsortium für Translationale Krebsforschung/ ; SyTASC//Deutsche Krebshilfe/ ; },
mesh = {3' Untranslated Regions ; Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Hematopoietic Stem Cells/metabolism ; Mice ; *Poly(A)-Binding Protein I/metabolism ; Polyadenylation ; RNA, Messenger/genetics ; },
abstract = {Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.},
}
@article {pmid35502187,
year = {2022},
author = {Espinoza-Mellado, MDR and Vilchis-Rangel, RE},
title = {Review of CRISPR-Cas Systems in Listeria Species: Current Knowledge and Perspectives.},
journal = {International journal of microbiology},
volume = {2022},
number = {},
pages = {9829770},
pmid = {35502187},
issn = {1687-918X},
abstract = {Listeria spp. are pathogens widely distributed in the environment and Listeria monocytogenes is associated with food-borne illness in humans. Food facilities represent an adverse environment for this bacterium, mainly due to the disinfection and cleaning processes included in good hygiene practices, and its virulence is related to stress responses. One of the recently described stress-response systems is CRISPR-Cas. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes have been found in several bacteria. CRISPR-Cas has revolutionized biotechnology since it acts as an adaptive immune system of bacteria, which also helps in the evasion of the host immune response. There are three CRISPR systems described on Listeria species. Type II is present in many pathogenic bacteria and characterized by the presence of cas9 that becomes the main target of some anti-CRISPR proteins, such as AcrIIA1, encoded on Listeria phages. The presence of Cas9, either alone or in combination with anti-CRISPR proteins, suggests having a main role on the virulence of bacteria. In this review, we describe the most recent information on CRISPR-Cas systems in Listeria spp., particularly in L. monocytogenes, and their relationship with the virulence and pathogenicity of those bacteria. Besides, some applications of CRISPR systems and future challenges in the food processing industry, bacterial vaccination, antimicrobial resistance, pathogens biocontrol by phage therapy, and regulation of gene expression have been explored.},
}
@article {pmid35500195,
year = {2022},
author = {Zhao, M and Gao, M and Xiong, L and Liu, Y and Tao, X and Gao, B and Liu, M and Wang, FQ and Wei, DZ},
title = {CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00112},
pmid = {35500195},
issn = {2161-5063},
abstract = {Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.},
}
@article {pmid35499084,
year = {2022},
author = {Caruso, SM and Quinn, PM and da Costa, BL and Tsang, SH},
title = {CRISPR/Cas therapeutic strategies for autosomal dominant disorders.},
journal = {The Journal of clinical investigation},
volume = {132},
number = {9},
pages = {},
pmid = {35499084},
issn = {1558-8238},
support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; U01 EY030580/EY/NEI NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; R01 EY009076/EY/NEI NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R24 EY027285/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R24 EY028758/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; Mutation ; },
abstract = {Autosomal dominant disorders present unique challenges, as therapeutics must often distinguish between healthy and diseased alleles while maintaining high efficiency, specificity, and safety. For this task, CRISPR/Cas remains particularly promising. Various CRISPR/Cas systems, like homology-directed repair, base editors, and prime editors, have been demonstrated to selectively edit mutant alleles either by incorporating these mutations into sgRNA sequences (near the protospacer-adjacent motif ["near the PAM"]) or by targeting a novel PAM generated by the mutation ("in the PAM"). However, these probability-based designs are not always assured, necessitating generalized, mutation-agnostic strategies like ablate-and-replace and single-nucleotide polymorphism editing. Here, we detail recent advancements in CRISPR therapeutics to treat a wide range of autosomal dominant disorders and discuss how they are altering the landscape for future therapies.},
}
@article {pmid35499048,
year = {2022},
author = {Ibrahim, S and Saleem, B and Rehman, N and Zafar, SA and Naeem, MK and Khan, MR},
title = {CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.},
journal = {Journal of advanced research},
volume = {37},
number = {},
pages = {33-41},
pmid = {35499048},
issn = {2090-1224},
mesh = {CRISPR-Cas Systems ; Edible Grain ; Humans ; Inositol Phosphates ; Iron ; *Phytic Acid/metabolism ; *Triticum/genetics ; Zinc/metabolism ; },
abstract = {Introduction: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis.<br><br>Objective: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9.<br><br>Methods: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons.<br><br>Results: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants.<br><br>Conclusion: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.},
}
@article {pmid35496799,
year = {2022},
author = {Picchi-Constante, GFA and Hiraiwa, PM and Marek, M and Rogerio, VZ and Guerra-Slompo, EP and Romier, C and Zanchin, NIT},
title = {Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101324},
pmid = {35496799},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/genetics ; *Chagas Disease/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, Essential ; Humans ; *Trypanosoma cruzi/genetics ; },
abstract = {This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).},
}
@article {pmid35496795,
year = {2022},
author = {Ling, X and Chang, L and Chen, H and Liu, T},
title = {Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101321},
pmid = {35496795},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; },
abstract = {We recently developed a system to create human chimeric antigen receptor (CAR)-T cells using conjugated Cas12a (cCas12a) in which Cas12a is covalently linked to its CRISPR RNA (crRNA). This protocol describes site-specific modification of Cas12a and the preparation of Cas12a-crRNA complex using bio-orthogonal chemistry, followed by CAR-T cell generation through electroporation and AAV infection. This system shows robust editing efficiency in human cells and can be used for precisely targeted, highly efficient integration of CAR genes into T cell genome. For complete details on the use and execution of this protocol, please refer to Ling et al. (2021).},
}
@article {pmid35495695,
year = {2022},
author = {Zhang, X and An, X},
title = {Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {876174},
pmid = {35495695},
issn = {1664-302X},
abstract = {CRISPR-Cas systems acquire heritable defense memory against invading nucleic acids through adaptation. Type III CRISPR-Cas systems have unique and intriguing features of defense and are important in method development for Genetics research. We started to understand the common and unique properties of type III CRISPR-Cas adaptation in recent years. This review summarizes our knowledge regarding CRISPR-Cas adaptation with the emphasis on type III systems and discusses open questions for type III adaptation studies.},
}
@article {pmid35495653,
year = {2022},
author = {Wörtz, J and Smith, V and Fallmann, J and König, S and Thuraisingam, T and Walther, P and Urlaub, H and Stadler, PF and Allers, T and Hille, F and Marchfelder, A},
title = {Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {822304},
pmid = {35495653},
issn = {1664-302X},
abstract = {CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.},
}
@article {pmid35491944,
year = {2022},
author = {Fremin, BJ and Kyrpides, NC},
title = {Identifying candidate structured RNAs in CRISPR operons.},
journal = {RNA biology},
volume = {19},
number = {1},
pages = {678-685},
pmid = {35491944},
issn = {1555-8584},
mesh = {*CRISPR-Cas Systems ; Genomics ; Operon ; *RNA/genetics ; Repetitive Sequences, Nucleic Acid ; },
abstract = {Noncoding RNAs with secondary structures play important roles in CRISPR-Cas systems. Many of these structures likely remain undiscovered. We used a large-scale comparative genomics approach to predict 156 novel candidate structured RNAs from 36,111 CRISPR-Cas systems. A number of these were found to overlap with coding genes, including palindromic candidates that overlapped with a variety of Cas genes in type I and III systems. Among these 156 candidates, we identified 46 new models of CRISPR direct repeats and 1 tracrRNA. This tracrRNA model occasionally overlapped with predicted cas9 coding regions, emphasizing the importance of expanding our search windows for novel structure RNAs in coding regions. We also demonstrated that the antirepeat sequence in this tracrRNA model can be used to accurately assign thousands of predicted CRISPR arrays to type II-C systems. This study highlights the importance of unbiased identification of candidate structured RNAs across CRISPR-Cas systems.},
}
@article {pmid35490782,
year = {2022},
author = {Tao, Y and Chaudhari, S and Shotorbani, PY and Ding, Y and Chen, Z and Kasetti, R and Zode, G and Ma, R},
title = {Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {101990},
doi = {10.1016/j.jbc.2022.101990},
pmid = {35490782},
issn = {1083-351X},
abstract = {Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-CAS9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/ SOCE/Calpain axis contributes to HG-induced podocyte injury.},
}
@article {pmid35487018,
year = {2022},
author = {Klose, SM and Wawegama, N and Sansom, FM and Marenda, MS and Browning, GF},
title = {Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.},
journal = {Veterinary microbiology},
volume = {269},
number = {},
pages = {109436},
doi = {10.1016/j.vetmic.2022.109436},
pmid = {35487018},
issn = {1873-2542},
abstract = {Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.},
}
@article {pmid35483740,
year = {2022},
author = {Herman, JA and Arora, S and Carter, L and Zhu, J and Biggins, S and Paddison, PJ},
title = {Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.},
journal = {Genes &amp; development},
volume = {36},
number = {7-8},
pages = {495-510},
doi = {10.1101/gad.349319.121},
pmid = {35483740},
issn = {1549-5477},
support = {R01 GM064386/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Mutagenesis ; },
abstract = {The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.},
}
@article {pmid35483732,
year = {2022},
author = {Shin, J and Miller, M and Wang, YC},
title = {Recent advances in CRISPR-based systems for the detection of foodborne pathogens.},
journal = {Comprehensive reviews in food science and food safety},
volume = {},
number = {},
pages = {},
doi = {10.1111/1541-4337.12956},
pmid = {35483732},
issn = {1541-4337},
support = {ILLU-698-353//U.S. Department of Agriculture/ ; //University of Illinois Urbana-Champaign/ ; R20-7704//Royal Society of Chemistry/ ; },
abstract = {There has long been a need for more advanced forms of pathogen detection in the food industry. Though in its infancy, biosensing based on clustered regularly interspaced short palindromic repeats (CRISPR) has the potential to solve many problems that cannot be addressed using conventional methods. In this review, we briefly introduce and classify the various CRISPR/Cas protein effectors that have thus far been used in biosensors. We then assess the current state of CRISPR technology in food-safety contexts; describe how each Cas effector is utilized in foodborne-pathogen detection; and discuss the limitations of the current technology, as well as how it might usefully be applied in other areas of the food industry. We conclude that, if the limitations of existing CRISPR/Cas-based detection methods are overcome, they can be deployed on a wide scale and produce a range of positive food-safety outcomes.},
}
@article {pmid35481139,
year = {2022},
author = {Li, Q and Feng, Q and Snouffer, A and Zhang, B and Rodríguez, GR and van der Knaap, E},
title = {Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {879642},
pmid = {35481139},
issn = {1664-462X},
abstract = {CRISPR/Cas-mediated genome editing is a powerful approach to accelerate yield enhancement to feed growing populations. Most applications focus on "negative regulators" by targeting coding regions and promoters to create nulls or weak loss-of-function alleles. However, many agriculturally important traits are conferred by gain-of-function alleles. Therefore, creating gain-of-function alleles for "positive regulators" by CRISPR will be of great value for crop improvement. CYP78A family members are the positive regulators of organ weight and size in crops. In this study, we engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) that is highly associated with fruit weight. The SNP was located in a conserved putative cis-regulatory element (CRE) as detected by the homology-based prediction and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Twenty-one mutant alleles with various insertion and deletion sizes were generated in the LA1589 background. Five mutant alleles (m2+4bp , m3+1bp , m5-1bp , m13-8bp , and m14-9bp) showed a consistent increase in fruit weight and a significant decrease in the proportion of small fruits in all experimental evaluations. Notably, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7-15.7 and 8.7-16.3%, respectively. Further analysis of fruit weight based on fruit position on the inflorescence indicated that the five beneficial alleles increase the weight of all fruits along inflorescence. We also found that allele types and transcriptional changes of SlKLUH were poor predictors of the changes in fruit weight. This study not only provides a way of identifying conserved CRE but also highlights enormous potential for CRISPR/Cas-mediated cis-engineering of CYP78A members in yield improvement.},
}
@article {pmid35478255,
year = {2022},
author = {Khairkhah, N and Bolhassani, A and Najafipour, R},
title = {Current and future direction in treatment of HPV-related cervical disease.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {},
number = {},
pages = {},
pmid = {35478255},
issn = {1432-1440},
abstract = {Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of therapeutic HPV vaccines using E6 and E7 oncogenes, or both as the target antigens remains essential. Also, knocking out the E6 and E7 oncogenes in host genome by genome-editing CRISPR/Cas system can result in tumor growth suppression. These methods have shown promising results in both preclinical and clinical trials and can be used for controlling the progression of HPV-related cervical diseases. This comprehensive review will detail the current treatment of HPV-related cervical precancerous and cancerous diseases. We also reviewed the future direction of treatment including different kinds of therapeutic methods and vaccines, genome-editing CRISPR/Cas system being studied in clinical trials. Although the progress in the development of therapeutic HPV vaccine has been slow, encouraging results from recent trials showed vaccine-induced regression in high-grade CIN lesions. CRISPR/Cas genome-editing system is also a promising strategy for HPV cancer therapy. However, its safety and specificity need to be optimized before it is used in clinical setting.},
}
@article {pmid35478023,
year = {2022},
author = {Mallapaty, S},
title = {China focuses on ethics to deter another 'CRISPR babies' scandal.},
journal = {Nature},
volume = {605},
number = {7908},
pages = {15-16},
doi = {10.1038/d41586-022-01051-0},
pmid = {35478023},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; China ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Ethics, Medical ; *Gene Editing ; Humans ; },
}
@article {pmid35475170,
year = {2022},
author = {Nemudraia, A and Nemudryi, A and Buyukyoruk, M and Scherffius, A and Zahl, T and Wiegand, T and Pandey, S and Nichols, J and Hall, L and McVey, A and Lee, H and Wilkinson, R and Snyder, L and Jones, J and Koutmou, K and Santiago-Frangos, A and Wiedenheft, B},
title = {Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.},
journal = {Research square},
volume = {},
number = {},
pages = {},
pmid = {35475170},
support = {R35 GM128836/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
abstract = {Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.},
}
@article {pmid35474904,
year = {2022},
author = {Cheng, L and Yang, F and Tang, L and Qian, L and Chen, X and Guan, F and Zhang, J and Li, G},
title = {Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage.},
journal = {Research (Washington, D.C.)},
volume = {2022},
number = {},
pages = {9826484},
pmid = {35474904},
issn = {2639-5274},
abstract = {Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.},
}
@article {pmid35473305,
year = {2022},
author = {Bindal, G and Amlinger, L and Lundgren, M and Rath, D},
title = {Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0003822},
doi = {10.1128/msphere.00038-22},
pmid = {35473305},
issn = {2379-5042},
abstract = {Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.},
}
@article {pmid35472302,
year = {2022},
author = {Cho, SI and Lee, S and Mok, YG and Lim, K and Lee, J and Lee, JM and Chung, E and Kim, JS},
title = {Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.},
journal = {Cell},
volume = {185},
number = {10},
pages = {1764-1776.e12},
doi = {10.1016/j.cell.2022.03.039},
pmid = {35472302},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems ; Cytosine/metabolism ; *DNA, Mitochondrial/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Humans ; Mitochondria/genetics/metabolism ; *Mitochondrial Diseases/genetics ; Purines ; },
abstract = {Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.},
}
@article {pmid35472287,
year = {2022},
author = {Horáčková, V and Voleman, L and Hagen, KD and Petrů, M and Vinopalová, M and Weisz, F and Janowicz, N and Marková, L and Motyčková, A and Najdrová, V and Tůmová, P and Dawson, SC and Doležal, P},
title = {Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.},
journal = {Open biology},
volume = {12},
number = {4},
pages = {210361},
pmid = {35472287},
issn = {2046-2441},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Giardia lamblia/genetics ; Humans ; RNA, Guide ; Tetraploidy ; },
abstract = {CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.},
}
@article {pmid35470620,
year = {2022},
author = {LE, Y and He, X and Sun, J},
title = {[Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1475-1489},
doi = {10.13345/j.cjb.210827},
pmid = {35470620},
issn = {1872-2075},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; },
abstract = {The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.},
}
@article {pmid35470618,
year = {2022},
author = {Chao, S and Hu, X},
title = {[Application of gene editing technology in Escherichia coli].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1446-1461},
doi = {10.13345/j.cjb.210680},
pmid = {35470618},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing ; Homologous Recombination ; Technology ; },
abstract = {Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.},
}
@article {pmid35470617,
year = {2022},
author = {Wang, C and Liu, Z and Tang, B and Yang, H and Sun, D},
title = {[Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1432-1445},
doi = {10.13345/j.cjb.210348},
pmid = {35470617},
issn = {1872-2075},
mesh = {Anti-Bacterial Agents ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; },
abstract = {Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.},
}
@article {pmid35469710,
year = {2022},
author = {Cury, J and Bernheim, A},
title = {CRISPR-Cas and restriction-modification team up to achieve long-term immunity.},
journal = {Trends in microbiology},
volume = {30},
number = {6},
pages = {513-514},
doi = {10.1016/j.tim.2022.04.001},
pmid = {35469710},
issn = {1878-4380},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; },
abstract = {Bacteria have been shown to harbor a growing arsenal of various defense systems against phages. Maguin et al. have uncovered how two of the most frequent defense systems interact: the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system recycles by-products of the restriction-modification (RM) system to increase bacterial defense in the long run.},
}
@article {pmid35468950,
year = {2022},
author = {Mitkas, AA and Valverde, M and Chen, W},
title = {Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.},
journal = {Nature chemical biology},
volume = {18},
number = {5},
pages = {492-500},
pmid = {35468950},
issn = {1552-4469},
support = {CBET1803008, MCB1817675 and MCB2013991//National Science Foundation (NSF)/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Endonucleases/metabolism ; RNA/metabolism ; RNA Processing, Post-Transcriptional ; },
abstract = {In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR-Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA-RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and 'Turn-On' assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.},
}
@article {pmid35468907,
year = {2022},
author = {Thean, DGL and Chu, HY and Fong, JHC and Chan, BKC and Zhou, P and Kwok, CCS and Chan, YM and Mak, SYL and Choi, GCG and Ho, JWK and Zheng, Z and Wong, ASL},
title = {Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2219},
pmid = {35468907},
issn = {2041-1723},
support = {32022089//National Natural Science Foundation of China (National Science Foundation of China)/ ; 17104619//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*Bacterial Proteins/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Humans ; Machine Learning ; Mutagenesis ; },
abstract = {The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.},
}
@article {pmid35468792,
year = {2022},
author = {Huang, H and Huang, G and Tan, Z and Hu, Y and Shan, L and Zhou, J and Zhang, X and Ma, S and Lv, W and Huang, T and Liu, Y and Wang, D and Zhao, X and Lin, Y and Rong, Z},
title = {Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {91},
pmid = {35468792},
issn = {1741-7007},
support = {2017YFA0105001//the National Key R&amp;D Program of China/ ; 82070002, 81872511//the National Natural Science Foundation of China/ ; 82072329//the National Natural Science Foundation of China/ ; 2021M701615//the Fellowship of China Postdoctoral Science Foundation/ ; 2021M691473//the Fellowship of China Postdoctoral Science Foundation/ ; 2021M701634//the Fellowship of China Postdoctoral Science Foundation/ ; },
mesh = {Acidaminococcus/genetics ; *CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing ; Proto-Oncogene Proteins B-raf/genetics ; },
abstract = {BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).<br><br>RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.<br><br>CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.},
}
@article {pmid35467081,
year = {2022},
author = {Sparmann, A and Beisel, CL},
title = {CRISPR memories in single cells.},
journal = {Molecular systems biology},
volume = {18},
number = {4},
pages = {e11011},
pmid = {35467081},
issn = {1744-4292},
mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas systems allow bacteria to memorize prior infections as a means to combat the same invader if it attempts another attack in the future. While the underlying mechanisms of this bacterial immunity have been intensely studied over the past decade, little attention has been paid to CRISPR defense at the single-cell level. In their recent work, Brouns and colleagues (McKenzie et al, 2022) track memory acquisition and defense in individual cells and find a wide range of temporal dynamics that shape how a cell population experiences and combats an active infection.},
}
@article {pmid35467080,
year = {2022},
author = {McKenzie, RE and Keizer, EM and Vink, JNA and van Lopik, J and Büke, F and Kalkman, V and Fleck, C and Tans, SJ and Brouns, SJJ},
title = {Single cell variability of CRISPR-Cas interference and adaptation.},
journal = {Molecular systems biology},
volume = {18},
number = {4},
pages = {e10680},
doi = {10.15252/msb.202110680},
pmid = {35467080},
issn = {1744-4292},
support = {801041//European Union Horizons 2020, CyGenTiG/ ; 101003229//EC|H2020|H2020 Priority Excellent Science|H2020 European Research Council (ERC)/ ; //NWO Frontiers of Nanoscience Nanofront/ ; VI.C.182.027//Netherlands Organisation for Scientific Research NWO vici/ ; },
mesh = {Adaptation, Physiological/genetics ; *Bacteriophages ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Escherichia coli/genetics/metabolism ; },
abstract = {While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.},
}
@article {pmid35466160,
year = {2022},
author = {Bajaj, A and Cuchel, M},
title = {Advancements in the Treatment of Homozygous Familial Hypercholesterolemia.},
journal = {Journal of atherosclerosis and thrombosis},
volume = {},
number = {},
pages = {},
doi = {10.5551/jat.RV17065},
pmid = {35466160},
issn = {1880-3873},
abstract = {Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder with extreme elevations of low-density lipoprotein cholesterol (LDL-C) leading to premature atherosclerotic cardiovascular disease (ASCVD) as early as in childhood. Management of HoFH centers around aggressive and adequate reduction of LDL-C levels to slow the trajectory of ASCVD development. Historically, lowering LDL-C levels in HoFH has been challenging because of both the markedly elevated LDL-C levels (often ＞400 mg/dL) and reduced response to treatment options, such as statins, for which the mechanism of action requires a functional LDL receptor. However, the treatment landscape for HoFH has rapidly progressed over the last decade. While statins and ezetimibe remain first-line treatment, patients often require addition of multiple therapies to achieve goal LDL-C levels. The PCSK9 inhibitors are an important recent addition to the available treatment options, along with lomitapide, bile acid sequestrants, and, possibly, bempedoic acid. Additionally, ANGPTL3 has emerged as an important therapeutic target, with evinacumab being the first available ANGPTL3 inhibitor on the market for the treatment of patients with HoFH. For patients who cannot achieve adequate LDL-C reduction, lipoprotein apheresis may be necessary, with the added benefit of reducing lipoprotein(a) levels that carries an added risk if also elevated in patients with HoFH. Finally, gene therapy and genome editing using CRISPR/Cas-9 are moving through clinical development and may dramatically alter the future landscape of treatment for HoFH.},
}
@article {pmid35465826,
year = {2022},
author = {Ghosh, A and Myacheva, K and Riester, M and Schmidt, C and Diederichs, S},
title = {Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing.},
journal = {RNA biology},
volume = {19},
number = {1},
pages = {588-593},
doi = {10.1080/15476286.2022.2067713},
pmid = {35465826},
issn = {1555-8584},
mesh = {CRISPR-Cas Systems ; Chimera/metabolism ; DNA, Single-Stranded/genetics ; *Gene Editing/methods ; Oligonucleotides/genetics ; *RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; },
abstract = {The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.},
}
@article {pmid35461863,
year = {2022},
author = {Yadav, M and Atala, A and Lu, B},
title = {Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.},
journal = {International journal of biological macromolecules},
volume = {209},
number = {Pt A},
pages = {1260-1270},
doi = {10.1016/j.ijbiomac.2022.04.114},
pmid = {35461863},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression ; Gene Products, gag/genetics ; Oligonucleotides ; *RNA, Guide/genetics ; RNA, Messenger/genetics ; },
abstract = {Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.},
}
@article {pmid35461662,
year = {2022},
author = {Le, Y and Sun, J},
title = {CRISPR/Cas genome editing systems in thermophiles: Current status, associated challenges, and future perspectives.},
journal = {Advances in applied microbiology},
volume = {118},
number = {},
pages = {1-30},
doi = {10.1016/bs.aambs.2022.02.001},
pmid = {35461662},
issn = {0065-2164},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Metabolic Engineering ; },
abstract = {Thermophiles, offering an attractive and unique platform for a broad range of applications in biofuels and environment protections, have received a significant attention and growing interest from academy and industry. However, the exploration and exploitation of thermophilic organisms have been hampered by the lack of a powerful genome manipulation tool to improve production efficiency. At current, the clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated (Cas) system has been successfully exploited as a competent, simplistic, and powerful tool for genome engineering both in eukaryotes and prokaryotes. Indeed, with the significant efforts made in recent years, some thermostable Cas9 proteins have been well identified and characterized and further, some thermostable Cas9-based editing tools have been successfully established in some representative obligate thermophiles. In this regard, we reviewed the current status and its progress in CRISPR/Cas-based genome editing system towards a variety of thermophilic organisms. Despite the potentials of these progresses, multiple factors/barriers still have to be overcome and optimized for improving its editing efficiency in thermophiles. Some insights into the roles of thermostable CRISPR/Cas technologies for the metabolic engineering of thermophiles as a thermophilic microbial cell factory were also fully analyzed and discussed.},
}
@article {pmid35461452,
year = {2022},
author = {Bieluszewski, T and Szymanska-Lejman, M and Dziegielewski, W and Zhu, L and Ziolkowski, PA},
title = {Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2484},
number = {},
pages = {161-182},
pmid = {35461452},
issn = {1940-6029},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Seeds/genetics ; },
abstract = {Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.},
}
@article {pmid35461116,
year = {2022},
author = {Li, Y and Deng, F and Goldys, EM},
title = {A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics.},
journal = {Talanta},
volume = {246},
number = {},
pages = {123469},
doi = {10.1016/j.talanta.2022.123469},
pmid = {35461116},
issn = {1873-3573},
abstract = {Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL-1 (∼50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-γ and EGFR, under 3-4 h. It has a 103 times higher sensitivity compared to a commercial IFN-γ ELISA kit, but uses the same experimental scheme. The same 1 fg mL-1 sensitivity along with 6 logs of linear range was realized for IFN-γ detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges.},
}
@article {pmid35460444,
year = {2022},
author = {Das, D and Singha, DL and Paswan, RR and Chowdhury, N and Sharma, M and Reddy, PS and Chikkaputtaiah, C},
title = {Recent advancements in CRISPR/Cas technology for accelerated crop improvement.},
journal = {Planta},
volume = {255},
number = {5},
pages = {109},
pmid = {35460444},
issn = {1432-2048},
support = {MLP-0007//Council of Scientific and Industrial Research, India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Technology ; },
abstract = {MAIN CONCLUSION: Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to support global production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world's increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.},
}
@article {pmid35458632,
year = {2022},
author = {Vojnits, K and Nakanishi, M and Porras, D and Kim, Y and Feng, Z and Golubeva, D and Bhatia, M},
title = {Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {8},
pages = {},
pmid = {35458632},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Gene Editing/methods ; Genes, Reporter ; Green Fluorescent Proteins ; Humans ; *Pluripotent Stem Cells ; },
abstract = {Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC-OCT4-EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC-AAVS1-EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC-AAVS1-EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.},
}
@article {pmid35458562,
year = {2022},
author = {Liu, J and Tao, D and Chen, X and Shen, L and Zhu, L and Xu, B and Liu, H and Zhao, S and Li, X and Liu, X and Xie, S and Niu, L},
title = {Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.},
journal = {Viruses},
volume = {14},
number = {4},
pages = {},
pmid = {35458562},
issn = {1999-4915},
support = {No. 32072685//the Natural Science Foundation of China/ ; No. SCSZTD-2021-09//the Sichuan Science and Technology Support Program/ ; No. CARS-36-05B//the earmarked fund for China Agriculture Research System/ ; },
mesh = {Alphacoronavirus ; Animals ; CRISPR-Cas Systems ; *Coronavirus/genetics ; *Coronavirus Infections/diagnosis/genetics/veterinary ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; *Porcine epidemic diarrhea virus/genetics ; RNA-Directed DNA Polymerase/genetics ; Sensitivity and Specificity ; Swine ; *Swine Diseases ; },
abstract = {Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.},
}
@article {pmid35458523,
year = {2022},
author = {Calderón, K and Rojas-Neyra, A and Carbajal-Lévano, B and Luján-Valenzuela, L and Ticona, J and Isasi-Rivas, G and Montalvan, A and Criollo-Orozco, M and Huaccachi-Gonzáles, E and Tataje-Lavanda, L and Alvarez, KLF and Fernández-Sánchez, M and Fernández-Díaz, M and Tang, N and Yao, Y and Nair, V},
title = {A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.},
journal = {Viruses},
volume = {14},
number = {4},
pages = {},
pmid = {35458523},
issn = {1999-4915},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; Contract N° 038-INNOVATEPERU-PIEC1-2020//Innovate Peru Program of the Ministry of Production/ ; BBS/E/I/00007039//Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {Animals ; Antibodies, Viral ; CRISPR-Cas Systems ; Chickens ; Genotype ; Herpesvirus 1, Meleagrid/genetics ; *Herpesvirus 2, Gallid ; Integrases ; *Newcastle Disease/prevention &amp; control ; Newcastle disease virus/genetics ; *Poultry Diseases ; Vaccines, Synthetic/genetics ; *Viral Vaccines/genetics ; },
abstract = {In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.},
}
@article {pmid35457940,
year = {2022},
author = {Song, W and Zhang, T and Lin, H and Yang, Y and Zhao, G and Huang, X},
title = {Conventional and Microfluidic Methods for the Detection of Nucleic Acid of SARS-CoV-2.},
journal = {Micromachines},
volume = {13},
number = {4},
pages = {},
pmid = {35457940},
issn = {2072-666X},
abstract = {Nucleic acid testing (NAT) played a crucial role in containing the spread of SARS-CoV-2 during the epidemic. The gold standard technique, the quantitative real-time polymerase chain reaction (qRT-PCR) technique, is currently used by the government and medical boards to detect SARS-CoV-2. Due to the limitations of this technology, it is not capable of meeting the needs of large-scale rapid detection. To solve this problem, many new techniques for detecting nucleic acids of SARS-CoV-2 have been reported. Therefore, a review that systematically and comprehensively introduces and compares various detection technologies is needed. In this paper, we not only review the traditional NAT but also provide an overview of microfluidic-based NAT technologies and summarize and discuss the characteristics and development prospects of these techniques.},
}
@article {pmid35457271,
year = {2022},
author = {Zegeye, WA and Tsegaw, M and Zhang, Y and Cao, L},
title = {CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457271},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology-being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modified crops face. Developing transgenic-free edited plants without introducing foreign DNA has received regulatory approval in a number of countries. Several ongoing efforts from various countries are rapidly expanding to adopt the innovations. This review covers the mechanisms of CRISPR/Cas9, comparisons of CRISPR/Cas9 with other gene-editing technologies-including newly emerged Cas variants-and focuses on CRISPR/Cas9-targeted genes for rice crop improvement. We have further highlighted CRISPR/Cas9 vector construction model design and different bioinformatics tools for target site selection.},
}
@article {pmid35457234,
year = {2022},
author = {Rozov, SM and Permyakova, NV and Sidorchuk, YV and Deineko, EV},
title = {Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457234},
issn = {1422-0067},
support = {21-14-00091//Russian Science Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Genome, Plant ; Mammals/genetics ; Plants, Genetically Modified/genetics ; Recombinant Proteins/genetics ; Transgenes ; },
abstract = {Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic "safe harbors") using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of "euchromatization" of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.},
}
@article {pmid35457228,
year = {2022},
author = {López-Márquez, A and Morín, M and Fernández-Peñalver, S and Badosa, C and Hernández-Delgado, A and Natera-de Benito, D and Ortez, C and Nascimento, A and Grinberg, D and Balcells, S and Roldán, M and Moreno-Pelayo, MÁ and Jiménez-Mallebrera, C},
title = {CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457228},
issn = {1422-0067},
support = {PI19/0122//Instituto de Salud Carlos III/ ; IMP/00009//Instituto de Salud Carlos III/ ; ER19P5AC728/2021//Centre for Biomedical Network Research on Rare Diseases/ ; ACCI2018-02//Centre for Biomedical Network Research on Rare Diseases/ ; CAM, B2017/ BMD3721//Comunidad de Madrid/ ; },
mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; *Collagen Type VI/genetics/metabolism ; Extracellular Matrix/metabolism ; Fibroblasts/metabolism ; Humans ; Mutation ; },
abstract = {Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.},
}
@article {pmid35457099,
year = {2022},
author = {Vanhoye, X and Janin, A and Caillaud, A and Rimbert, A and Venet, F and Gossez, M and Dijk, W and Marmontel, O and Nony, S and Chatelain, C and Durand, C and Lindenbaum, P and Rieusset, J and Cariou, B and Moulin, P and Di Filippo, M},
title = {APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457099},
issn = {1422-0067},
support = {Young Researcher Project//Hospices Civils de Lyon/ ; ANR-16-RHUS-0007//Agence Nationale de la Recherche/ ; Fondation Recherche Médicale//Fondation Recherche Médicale/ ; },
mesh = {Apolipoproteins B/metabolism ; CRISPR-Cas Systems ; *Fatty Liver/genetics ; Humans ; *Hypobetalipoproteinemia, Familial, Apolipoprotein B/genetics ; *Hypobetalipoproteinemias/diagnosis/genetics/metabolism ; },
abstract = {Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.},
}
@article {pmid35456898,
year = {2022},
author = {Dunbar, T and Tsakirpaloglou, N and Septiningsih, EM and Thomson, MJ},
title = {Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35456898},
issn = {1422-0067},
support = {2020-67013-31811//United States Department of Agriculture/ ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA ; Gene Editing/methods ; Genome, Plant ; *Nanotubes, Carbon ; *Oryza/genetics ; Plant Leaves/genetics ; Plants/genetics ; Plants, Genetically Modified/genetics ; Plasmids/genetics ; Seeds/genetics ; },
abstract = {CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.},
}
@article {pmid35453874,
year = {2022},
author = {Li, Y and Shi, Z and Hu, A and Cui, J and Yang, K and Liu, Y and Deng, G and Zhu, C and Zhu, L},
title = {Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {4},
pages = {},
pmid = {35453874},
issn = {2075-4418},
support = {82002189//National Natural Science Foundation of China/ ; 2008085QF311//Natural Science Foundation of Anhui Province of China/ ; 202004d07020014//The Key Research and Development Program of Anhui Province of China/ ; 2018489, 2021442//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; YZJJ2020QN35, YZJJ2021QN03//Dean's Fund of Hefei Institute of Physical Science, China/ ; },
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.},
}
@article {pmid35453665,
year = {2022},
author = {Azlan, A and Rajasegaran, Y and Kang Zi, K and Rosli, AA and Yik, MY and Yusoff, NM and Heidenreich, O and Moses, EJ},
title = {Elucidating miRNA Function in Cancer Biology via the Molecular Genetics' Toolbox.},
journal = {Biomedicines},
volume = {10},
number = {4},
pages = {},
pmid = {35453665},
issn = {2227-9059},
support = {FRGS/1/2018/SKK08/USM/02/8 (203/CIPPT/6711672)//Ministry of Higher Education/ ; USM-RUI: 1001/CIPPT/8012265//Universiti Sains Malaysia/ ; },
abstract = {Micro-RNA (miRNAs) are short non-coding RNAs of about 18-20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments.},
}
@article {pmid35452778,
year = {2022},
author = {Ceasar, SA and Maharajan, T and Hillary, VE and Ajeesh Krishna, TP},
title = {Insights to improve the plant nutrient transport by CRISPR/Cas system.},
journal = {Biotechnology advances},
volume = {59},
number = {},
pages = {107963},
doi = {10.1016/j.biotechadv.2022.107963},
pmid = {35452778},
issn = {1873-1899},
abstract = {We need to improve food production to feed the ever growing world population especially in a changing climate. Nutrient deficiency in soils is one of the primary bottlenecks affecting the crop production both in developed and developing countries. Farmers are forced to apply synthetic fertilizers to improve the crop production to meet the demand. Understanding the mechanism of nutrient transport is helpful to improve the nutrient-use efficiency of crops and promote the sustainable agriculture. Many transporters involved in the acquisition, export and redistribution of nutrients in plants are characterized. In these studies, heterologous systems like yeast and Xenopus were most frequently used to study the transport function of plant nutrient transporters. CRIPSR/Cas system introduced recently has taken central stage for efficient genome editing in diverse organisms including plants. In this review, we discuss the key nutrient transporters involved in the acquisition and redistribution of nutrients from soil. We draw insights on the possible application CRISPR/Cas system for improving the nutrient transport in plants by engineering key residues of nutrient transporters, transcriptional regulation of nutrient transport signals, engineering motifs in promoters and transcription factors. CRISPR-based engineering of plant nutrient transport not only helps to study the process in native plants with conserved regulatory system but also aid to develop non-transgenic crops with better nutrient use-efficiency. This will reduce the application of synthetic fertilizers and promote the sustainable agriculture strengthening the food and nutrient security.},
}
@article {pmid35452274,
year = {2022},
author = {Anliker, B and Childs, L and Rau, J and Renner, M and Schüle, S and Schuessler-Lenz, M and Sebe, A},
title = {Regulatory Considerations for Clinical Trial Applications with CRISPR-Based Medicinal Products.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2021.0148},
pmid = {35452274},
issn = {2573-1602},
abstract = {Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.},
}
@article {pmid35452075,
year = {2022},
author = {Oh, SA and Senger, K and Madireddi, S and Akhmetzyanova, I and Ishizuka, IE and Tarighat, S and Lo, JH and Shaw, D and Haley, B and Rutz, S},
title = {High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA.},
journal = {The Journal of experimental medicine},
volume = {219},
number = {5},
pages = {},
pmid = {35452075},
issn = {1540-9538},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Humans ; Plasmids/genetics ; T-Lymphocytes ; },
abstract = {Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.},
}
@article {pmid35451949,
year = {2022},
author = {Han, X and Zhou, X and Pei, Z and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Yang, B and Chen, W},
title = {Characterization of CRISPR-Cas systems in Bifidobacterium breve.},
journal = {Microbial genomics},
volume = {8},
number = {4},
pages = {},
doi = {10.1099/mgen.0.000812},
pmid = {35451949},
issn = {2057-5858},
mesh = {*Bifidobacterium breve/genetics ; *CRISPR-Cas Systems/genetics ; DNA ; Sequence Analysis, DNA ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system is an important adaptive immune system for bacteria to resist foreign DNA infection, which has been widely used in genotyping and gene editing. To provide a theoretical basis for the application of the CRISPR-Cas system in Bifidobacterium breve, the occurrence and diversity of CRISPR-Cas systems were analysed in 150 B. breve strains. Specifically, 47 % (71/150) of B. breve genomes possessed the CRISPR-Cas system, and type I-C CRISPR-Cas system was the most widely distributed among those strains. The spacer sequences present in B. breve can be used as a genotyping marker. Additionally, the phage assembly-related proteins were important targets of the type I-C CRISPR-Cas system in B. breve, and the protospacer adjacent motif sequences were further characterized in B. breve type I-C system as 5'-TTC-3'. All these results might provide a molecular basis for the development of endogenous genome editing tools in B. breve.},
}
@article {pmid35451838,
year = {2022},
author = {Luo, T and Li, J and He, Y and Liu, H and Deng, Z and Long, X and Wan, Q and Ding, J and Gong, Z and Yang, Y and Zhong, S},
title = {Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.},
journal = {Analytical chemistry},
volume = {94},
number = {17},
pages = {6566-6573},
doi = {10.1021/acs.analchem.2c00401},
pmid = {35451838},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded/genetics ; *MicroRNAs/genetics ; },
abstract = {Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.},
}
@article {pmid35451132,
year = {2022},
author = {Kershanskaya, OI and Yessenbaeva, GL and Nelidova, DS and Karabekova, AN and Sadullaeva, ZN},
title = {CRISPR/Cas genome editing perspectives for barley breeding.},
journal = {Physiologia plantarum},
volume = {},
number = {},
pages = {e13686},
doi = {10.1111/ppl.13686},
pmid = {35451132},
issn = {1399-3054},
abstract = {The CRISPR/Cas9 technology shows potential to improve crop breeding efficiency and antiviral defense. The interest in DNA editing in crops has grown due to the possibility of increasing the resistance of different plants to many viruses. Our aim was to create an elite disease-resistant local barley cultivar using CRISPR/Cas9 biotechnology. For this purpose, we used CRISPR/Cas 9-eIF4E with the eukaryotic translation initiation factor 4E (eIF4E) barley gene to edit the genomes of five local Kazakhstan barley cultivars. After identifying the single guide RNA (sgRNA) target sequences, they were synthesized and cloned into the CRISPR-plant vector before being introduced into barley cells via our own patented Agrobacterium germ-line transformation technique. Barley plants eIF4E-modified were successfully obtained and were resistant to virus infection. Based on our research, the CRISPR/Cas9 system for plant genome editing could be a prospect for applying this breakthrough biotechnology in barley breeding. This article is protected by copyright. All rights reserved.},
}
@article {pmid35448780,
year = {2022},
author = {Haroon, M and Wang, X and Afzal, R and Zafar, MM and Idrees, F and Batool, M and Khan, AS and Imran, M},
title = {Novel Plant Breeding Techniques Shake Hands with Cereals to Increase Production.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {8},
pages = {},
pmid = {35448780},
issn = {2223-7747},
support = {2018JQ5218//National Natural Science Foundation of China/ ; },
abstract = {Cereals are the main source of human food on our planet. The ever-increasing food demand, continuously changing environment, and diseases of cereal crops have made adequate production a challenging task for feeding the ever-increasing population. Plant breeders are striving their hardest to increase production by manipulating conventional breeding methods based on the biology of plants, either self-pollinating or cross-pollinating. However, traditional approaches take a decade, space, and inputs in order to make crosses and release improved varieties. Recent advancements in genome editing tools (GETs) have increased the possibility of precise and rapid genome editing. New GETs such as CRISPR/Cas9, CRISPR/Cpf1, prime editing, base editing, dCas9 epigenetic modification, and several other transgene-free genome editing approaches are available to fill the lacuna of selection cycles and limited genetic diversity. Over the last few years, these technologies have led to revolutionary developments and researchers have quickly attained remarkable achievements. However, GETs are associated with various bottlenecks that prevent the scaling development of new varieties that can be dealt with by integrating the GETs with the improved conventional breeding methods such as speed breeding, which would take plant breeding to the next level. In this review, we have summarized all these traditional, molecular, and integrated approaches to speed up the breeding procedure of cereals.},
}
@article {pmid35448772,
year = {2022},
author = {Niazian, M and Belzile, F and Torkamaneh, D},
title = {CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {8},
pages = {},
pmid = {35448772},
issn = {2223-7747},
support = {6548//Genome Canada/ ; },
abstract = {Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene's function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective "in root" functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean.},
}
@article {pmid35447246,
year = {2022},
author = {Kanafi, MM and Tavallaei, M},
title = {Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases.},
journal = {Gene},
volume = {830},
number = {},
pages = {146518},
doi = {10.1016/j.gene.2022.146518},
pmid = {35447246},
issn = {1879-0038},
abstract = {Prokaryotes possess an adaptive immune system using various CRISPR associated (Cas) genes to make an archive of records from invading phages and eliminate them upon re-exposure when specialized Cas proteins cut foreign DNA into small pieces. On the basis of the different types of Cas proteins, CRISPR systems seen in some prokaryotic genomes, are different to each other. It has been proved that CRISPR has a great potential for genome engineering. Studies have also demonstrated that in comparison to the preceding genome engineering tools CRISPR/Cas systems can be harnessed as a flexible tool with easy multiplexing and scaling ability. Recent studies suggest that CRISPR/Cas systems can also be used for non-genome engineering roles. Isolation and identification of new Cas proteins or modification of existing ones are effectively increasing the number of CRISPR applications and helps its development. D10A and H840A mutations at RuvC and HNH endonuclease domains of wild type Streptococcus pyogenes Cas9 (SpCas9) respectively creates a nuclease, dead Cas9 (dCas9) molecule, that does not cut target DNA but still retains its capability for binding to target DNA based on the gRNA targeting sequence. In this article we review the potentials of this enzyme, dCas9, toward development of the applications of CRISPR/dCas9 technology in fields such as; visualization of genomic loci, disease diagnosis and transcriptional repression and activation.},
}
@article {pmid35446558,
year = {2022},
author = {Chen, Z and Devi, G and Arif, A and Zamore, PD and Sontheimer, EJ and Watts, JK},
title = {Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.2c00116},
pmid = {35446558},
issn = {1554-8937},
abstract = {CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.},
}
@article {pmid35446391,
year = {2022},
author = {Lind Gleerup, J and Mogensen, TH},
title = {CRISPR-Cas in diagnostics and therapy of infectious diseases.},
journal = {The Journal of infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1093/infdis/jiac145},
pmid = {35446391},
issn = {1537-6613},
abstract = {Infectious diseases are a major threat to the global health. The rise in antimicrobial resistant organisms, incurable chronic infections and an increasing demand for rapid accurate diagnostics have prompted researchers to experiment with new approaches. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein is a naturally occurring adaptive immune system in bacteria, that has been developed as a tool for performing genomic alterations in any genome of interest, including humans and microbes. Accordingly, several studies have been conducted to investigate how the technology can be utilized in infectious diseases to improve diagnostics, disrupt antimicrobial resistance, and cure chronic infections. This review will provide an overview of the CRISPR-Cas system, and how it has been applied in studies on infectious diseases. The review will also investigate the current challenges of the technology and the improvements that are needed for the platform to be adopted for clinical use in patients.},
}
@article {pmid35445018,
year = {2022},
author = {Hu, Y and Li, W},
title = {Development and Application of CRISPR-Cas Based Tools.},
journal = {Frontiers in cell and developmental biology},
volume = {10},
number = {},
pages = {834646},
pmid = {35445018},
issn = {2296-634X},
abstract = {Abundant CRISPR-Cas systems in nature provide us with unlimited valuable resources to develop a variety of versatile tools, which are powerful weapons in biological discovery and disease treatment. Here, we systematically review the development of CRISPR-Cas based tools from DNA nuclease to RNA nuclease, from nuclease dependent-tools to nucleic acid recognition dependent-tools. Also, considering the limitations and challenges of current CRISPR-Cas based tools, we discuss the potential directions for development of novel CRISPR toolkits in the future.},
}
@article {pmid35442747,
year = {2022},
author = {Zou, Y and Sun, X and Yang, Q and Zheng, M and Shimoni, O and Ruan, W and Wang, Y and Zhang, D and Yin, J and Huang, X and Tao, W and Park, JB and Liang, XJ and Leong, KW and Shi, B},
title = {Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.},
journal = {Science advances},
volume = {8},
number = {16},
pages = {eabm8011},
pmid = {35442747},
issn = {2375-2548},
mesh = {Animals ; Blood-Brain Barrier ; CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Glioblastoma/genetics/therapy ; Mice ; *Nanocapsules ; RNA, Guide/genetics ; },
abstract = {We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.},
}
@article {pmid35440677,
year = {2022},
author = {de Souza Pacheco, I and Doss, AA and Vindiola, BG and Brown, DJ and Ettinger, CL and Stajich, JE and Redak, RA and Walling, LL and Atkinson, PW},
title = {Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {6428},
pmid = {35440677},
issn = {2045-2322},
support = {01170-002//California Department of Food and Agriculture/ ; 012604-002//Animal and Plant Health Inspection Service/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Hemiptera/genetics ; Pigmentation/genetics ; },
abstract = {CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.},
}
@article {pmid35440579,
year = {2022},
author = {de Rooij, MFM and Thus, YJ and Swier, N and Beijersbergen, RL and Pals, ST and Spaargaren, M},
title = {A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2136},
pmid = {35440579},
issn = {2041-1723},
support = {12539//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 7873//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 10275//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {Agammaglobulinaemia Tyrosine Kinase/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Adhesion/genetics ; Humans ; Integrins/metabolism ; *Leukemia/drug therapy ; Protein Kinase Inhibitors/therapeutic use ; Signal Transduction ; Tumor Microenvironment ; },
abstract = {The clinical introduction of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which targets B-cell antigen-receptor (BCR)-controlled integrin-mediated retention of malignant B cells in their growth-supportive lymphoid organ microenvironment, provided a major breakthrough in lymphoma and leukemia treatment. Unfortunately, a significant subset of patients is intrinsically resistant or acquires resistance against ibrutinib. Here, to discover novel therapeutic targets, we present an unbiased loss-of-adhesion CRISPR-Cas9 knockout screening method to identify proteins involved in BCR-controlled integrin-mediated adhesion. Illustrating the validity of our approach, several kinases with an established role in BCR-controlled adhesion, including BTK and PI3K, both targets for clinically applied inhibitors, are among the top hits of our screen. We anticipate that pharmacological inhibitors of the identified targets, e.g. PAK2 and PTK2B/PYK2, may have great clinical potential as therapy for lymphoma and leukemia patients. Furthermore, this screening platform is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.},
}
@article {pmid35440051,
year = {2022},
author = {Tao, R and Wang, Y and Hu, Y and Jiao, Y and Zhou, L and Jiang, L and Li, L and He, X and Li, M and Yu, Y and Chen, Q and Yao, S},
title = {WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {108},
pmid = {35440051},
issn = {2059-3635},
support = {No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No.81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.},
}
@article {pmid35438517,
year = {2022},
author = {Sherkow, JS},
title = {Immaculate Conception? Priority and Invention in the CRISPR Patent Dispute.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {174-180},
doi = {10.1089/crispr.2022.0033},
pmid = {35438517},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Dissent and Disputes ; *Gene Editing ; Inventions ; RNA, Guide/genetics ; },
abstract = {The U.S. Patent Trial and Appeal Board (PTAB), in an interference proceeding decided in February 2022, concluded that researchers at the Broad Institute (Cambridge, MA) were the first to "conceive" of using single-guide RNA CRISPR-Cas9 genome editing in eukaryotic cells in 2012. The PTAB reached this verdict even though competing researchers at the University of California, Berkeley, among other institutions, could document the idea 7 months earlier. Understanding the basis for the PTAB's decision turns on patent law's particular "conception" requirement. In this study, I explain that requirement, detail the PTAB's interference decision, and discuss the decision's practical effects on CRISPR technology and routine science.},
}
@article {pmid35438515,
year = {2022},
author = {Zuo, Z and Babu, K and Ganguly, C and Zolekar, A and Newsom, S and Rajan, R and Wang, YC and Liu, J},
title = {Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {329-340},
doi = {10.1089/crispr.2021.0076},
pmid = {35438515},
issn = {2573-1602},
support = {R15 HL147265/HL/NHLBI NIH HHS/United States ; P20 GM103640/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; Endonucleases/genetics ; *Gene Editing ; Humans ; RNA/chemistry ; },
abstract = {The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.},
}
@article {pmid35438514,
year = {2022},
author = {Sun, W and Wang, Y},
title = {SuperFi-Cas9: High Fidelity Meets High Activity.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {171-173},
doi = {10.1089/crispr.2022.29146.ywa},
pmid = {35438514},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide ; },
}
@article {pmid35438513,
year = {2022},
author = {Barrangou, R},
title = {CRISPR à la carte.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {170},
doi = {10.1089/crispr.2022.29147.rba},
pmid = {35438513},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid35437598,
year = {2022},
author = {Yagita, Y and Abe, Y and Fujiki, Y},
title = {De novo formation and maintenance of mammalian peroxisomes in cultured PEX16-knockout cells generated by CRISPR/Cas9.},
journal = {Journal of cell science},
volume = {135},
number = {9},
pages = {},
doi = {10.1242/jcs.258377},
pmid = {35437598},
issn = {1477-9137},
support = {JP26116007//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; //Naito Foundation/ ; //Japan Foundation for Applied Enzymology/ ; //Novartis Foundation (Japan) for the Promotion of Science/ ; JP19K07386//Ministry of Education, Culture, Sports, Science, and Technology/ ; JP19K07386//Japan Society for the Promotion of Science/ ; //Novartis Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; Intracellular Membranes/metabolism ; Mammals/metabolism ; Membrane Proteins/metabolism ; *Peroxisomes/metabolism ; },
abstract = {Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.},
}
@article {pmid35435904,
year = {2022},
author = {Hu, C and Doerksen, T and Bugbee, T and Wallace, NA and Palinski, R},
title = {Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {181},
pages = {},
doi = {10.3791/62583},
pmid = {35435904},
issn = {1940-087X},
support = {P20 GM130448/GM/NIGMS NIH HHS/United States ; R15 CA242057/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; High-Throughput Nucleotide Sequencing ; Mutation ; },
abstract = {Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.},
}
@article {pmid35435766,
year = {2022},
author = {Vaca, DJ and Thibau, A and Leisegang, MS and Malmström, J and Linke, D and Eble, JA and Ballhorn, W and Schaller, M and Happonen, L and Kempf, VAJ},
title = {Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0059822},
doi = {10.1128/spectrum.00598-22},
pmid = {35435766},
issn = {2165-0497},
abstract = {Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.},
}
@article {pmid35433512,
year = {2022},
author = {Lu, P and Chen, J and Li, Z and Li, Z and Zhang, J and Kan, B and Pang, B},
title = {Visual Identification and Serotyping of Toxigenic Vibrio cholerae Serogroups O1 and O139 With CARID.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {863435},
pmid = {35433512},
issn = {2235-2988},
mesh = {*Cholera/diagnosis/microbiology ; Cholera Toxin ; Humans ; Serogroup ; Serotyping ; *Vibrio cholerae O1/genetics ; },
abstract = {There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The results can be determined by fluorescence signal and visualized by lateral flow dipstick. We identified 154 V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. The limit of detection of CARID was 20 copies/reaction of V. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. Simulated sample tests showed that CARID is suitable for complex samples. In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. cholerae, which makes it suitable for field responses to cholera.},
}
@article {pmid35432328,
year = {2022},
author = {Moraes, L and Trentini, MM and Fousteris, D and Eto, SF and Chudzinski-Tavassi, AM and Leite, LCC and Kanno, AI},
title = {CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {867195},
pmid = {35432328},
issn = {1664-3224},
mesh = {Adjuvants, Immunologic ; Adjuvants, Pharmaceutic ; Animals ; BCG Vaccine/genetics ; CRISPR-Cas Systems ; Escherichia coli ; Mice ; *Tuberculosis ; *Tuberculosis Vaccines/genetics ; },
abstract = {Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.},
}
@article {pmid35430708,
year = {2022},
author = {Bhoria, S and Yadav, J and Yadav, H and Chaudhary, D and Jaiwal, R and Jaiwal, PK},
title = {Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus.},
journal = {Biotechnology letters},
volume = {},
number = {},
pages = {},
pmid = {35430708},
issn = {1573-6776},
abstract = {Diabetes mellitus is the most prevalent deadly disease caused by the destruction and dysfunction of pancreatic β cells that consequentially increased blood glucose levels. The management of this disease via external administration of insulin/insulin analogs has been difficult and challenging due to their limited production and accessibility at affordable prices. The conventional insulin production platforms (Escherichia coli, Saccharomyces cerevisiae and mammalian cell lines) with limited scalability and high upstream process costs have not been successful in meeting the rapidly increasing insulin demands. However, plants have been used as safe, scalable, environmentally friendly and cost-effective high capacity production platforms for recombinant orally delivered insulin. Recent technological advances in genome engineering and editing technologies for adequate insulin and insulin analogs production, renewable cellular sources of insulin through transplantation of islets or insulin-producing cells and reprogramming or differentiation of non β cells into β-like cells, used either alone or in combination, for diabetes containment are reviewed here along with their future prospects.},
}
@article {pmid35430408,
year = {2022},
author = {Moon, J and Song, J and Jang, H and Kang, H and Huh, YM and Son, HY and Rho, HW and Park, M and Talwar, CS and Park, KH and Woo, E and Lim, J and Lim, EK and Jung, J and Jung, Y and Park, HG and Kang, T},
title = {Ligation-free isothermal nucleic acid amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {209},
number = {},
pages = {114256},
doi = {10.1016/j.bios.2022.114256},
pmid = {35430408},
issn = {1873-4235},
mesh = {Animals ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Mice ; Nucleic Acid Amplification Techniques/methods ; RNA ; RNA, Messenger ; },
abstract = {In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.},
}
@article {pmid35429769,
year = {2022},
author = {Pan, Y and Luan, X and Zeng, F and Xu, Q and Li, Z and Gao, Y and Liu, X and Li, X and Han, X and Shen, J and Song, Y},
title = {Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging.},
journal = {Biosensors &amp; bioelectronics},
volume = {209},
number = {},
pages = {114239},
doi = {10.1016/j.bios.2022.114239},
pmid = {35429769},
issn = {1873-4235},
mesh = {Adenosine Triphosphate ; Animals ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Gene Editing/methods ; *Metal-Organic Frameworks ; Oligonucleotides ; },
abstract = {In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.},
}
@article {pmid35429445,
year = {2022},
author = {Chen, Q and Wen, Y},
title = {Target recognition triggered CRISPR-Cas12a assisted allosteric scaffold for sensitively analyzing bacterial infection after dental implantation.},
journal = {Analytical biochemistry},
volume = {647},
number = {},
pages = {114666},
doi = {10.1016/j.ab.2022.114666},
pmid = {35429445},
issn = {1096-0309},
mesh = {*Bacterial Infections/diagnosis/genetics ; *CRISPR-Cas Systems ; DNA ; Dental Implantation ; Humans ; Limit of Detection ; },
abstract = {Accurate identification and sensitive quantification of infected bacteria after dental implant both play crucial roles in early-diagnosis of bacterial infection and guiding medicine applications. Herein, we propose a sensitive and accurate bacteria detection method based on CRISPR-Cas12a system assisted allosteric scaffold. In the method, the allosteric scaffold takes the responsibility of specifically identifying target bacteria and inducing CRISPR-Cas12a based signal amplification. Eventually, the method exhibits a wide detection range from 6 × 106 cfu/mL to 6 × 102 cfu/mL with the limit of detection (LOD) of 47 cfu/mL. Furthermore, the established approach also possesses a high specificity due to high selectivity of aptamer and robust accuracy in recognizing double strand DNA by CRISPR-Cas12a system. We believe that this work can provide new strategies in the field of diagnosing bacterial infections after dental implantation.},
}
@article {pmid35427904,
year = {2022},
author = {Lee, HM and Kim, AH and Hwang, S and Jung, J and Seol, H and Sung, JJ and Jeong, SM and Choi, YM and Jun, JK and Kim, HS and Jang, J},
title = {Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102779},
doi = {10.1016/j.scr.2022.102779},
pmid = {35427904},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Gene Targeting ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Myosin Heavy Chains/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.},
}
@article {pmid35427438,
year = {2022},
author = {Kelkar, A and Groth, T and Neelamegham, S},
title = {Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library.},
journal = {Current protocols},
volume = {2},
number = {4},
pages = {e402},
doi = {10.1002/cpz1.402},
pmid = {35427438},
issn = {2691-1299},
support = {HL103411/GF/NIH HHS/United States ; GM133195/GF/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; *Genetic Testing ; Glycosylation ; Humans ; Lectins/genetics ; },
abstract = {CRISPR-Cas9-based forward genetic screens represent a powerful discovery platform to uncover genes regulating specific biological processes. This article describes a method for utilizing a freely available GlycoGene CRISPR library to knock out any gene participating in human glycosylation in arbitrary cell types. The end product is a stable GlycoGene CRISPR knockout cell library, where each cell contains one or more sgRNA and lacks corresponding function. The cell library can be screened using various lectin/antibody reagents. It can also be applied in functional assays to establish glycan structure-glycogene-glycopathway relationships. This is a powerful systems glycobiology strategy for dissecting glycosylation pathways and processes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Scale-up and NGS validation of the GlycoGene CRISPR plasmid library Basic Protocol 2: Preparation of a GlycoGene CRISPR lentivirus pool and an isogenic cell line stably expressing Cas9 nuclease Basic Protocol 3: Preparation of a GlycoGene CRISPR cell library, self-inactivation of Cas9, and library validation by NGS Basic Protocol 4: Enrichment of lectin-binding or non-binding cells and related multiplex NGS data acquisition Basic Protocol 5: Bioinformatics pathway analysis.},
}
@article {pmid35426306,
year = {2022},
author = {Gao, ZF and Zheng, LL and Dong, LM and Li, JZ and Shen, Y and Chen, P and Xia, F},
title = {Label-Free Resonance Rayleigh Scattering Amplification for Lipopolysaccharide Detection and Logical Circuit by CRISPR/Cas12a-Driven Guanine Nanowire Assisted Non-Cross-Linking Hybridization Chain Reaction.},
journal = {Analytical chemistry},
volume = {94},
number = {16},
pages = {6371-6379},
doi = {10.1021/acs.analchem.2c00848},
pmid = {35426306},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; Guanine ; Lipopolysaccharides ; Logic ; *Nanowires ; },
abstract = {Although the CRISPR/Cas system has pioneered a new generation of analytical techniques, there remain many challenges in developing a label-free, accurate, and reliable CRISPR/Cas-based assay for reporting the levels of low abundance biomolecules in complex biological samples. Here, we reported a novel CRISPR-derived resonance Rayleigh scattering (RRS) amplification strategy and logical circuit based on a guanine nanowire (G-wire) assisted non-cross-linking hybridization chain reaction (GWancHCR) for label-free detection of lipopolysaccharide (LPS). In the presence of a target, the protospacer-adjacent motif-inserted aptamer is rationally designed to specifically combine with LPS rather than Cas12a, suppressing the trans-cleavage activity of CRISPR/Cas12a and retaining the reporter probes to trigger non-cross-linking aggregation. Owing to the automatic hybridization chain reaction (HCR), in the presence of Mg2+, the released G-quadruplex sequence aggregated to assemble the G-wire superstructure through non-cross-linking. As a result, a dramatically amplified RRS intensity is observed, allowing for reporting LPS levels in a low detection limit of 0.17 pg/mL and a wide linear range among 1.0-100.0 ng/mL. Moreover, this reaction event is capable of programming to perform classical Boolean logic tree analysis, including basic logic computing and complex integrated logic circuits. This study comprehensively analyzed with respect to information flow, matter (molecular events), and energy (RRS), revealing the potential promise in designing of molecular-level "Internet of Things", intelligent computing, and sensing systems.},
}
@article {pmid35422794,
year = {2022},
author = {Kamruzzaman, M and Yan, A and Castro-Escarpulli, G},
title = {Editorial: CRISPR-Cas Systems in Bacteria and Archaea.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {887778},
pmid = {35422794},
issn = {1664-302X},
}
@article {pmid35422517,
year = {2022},
author = {Sinan, S and Russell, R},
title = {A tweak and a peek: How Cas9 pries open double-stranded DNA to check its sequence.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {4},
pages = {286-288},
pmid = {35422517},
issn = {1545-9985},
mesh = {*CRISPR-Cas Systems ; *DNA ; },
}
@article {pmid35422516,
year = {2022},
author = {Cofsky, JC and Soczek, KM and Knott, GJ and Nogales, E and Doudna, JA},
title = {CRISPR-Cas9 bends and twists DNA to read its sequence.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {4},
pages = {395-402},
pmid = {35422516},
issn = {1545-9985},
support = {U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/metabolism ; Endonucleases/metabolism ; Gene Editing ; *RNA, Guide/metabolism ; },
abstract = {In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.},
}
@article {pmid35422284,
year = {2022},
author = {Li, Q and Lv, X and Tang, C and Yin, C},
title = {Co-delivery of doxorubicin and CRISPR/Cas9 or RNAi-expressing plasmid by chitosan-based nanoparticle for cancer therapy.},
journal = {Carbohydrate polymers},
volume = {287},
number = {},
pages = {119315},
doi = {10.1016/j.carbpol.2022.119315},
pmid = {35422284},
issn = {1879-1344},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Chitosan ; Doxorubicin/pharmacology ; Drug Carriers ; Drug Delivery Systems/methods ; Folic Acid ; *Frontotemporal Dementia ; Humans ; *Nanoparticles ; *Neoplasms/drug therapy/genetics ; Plasmids/genetics ; RNA Interference ; Survivin/genetics/metabolism ; },
abstract = {Folic acid (FA) and 2-(Diisopropylamino) ethyl methacrylate (DPA) double grafted trimethyl chitosan (TMC) nanoparticles (FTD NPs) were synthesized for the co-delivery of doxorubicin (DOX) and Survivin CRISPR/Cas9-expressing plasmid (sgSurvivin pDNA) or Survivin shRNA-expressing plasmid (iSur pDNA). FA modification enhanced the uptake of DOX and pDNA loaded into FTD NPs in tumor cells. A rapid release of DOX was triggered under acidic conditions due to pH-sensitiveness of FTD NPs arising from DPA conjugation. Negligible differences between FTD/sgSurvivin pDNA NPs and FTD/iSur pDNA NPs demonstrated that RNA interference (RNAi) and CRISPR/Cas9 technologies possessed comparable antitumor efficiency. Notably, the in vitro and in vivo antitumor efficacies of FTD/DOX/sgSurvivin pDNA NPs were superior to those of single delivery of DOX or sgSurvivin pDNA, while were comparable to those of FTD/DOX/iSur pDNA NPs. These results suggested that the combination of chemotherapeutics and CRISPR/Cas9 systems would provide a potential modality for cancer therapy.},
}
@article {pmid35421847,
year = {2022},
author = {Zhang, F and Meier, AB and Sinnecker, D and Engelhardt, S and Lipp, P and Laugwitz, KL and Dorn, T and Moretti, A},
title = {Generation of heterozygous (MRli003-A-5) and homozygous (MRli003-A-6) voltage-sensing knock-in human iPSC lines by CRISPR/Cas9 editing of the AAVS1 locus.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102785},
doi = {10.1016/j.scr.2022.102785},
pmid = {35421847},
issn = {1876-7753},
support = {788381/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; },
abstract = {Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.},
}
@article {pmid35421656,
year = {2022},
author = {Fu, R and Wang, Y and Liu, Y and Liu, H and Zhao, Q and Zhang, Y and Wang, C and Li, Z and Jiao, B and He, Y},
title = {CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.},
journal = {Food chemistry},
volume = {387},
number = {},
pages = {132919},
doi = {10.1016/j.foodchem.2022.132919},
pmid = {35421656},
issn = {1873-7072},
mesh = {Acetylcholinesterase/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems ; *DNA, Catalytic ; Manganese Compounds ; Organophosphorus Compounds ; Oxides ; *Pesticides ; },
abstract = {Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.},
}
@article {pmid35421352,
year = {2022},
author = {Bari, SMN and Chou-Zheng, L and Howell, O and Hossain, M and Hill, CM and Boyle, TA and Cater, K and Dandu, VS and Thomas, A and Aslan, B and Hatoum-Aslan, A},
title = {A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {4},
pages = {570-582.e7},
doi = {10.1016/j.chom.2022.03.001},
pmid = {35421352},
issn = {1934-6069},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Multifunctional Enzymes/genetics ; *Nucleic Acids ; Staphylococcus Phages/genetics ; },
abstract = {The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve antiphage defense. However, the prevalence and effectiveness of minimal, single-component systems that cleave DNA remain unknown. Here, we describe a unique mode of nucleic acid immunity mediated by a single enzyme with nuclease and helicase activities, herein referred to as Nhi (nuclease-helicase immunity). This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi targets and degrades phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in antiphage defense.},
}
@article {pmid35421333,
year = {2022},
author = {Huiting, E and Bondy-Denomy, J},
title = {A single bacterial enzyme i(NHI)bits phage DNA replication.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {4},
pages = {417-419},
doi = {10.1016/j.chom.2022.03.025},
pmid = {35421333},
issn = {1934-6069},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; DNA Helicases/genetics ; DNA Replication ; },
abstract = {In this issue of Cell Host &amp; Microbe, Nayeemul Bari et al. discover an anti-phage immune system in bacteria that uses a single enzyme to accomplish the challenging feat of detecting phage DNA and limiting its replication. Unlike CRISPR-Cas and restriction modification (R-M) systems, which use sequence motifs, nuclease-helicase immunity (Nhi) is proposed to target phage-specific replication intermediates.},
}
@article {pmid35420793,
year = {2022},
author = {Wang, J and Skeens, E and Arantes, PR and Maschietto, F and Allen, B and Kyro, GW and Lisi, GP and Palermo, G and Batista, VS},
title = {Structural Basis for Reduced Dynamics of Three Engineered HNH Endonuclease Lys-to-Ala Mutants for the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Associated 9 (CRISPR/Cas9) Enzyme.},
journal = {Biochemistry},
volume = {61},
number = {9},
pages = {785-794},
pmid = {35420793},
issn = {1520-4995},
support = {R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics ; *Endonucleases/chemistry ; },
abstract = {Many bacteria possess type-II immunity against invading phages or plasmids known as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system to detect and degrade the foreign DNA sequences. The Cas9 protein has two endonucleases responsible for double-strand breaks (the HNH domain for cleaving the target strand of DNA duplexes and RuvC domain for the nontarget strand, respectively) and a single-guide RNA-binding domain where the RNA and target DNA strands are base-paired. Three engineered single Lys-to-Ala HNH mutants (K810A, K848A, and K855A) exhibit an enhanced substrate specificity for cleavage of the target DNA strand. We report in this study that in the wild-type (wt) enzyme, D835, Y836, and D837 within the Y836-containing loop (comprising E827-D837) adjacent to the catalytic site have uncharacterizable broadened 1H15N nuclear magnetic resonance (NMR) features, whereas remaining residues in the loop have different extents of broadened NMR spectra. We find that this loop in the wt enzyme exhibits three distinct conformations over the duration of the molecular dynamics simulations, whereas the three Lys-to-Ala mutants retain only one conformation. The versatility of multiple alternate conformations of this loop in the wt enzyme could help to recruit noncognate DNA substrates into the HNH active site for cleavage, thereby reducing its substrate specificity relative to the three mutants. Our study provides further experimental and computational evidence that Lys-to-Ala substitutions reduce dynamics of proteins and thus increase their stability.},
}
@article {pmid35419381,
year = {2022},
author = {Happi Mbakam, C and Lamothe, G and Tremblay, JP},
title = {Therapeutic Strategies for Dystrophin Replacement in Duchenne Muscular Dystrophy.},
journal = {Frontiers in medicine},
volume = {9},
number = {},
pages = {859930},
pmid = {35419381},
issn = {2296-858X},
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease characterized by progressive muscle wasting due to modifications in the DMD gene (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) that result in a lack of functional dystrophin expression. Many therapeutic approaches have so far been attempted to induce dystrophin expression and improve the patient phenotype. In this manuscript, we describe the relevant updates for some therapeutic strategies for DMD aiming to restore dystrophin expression. We also present and analyze in vitro and in vivo ongoing experimental approaches to treat the disease.},
}
@article {pmid35418239,
year = {2022},
author = {Orazi, G and Collins, AJ and Whitaker, RJ},
title = {Prediction of Prophages and Their Host Ranges in Pathogenic and Commensal Neisseria Species.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0008322},
doi = {10.1128/msystems.00083-22},
pmid = {35418239},
issn = {2379-5077},
abstract = {The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one another, primarily via transformation and homologous recombination and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified, and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1,302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from prophages and other MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE Drug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) are known to infect Neisseria species. In this study, we identified a large number of candidate phages integrated in the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.},
}
@article {pmid35415942,
year = {2022},
author = {Bao, YY and Zhong, JT and Shen, LF and Dai, LB and Zhou, SH and Fan, J and Yao, HT and Lu, ZJ},
title = {Effect of Glut-1 and HIF-1α double knockout by CRISPR/CAS9 on radiosensitivity in laryngeal carcinoma via the PI3K/Akt/mTOR pathway.},
journal = {Journal of cellular and molecular medicine},
volume = {26},
number = {10},
pages = {2881-2894},
doi = {10.1111/jcmm.17303},
pmid = {35415942},
issn = {1582-4934},
support = {81372903//National Natural Science Foundation of China/ ; 2016C33144//Science and Technology Department of Zhejiang Province, China/ ; 2016ZA127//Zhejiang TCM Science and technology program of Zhejiang provincial administration of traditional Chinese medicine/ ; },
mesh = {CRISPR-Cas Systems ; *Carcinoma ; Cell Line, Tumor ; Glucose ; Humans ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Laryngeal Neoplasms/genetics/metabolism/radiotherapy ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; Proto-Oncogene Proteins c-akt/genetics/metabolism ; Radiation Tolerance/genetics ; TOR Serine-Threonine Kinases/genetics/metabolism ; Wortmannin ; },
abstract = {Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of 18 F-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1α and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1α and/or Glut-1 in the presence of irradiation. HIF-1α and/or Glut-1 knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1α and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the PI3K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1α and Glut-1. HIF-1α and/or Glut-1 knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1α and/or Glut-1 knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.},
}
@article {pmid35414398,
year = {2022},
author = {López-Valls, M and Escalona-Noguero, C and Rodríguez-Díaz, C and Pardo, D and Castellanos, M and Milán-Rois, P and Martínez-Garay, C and Coloma, R and Abreu, M and Cantón, R and Galán, JC and Miranda, R and Somoza, Á and Sot, B},
title = {CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles.},
journal = {Analytica chimica acta},
volume = {1205},
number = {},
pages = {339749},
pmid = {35414398},
issn = {1873-4324},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Gold ; Humans ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; },
abstract = {The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.},
}
@article {pmid35414381,
year = {2022},
author = {Sohail, M and Xie, S and Zhang, X and Li, B},
title = {Methodologies in visualizing the activation of CRISPR/Cas: The last mile in developing CRISPR-Based diagnostics and biosensing - A review.},
journal = {Analytica chimica acta},
volume = {1205},
number = {},
pages = {339541},
doi = {10.1016/j.aca.2022.339541},
pmid = {35414381},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Colorimetry ; Molecular Probes ; *Nucleic Acids ; },
abstract = {CRISPR/Cas-based analytical procedures have revolutionized the sensing platform to fulfill the requirements of the current era in terms of sensitivity, selectivity, robustness, user-friendly feature, and cost-effectiveness for the detection of nucleic acid as well as non-nucleic acid analytes. Molecular target monitoring and transduction of the signals is a crucial prerequisite for precise molecular sensing tools. Besides, the reporting systems have become the last milestone for fabricating Cas-based molecular probes to visualize the activation of CRISPR/Cas enzymes. In this review, we have highlighted various CRISPR/Cas reporters, their mechanisms, sensing strategies, merits, and demerits. Moreover, signal transducers, i.e. fluorescent, colorimetric, and electrochemical, have also been discussed in detail along with various sensing strategies to generate recordable signals. It was concluded that there is still a need to overcome issues offered by the reported sensing devices, such as off-target effect, target sequence limitation, multiplexed quantitative detection, the influence of the inhibitor, and reaction kinetic constraint. Additionally, it is required to make them available for commercial use by validating their stability, robustness, safety profile in an off-lab environment as most of the probes have been tested in the controlled atmosphere of the laboratories. We believe that this novel critical interpretation and summary will assist the researchers in designing and validating new CRISPR/Cas reporters and probes for practical applications on a commercial scale.},
}
@article {pmid35414240,
year = {2022},
author = {Liu, Y and Champer, J},
title = {Modelling homing suppression gene drive in haplodiploid organisms.},
journal = {Proceedings. Biological sciences},
volume = {289},
number = {1972},
pages = {20220320},
pmid = {35414240},
issn = {1471-2954},
mesh = {Alleles ; CRISPR-Cas Systems ; Female ; *Gene Drive Technology/methods ; Germ Cells ; Humans ; Male ; RNA, Guide/genetics ; },
abstract = {Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo owing to maternally deposited Cas9 and guide RNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly owing to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.},
}
@article {pmid35414130,
year = {2022},
author = {Tornabene, P and Ferla, R and Llado-Santaeularia, M and Centrulo, M and Dell'Anno, M and Esposito, F and Marrocco, E and Pone, E and Minopoli, R and Iodice, C and Nusco, E and Rossi, S and Lyubenova, H and Manfredi, A and Di Filippo, L and Iuliano, A and Torella, A and Piluso, G and Musacchia, F and Surace, EM and Cacchiarelli, D and Nigro, V and Auricchio, A},
title = {Therapeutic homology-independent targeted integration in retina and liver.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1963},
pmid = {35414130},
issn = {2041-1723},
support = {TA-GT-0619-0762-FED//Foundation Fighting Blindness (Foundation Fighting Blindness, Inc.)/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Dependovirus/genetics ; *Gene Editing/methods ; Genetic Vectors/genetics ; Liver ; Mice ; Retina/metabolism ; Swine ; },
abstract = {Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.},
}
@article {pmid35414049,
year = {2022},
author = {Shi, H and Xu, Y and Tian, N and Yang, M and Liang, FS},
title = {Inducible and reversible RNA N6-methyladenosine editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1958},
pmid = {35414049},
issn = {2041-1723},
support = {R21 CA247638/CA/NCI NIH HHS/United States ; R21CA247638//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Adenosine/analogs &amp; derivatives ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenesis, Genetic ; Humans ; *RNA/genetics ; },
abstract = {RNA modifications, including N6-methyladenosine (m6A), have been reported to regulate fundamental RNA processes and properties, and directly linked to various human diseases. Methods enabling temporal and transcript/locus-specific editing of specific RNA modifications are essential, but still limited, to dissect the dynamic and context-dependent functions of these epigenetic modifications. Here, we develop a chemically inducible and reversible RNA m6A modification editing platform integrating chemically induced proximity (CIP) and CRISPR methods. We show that m6A editing can be temporally controlled at specific sites of individual RNA transcripts by the addition or removal of the CIP inducer, abscisic acid (ABA), in the system. By incorporating a photo-caged ABA, a light-controlled version of m6A editing platform can be developed. We expect that this platform and strategy can be generally applied to edit other RNA modifications in addition to m6A.},
}
@article {pmid35414015,
year = {2022},
author = {Guo, LY and Bian, J and Davis, AE and Liu, P and Kempton, HR and Zhang, X and Chemparathy, A and Gu, B and Lin, X and Rane, DA and Xu, X and Jamiolkowski, RM and Hu, Y and Wang, S and Qi, LS},
title = {Multiplexed genome regulation in vivo with hyper-efficient Cas12a.},
journal = {Nature cell biology},
volume = {24},
number = {4},
pages = {590-600},
pmid = {35414015},
issn = {1476-4679},
support = {2046650//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; P30 EY026877/EY/NEI NIH HHS/United States ; T32 EY020485/EY/NEI NIH HHS/United States ; DISC2-12669//California Institute for Regenerative Medicine (CIRM)/ ; R01 EY028106/EY/NEI NIH HHS/United States ; R01 EY032518/EY/NEI NIH HHS/United States ; R01 EY023295/EY/NEI NIH HHS/United States ; P30-EY026877//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; R01 NS109990/NS/NINDS NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Mice ; RNA/metabolism ; },
abstract = {Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.},
}
@article {pmid35413320,
year = {2022},
author = {Chauhan, N and Saxena, K and Jain, U},
title = {Single molecule detection; from microscopy to sensors.},
journal = {International journal of biological macromolecules},
volume = {209},
number = {Pt A},
pages = {1389-1401},
doi = {10.1016/j.ijbiomac.2022.04.038},
pmid = {35413320},
issn = {1879-0003},
mesh = {*Biosensing Techniques/methods ; Microscopy ; *Nanopores ; Nanotechnology/methods ; },
abstract = {Single molecule detection is necessary to find out physical, chemical properties and their mechanism involved in the normal functioning of body cells. In this way, they can provide a new direction to the healthcare system. Various techniques have been developed and employed for their successful detection. Herein, we have emphasized various traditional methods as well as biosensing technology which offer single molecule sensitivity. The various methods including plasmonic resonance, nanopores, whispering gallery mode, Simoa assay and recognition tunneling are discussed in the initial part which has been followed by a discussion about biosensor-based detection. Plasmonic, SERS, CRISPR/Cas, and other types of biosensors are focused in this review and found to be highly sensitive for single molecule detection. This review provides an overview of progression in different techniques employed for single molecule detection.},
}
@article {pmid35412632,
year = {2022},
author = {Pan, X and Li, H and Zhang, X},
title = {TedSim: temporal dynamics simulation of single-cell RNA sequencing data and cell division history.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {4272-4288},
pmid = {35412632},
issn = {1362-4962},
support = {R35 GM143070/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Division/genetics ; Cell Lineage/genetics ; Sequence Analysis, RNA/methods ; *Single-Cell Analysis/methods ; },
abstract = {Recently, lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree and makes it possible to reconstruct ancestral cell types and trace the origin of each cell type. Meanwhile, trajectory inference methods are widely used to infer cell trajectories and pseudotime in a dynamic process using gene expression data of present-day cells. Here, we present TedSim (single-cell temporal dynamics simulator), which simulates the cell division events from the root cell to present-day cells, simultaneously generating two data modalities for each single cell: the lineage barcode and gene expression data. TedSim is a framework that connects the two problems: lineage tracing and trajectory inference. Using TedSim, we conducted analysis to show that (i) TedSim generates realistic gene expression and barcode data, as well as realistic relationships between these two data modalities; (ii) trajectory inference methods can recover the underlying cell state transition mechanism with balanced cell type compositions; and (iii) integrating gene expression and barcode data can provide more insights into the temporal dynamics in cell differentiation compared to using only one type of data, but better integration methods need to be developed.},
}
@article {pmid35412236,
year = {2022},
author = {Aksenova, V and Arnaoutov, A and Dasso, M},
title = {Analysis of Nucleoporin Function Using Inducible Degron Techniques.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2502},
number = {},
pages = {129-150},
pmid = {35412236},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Gene Targeting/methods ; *Indoleacetic Acids/pharmacology ; Nuclear Pore ; *Nuclear Pore Complex Proteins/chemistry/genetics/metabolism ; Proteins ; Proteolysis ; },
abstract = {Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.},
}
@article {pmid35411846,
year = {2022},
author = {Rangarajan, AA and Yilmaz, C and Schnetz, K},
title = {Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli.},
journal = {Microbiology (Reading, England)},
volume = {168},
number = {4},
pages = {},
doi = {10.1099/mic.0.001173},
pmid = {35411846},
issn = {1465-2080},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; *Escherichia coli/genetics ; Genetic Engineering/methods ; },
abstract = {Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.},
}
@article {pmid35411005,
year = {2022},
author = {Yang, L and Zhang, L and Yin, P and Ding, H and Xiao, Y and Zeng, J and Wang, W and Zhou, H and Wang, Q and Zhang, Y and Chen, Z and Yang, M and Feng, Y},
title = {Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1931},
pmid = {35411005},
issn = {2041-1723},
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Viral Proteins/metabolism ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.},
}
@article {pmid35410430,
year = {2022},
author = {Che, P and Wu, E and Simon, MK and Anand, A and Lowe, K and Gao, H and Sigmund, AL and Yang, M and Albertsen, MC and Gordon-Kamm, W and Jones, TJ},
title = {Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {344},
pmid = {35410430},
issn = {2399-3642},
mesh = {CRISPR-Cas Systems ; Edible Grain/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Regeneration/genetics ; *Sorghum/genetics ; },
abstract = {For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.},
}
@article {pmid35410423,
year = {2022},
author = {Liu, Y and Pinto, F and Wan, X and Yang, Z and Peng, S and Li, M and Cooper, JM and Xie, Z and French, CE and Wang, B},
title = {Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1937},
pmid = {35410423},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; MR/V035401/1/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; },
abstract = {In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.},
}
@article {pmid35410288,
year = {2022},
author = {Oh, Y and Lee, WJ and Hur, JK and Song, WJ and Lee, Y and Kim, H and Gwon, LW and Kim, YH and Park, YH and Kim, CH and Lim, KS and Song, BS and Huh, JW and Kim, SU and Jun, BH and Jung, C and Lee, SH},
title = {Expansion of the prime editing modality with Cas9 from Francisella novicida.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {92},
pmid = {35410288},
issn = {1474-760X},
support = {NRF-2019R1C1C1006603//National Research Foundation/ ; NRF-2020R1I1A2075393//National Research Foundation/ ; NRF-2021M3A9I4024452//National Research Foundation/ ; 20009707//Ministry of Trade, Industry and Energy/ ; 9991006929, KMDF_PR_20200901_ 0264//Ministry of Trade, Industry and Energy/ ; HY-2020//the research fund of Hanyang University/ ; KGM1051911//Korea Research Institute of Bioscience and Biotechnology/ ; KGM4252122//Korea Research Institute of Bioscience and Biotechnology/ ; KGM5382113//Korea Research Institute of Bioscience and Biotechnology/ ; KGM4562121//Korea Research Institute of Bioscience and Biotechnology/ ; KGM5282113//Korea Research Institute of Bioscience and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; Francisella ; *Gene Editing ; Humans ; RNA-Directed DNA Polymerase ; },
abstract = {Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.},
}
@article {pmid35409000,
year = {2022},
author = {Huang, L and Luo, J and Song, N and Gao, W and Zhu, L and Yao, W},
title = {CRISPR/Cas9-Mediated Knockout of miR-130b Affects Mono- and Polyunsaturated Fatty Acid Content via PPARG-PGC1α Axis in Goat Mammary Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {7},
pages = {},
pmid = {35409000},
issn = {1422-0067},
support = {31772575//National Natural Science Foundation of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/genetics/metabolism ; Mammary Glands, Animal/metabolism ; *MicroRNAs/genetics/metabolism ; PPAR gamma/metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics/metabolism ; Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; },
abstract = {MicroRNA (miRNA)-130b, as a regulator of lipid metabolism in adipose and mammary gland tissues, is actively involved in lipogenesis, but its endogenous role in fatty acid synthesis remains unclear. Here, we aimed to explore the function and underlying mechanism of miR-130b in fatty acid synthesis using the CRISPR/Cas9 system in primary goat mammary epithelial cells (GMEC). A single clone with deletion of 43 nucleotides showed a significant decrease in miR-130b-5p and miR-130b-3p abundances and an increase of target genes PGC1α and PPARG. In addition, knockout of miR-130b promoted triacylglycerol (TAG) and cholesterol accumulation, and decreased the proportion of monounsaturated fatty acids (MUFA) C16:1, C18:1 and polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4, C20:5, C22:6. Similarly, the abundance of fatty acid synthesis genes ACACA and FASN and transcription regulators SREBP1c and SREBP2 was elevated. Subsequently, interference with PPARG instead of PGC1α in knockout cells restored the effect of miR-130b knockout, suggesting that PPARG is responsible for miR-130b regulating fatty acid synthesis. Moreover, disrupting PPARG inhibits PGC1α transcription and translation. These results reveal that miR-130b directly targets the PPARG-PGC1α axis, to inhibit fatty acid synthesis in GMEC. In conclusion, miR-130b could be a potential molecular regulator for improving the beneficial fatty acids content in goat milk.},
}
@article {pmid35408979,
year = {2022},
author = {Tripathi, L and Ntui, VO and Tripathi, JN},
title = {Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing.},
journal = {International journal of molecular sciences},
volume = {23},
number = {7},
pages = {},
pmid = {35408979},
issn = {1422-0067},
support = {CGIAR Window//United States Agency for International Development (USAID)/ ; CRP-RTB//CGIAR Research Program on Roots, Tubers and Bananas/ ; },
mesh = {*Bacterial Infections/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Musa/genetics/microbiology ; Plant Diseases/genetics/microbiology/prevention &amp; control ; *Xanthomonas/genetics ; },
abstract = {Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article.},
}
@article {pmid35403388,
year = {2022},
author = {Pan, W and Cheng, Z and Han, Z and Yang, H and Zhang, W and Zhang, H},
title = {Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {4},
pages = {339-344},
pmid = {35403388},
issn = {1862-1783},
support = {ZR202103010168//the Excellent Youth Foundation of Shandong Scientific Committee/ ; 2021T140017//the Shandong Science and Technology Innovation Funds, and the China Postdoctoral Science Foundation/ ; },
mesh = {CRISPR-Cas Systems ; *Citrullus/genetics ; *Cucurbitaceae/genetics ; Gene Editing ; Plant Breeding ; Transformation, Genetic ; },
abstract = {Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.},
}
@article {pmid35400359,
year = {2022},
author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD},
title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002},
pmid = {35400359},
issn = {1009-2137},
mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; },
abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.},
}
@article {pmid35398275,
year = {2022},
author = {Kreuter, J and Stark, G and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.},
journal = {Journal of biotechnology},
volume = {350},
number = {},
pages = {11-16},
doi = {10.1016/j.jbiotec.2022.03.017},
pmid = {35398275},
issn = {1873-4863},
mesh = {*Ascomycota/genetics ; Aureobasidium ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.},
}
@article {pmid35396804,
year = {2022},
author = {Raghav, D and Jyoti, A and Siddiqui, AJ and Saxena, J},
title = {Plant-associated endophytic fungi as potential bio-factories for extracellular enzymes: Progress, Challenges and Strain improvement with precision approaches.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jam.15574},
pmid = {35396804},
issn = {1365-2672},
abstract = {There is an intricate network of relations between endophytic fungi and their hosts that affects the production of various bioactive compounds. Plant-associated endophytic fungi contain industrially important enzymes and have the potential to fulfil their rapid demand in the international market to boost business in technology. Being safe and metabolically active, they have replaced the usage of toxic and harmful chemicals and hold a credible application in biotransformation, bioremediation and industrial processes. Despite these, there are limited reports on fungal endophytes that can directly cater to the demand and supply of industrially stable enzymes. The underlying reasons include low endogenous production and secretion of enzymes from fungal endophytes which have raised concern for widely accepted applications. Hence, it is imperative to augment the biosynthetic and secretory potential of fungal endophytes. Modern state-of-the-art biotechnological technologies aiming at strain improvement using cell factory engineering as well as precise gene editing like Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its Associated proteins (Cas) systems which can provide a boost in fungal endophyte enzyme production. Additionally, it is vital to characterize optimum conditions to grow one strain with multiple enzymes (OSME). The present review encompasses various plants-derived endophytic fungal enzymes and their applications in various sectors. Furthermore, we postulate the feasibility of new precision approaches with an aim for strain improvement and enhanced enzyme production.},
}
@article {pmid35396760,
year = {2022},
author = {Bhatt, A and Fatima, Z and Ruwali, M and Misra, CS and Rangu, SS and Rath, D and Rattan, A and Hameed, S},
title = {CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jam.15571},
pmid = {35396760},
issn = {1365-2672},
support = {BRNS/37080//Board of Research in Nuclear Sciences/ ; },
abstract = {AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved.<br><br>METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively.<br><br>CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis.<br><br>To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.},
}
@article {pmid35395997,
year = {2022},
author = {Yang, F and Jiang, M and Lin, ZH and Xie, ZL and Ma, ZN and Yang, L and Liu, H and Wang, ZY and Zhou, L},
title = {[Effects of the ITGA2B Nonsense Mutation (c.2659C &gt; T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {559-564},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.040},
pmid = {35395997},
issn = {1009-2137},
mesh = {Animals ; CRISPR-Cas Systems ; *Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics ; Humans ; *Integrin alpha2/genetics ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics ; RNA, Guide ; *Thrombasthenia/diagnosis/genetics ; Thrombin/genetics ; },
abstract = {OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.<br><br>METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet.<br><br>RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05).<br><br>CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.},
}
@article {pmid35395959,
year = {2022},
author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD},
title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {334-340},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002},
pmid = {35395959},
issn = {1009-2137},
mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; },
abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.},
}
@article {pmid35394860,
year = {2022},
author = {Mamontov, V and Martynov, A and Morozova, N and Bukatin, A and Staroverov, DB and Lukyanov, KA and Ispolatov, Y and Semenova, E and Severinov, K},
title = {Persistence of plasmids targeted by CRISPR interference in bacterial populations.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {15},
pages = {e2114905119},
doi = {10.1073/pnas.2114905119},
pmid = {35394860},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems/genetics/physiology ; *Escherichia coli/genetics ; Gene-Environment Interaction ; *Interspersed Repetitive Sequences/genetics ; Models, Genetic ; *Plasmids/genetics ; },
abstract = {SignificanceRNA-guided CRISPR-Cas nucleases efficiently protect bacterial cells from phage infection and plasmid transformation. Yet, the efficiency of CRISPR-Cas defense is not absolute. Mutations in either CRISPR-Cas components of the host or mobile genetic elements regions targeted by CRISPR-Cas inactivate the defensive action. Here, we show that even at conditions of active CRISPR-Cas and unaltered targeted plasmids, a kinetic equilibrium between CRISPR-Cas nucleases action and plasmid replication processes allows for existence of a small subpopulation of plasmid-bearing cells on the background of cells that have been cured from the plasmid. In nature, the observed diversification of phenotypes may allow rapid changes in the population structure to meet the demands of the environment.},
}
@article {pmid35390717,
year = {2022},
author = {Ivanov, AV and Safenkova, IV and Zherdev, AV and Dzantiev, BB},
title = {DIRECT2: A novel platform for a CRISPR-Cas12-based assay comprising universal DNA-IgG probe and a direct lateral flow test.},
journal = {Biosensors &amp; bioelectronics},
volume = {208},
number = {},
pages = {114227},
doi = {10.1016/j.bios.2022.114227},
pmid = {35390717},
issn = {1873-4235},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA Probes/genetics ; DNA, Single-Stranded ; Gold ; Immunoglobulin G ; *Metal Nanoparticles ; Mice ; },
abstract = {CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT2). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR-Cas12 activity detection and its bioanalytical applications.},
}
@article {pmid35389616,
year = {2022},
author = {Abdullah, and Wang, P and Han, T and Liu, W and Ren, W and Wu, Y and Xiao, Y},
title = {Adenine Base Editing System for Pseudomonas and Prediction Workflow for Protein Dysfunction via ABE.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1650-1657},
doi = {10.1021/acssynbio.2c00066},
pmid = {35389616},
issn = {2161-5063},
mesh = {Adenine/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Pseudomonas/genetics/metabolism ; Workflow ; },
abstract = {Pseudomonas is a large genus that inhabits diverse environments due to its distinct metabolic versatility. Its applications range from environmental to industrial biotechnology. Molecular tools that allow precise and efficient genetic manipulation are required to understand and harness its full potential. Here, we report the development of a highly efficient adenine base editing system, i.e., dxABE-PS, for Pseudomonas species. The system allows A:T → G:C transition with up to 100% efficiency along a broad target spectrum because we use xCas9 3.7, which recognizes NG PAM. To enhance the dxABE-PS utility, we develop a prediction workflow for protein dysfunction using ABE, namely, DABE-CSP (dysfunction via ABE through CRISPOR-SIFT prediction). We applied DABE-CSP to inactivate several genes in Pseudomonas putida KT2440 to accumulate a nylon precursor, i.e., muconic acid from catechol with 100% yield. Moreover, we expanded the ABE to non-model Pseudomonas species by developing an nxABE system for P. chengduensisDY56-96, isolated from sediment samples from the seamount area in the West Pacific Ocean. Taken together, the establishment of the ABE systems along with DABE-CSP will fast-track research on Pseudomonas species.},
}
@article {pmid35389262,
year = {2022},
author = {Martinez, MG and Combe, E and Inchauspe, A and Mangeot, PE and Delberghe, E and Chapus, F and Neveu, G and Alam, A and Carter, K and Testoni, B and Zoulim, F},
title = {CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants.},
journal = {mBio},
volume = {13},
number = {2},
pages = {e0288821},
pmid = {35389262},
issn = {2150-7511},
support = {ANR-10-LABX-61//Labex DevWeCan/ ; },
mesh = {CRISPR-Cas Systems ; DNA, Circular/genetics ; DNA, Viral/genetics ; *Hepatitis B virus/genetics ; *Hepatitis B, Chronic/drug therapy ; Humans ; RNA, Guide/genetics ; },
abstract = {Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. IMPORTANCE Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.},
}
@article {pmid35389256,
year = {2022},
author = {Govindarajan, S and Borges, A and Karambelkar, S and Bondy-Denomy, J},
title = {Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria.},
journal = {Journal of bacteriology},
volume = {204},
number = {5},
pages = {e0010522},
doi = {10.1128/jb.00105-22},
pmid = {35389256},
issn = {1098-5530},
support = {DP5-OD021344//NIH Director's Early Independence award/ ; //Sandler Foundation/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex. IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.},
}
@article {pmid35389053,
year = {2022},
author = {Töpfer, R and Trapp, O},
title = {A cool climate perspective on grapevine breeding: climate change and sustainability are driving forces for changing varieties in a traditional market.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {},
number = {},
pages = {},
pmid = {35389053},
issn = {1432-2242},
abstract = {A multitude of diverse breeding goals need to be combined in a new cultivar, which always forces to compromise. The biggest challenge grapevine breeders face is the extraordinarily complex trait of wine quality, which is the all-pervasive and most debated characteristic. Since the 1920s, Germany runs continuous grapevine breeding programmes. This continuity was the key to success and lead to various new cultivars on the market, so called PIWIs. Initially, introduced pests and diseases such as phylloxera, powdery and downy mildew were the driving forces for breeding. However, preconceptions about the wine quality of new resistant selections impeded the market introduction. These preconceptions are still echoing today and may be the reason in large parts of the viticultural community for: (1) ignoring substantial breeding progress, and (2) sticking to successful markets of well-known varietal wines or blends (e.g. Chardonnay, Cabernet Sauvignon, Riesling). New is the need to improve viticulture´s sustainability and to adapt to changing environmental conditions. Climate change with its extreme weather will impose the need for a change in cultivars in many wine growing regions. Therefore, a paradigm shift is knocking on the door: new varieties (PIWIs) versus traditional varieties for climate adapted and sustainable viticulture. However, it will be slow process and viticulture is politically well advised to pave the way to variety innovation. In contrast to the widely available PIWIs, competitive cultivars created by means of new breeding technologies (NBT, e.g. through CRISPR/Cas) are still decades from introduction to the market.},
}
@article {pmid35388178,
year = {2022},
author = {Rönspies, M and Schindele, P and Wetzel, R and Puchta, H},
title = {CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.},
journal = {Nature protocols},
volume = {17},
number = {5},
pages = {1332-1358},
pmid = {35388178},
issn = {1750-2799},
mesh = {*Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Chromosomes ; Gene Editing/methods ; Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.},
}
@article {pmid35388146,
year = {2022},
author = {Fuchs, RT and Curcuru, JL and Mabuchi, M and Noireterre, A and Weigele, PR and Sun, Z and Robb, GB},
title = {Characterization of Cme and Yme thermostable Cas12a orthologs.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {325},
pmid = {35388146},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *DNA Cleavage ; Nucleic Acid Conformation ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.},
}
@article {pmid35387989,
year = {2022},
author = {Clow, PA and Du, M and Jillette, N and Taghbalout, A and Zhu, JJ and Cheng, AW},
title = {CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1871},
pmid = {35387989},
issn = {2041-1723},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics ; *RNA, Guide/genetics ; },
abstract = {Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.},
}
@article {pmid35387980,
year = {2022},
author = {Zhang, G and Liu, Y and Huang, S and Qu, S and Cheng, D and Yao, Y and Ji, Q and Wang, X and Huang, X and Liu, J},
title = {Enhancement of prime editing via xrRNA motif-joined pegRNA.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1856},
pmid = {35387980},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Genome ; },
abstract = {The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3'-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.},
}
@article {pmid35387453,
year = {2022},
author = {Liu, FX and Cui, JQ and Park, H and Chan, KW and Leung, T and Tang, BZ and Yao, S},
title = {Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics.},
journal = {Analytical chemistry},
volume = {94},
number = {15},
pages = {5883-5892},
doi = {10.1021/acs.analchem.2c00067},
pmid = {35387453},
issn = {1520-6882},
mesh = {Biological Assay ; CRISPR-Cas Systems ; *Microfluidics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Recombinases/metabolism ; },
abstract = {High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.},
}
@article {pmid35386853,
year = {2022},
author = {He, X and Zeng, XX},
title = {Immunotherapy and CRISPR Cas Systems: Potential Cure of COVID-19?.},
journal = {Drug design, development and therapy},
volume = {16},
number = {},
pages = {951-972},
pmid = {35386853},
issn = {1177-8881},
mesh = {Antibodies, Neutralizing ; *COVID-19/drug therapy/therapy ; *CRISPR-Cas Systems ; Disease Progression ; Humans ; Immunization, Passive ; Immunologic Factors ; SARS-CoV-2 ; },
abstract = {The COVID-19 has plunged the world into a pandemic that affected millions. The continually emerging new variants of concern raise the question as to whether the existing vaccines will continue to provide sufficient protection for individuals from SARS-CoV-2 during natural infection. This narrative review aims to briefly outline various immunotherapeutic options and discuss the potential of clustered regularly interspaced short palindromic repeat (CRISPR Cas system technology against COVID-19 treatment as specific cure. As the development of vaccine, convalescent plasma, neutralizing antibodies are based on the understanding of human immune responses against SARS-CoV-2, boosting human body immune responses in case of SARS-CoV-2 infection, immunotherapeutics seem feasible as specific cure against COVID-19 if the present challenges are overcome. In cell based therapeutics, apart from the high costs, risks and side effects, there are technical problems such as the production of sufficient potent immune cells and antibodies under limited time to treat the COVID-19 patients in mild conditions prior to progression into a more severe case. The CRISPR Cas technology could be utilized to refine the specificity and safety of CAR-T cells, CAR-NK cells and neutralizing antibodies against SARS-CoV-2 during various stages of the COVID-19 disease progression in infected individuals. Moreover, CRISPR Cas technology are proposed in hypotheses to degrade the viral RNA in order to terminate the infection caused by SARS-CoV-2. Thus personalized cocktails of immunotherapeutics and CRISPR Cas systems against COVID-19 as a strategy might prevent further disease progression and circumvent immunity escape.},
}
@article {pmid35386818,
year = {2022},
author = {Jiang, Y and Hoenisch, RC and Chang, Y and Bao, X and Cameron, CE and Lian, XL},
title = {Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems.},
journal = {Bioactive materials},
volume = {14},
number = {},
pages = {313-320},
pmid = {35386818},
issn = {2452-199X},
abstract = {CRISPR/Cas-mediated genome editing in human pluripotent stem cells (hPSCs) offers unprecedented opportunities for developing in vitro disease modeling, drug screening and cell-based therapies. To efficiently deliver the CRISPR components, here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively. These vectors utilized the PiggyBac transposon system, which allows stable expression of CRISPR components in hPSCs. The Cas9 vector PB-CRISPR exhibited high efficiency (up to 99%) of inducing gene knockout in both protein-coding genes and long non-coding RNAs. The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown (98% knockdown for CD90) with optimized gRNA designs. Taken together, our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.},
}
@article {pmid35386712,
year = {2022},
author = {Scheller, SH and Rashad, Y and Saleh, FM and Willingham, KA and Reilich, A and Lin, D and Izadpanah, R and Alt, EU and Braun, SE},
title = {Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {821190},
pmid = {35386712},
issn = {1664-3224},
mesh = {Alleles ; CRISPR-Cas Systems ; *HIV Infections/genetics ; *HIV Seropositivity/genetics ; *HIV-1/genetics ; Humans ; Receptors, CCR5/genetics ; Virus Replication ; },
abstract = {Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.},
}
@article {pmid35386307,
year = {2022},
author = {Haider, MZ and Shabbir, MAB and Yaqub, T and Sattar, A and Maan, MK and Mahmood, S and Mehmood, T and Aslam, HB},
title = {CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.},
journal = {BioMed research international},
volume = {2022},
number = {},
pages = {9080396},
pmid = {35386307},
issn = {2314-6141},
mesh = {Ampicillin ; Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial ; Microbial Sensitivity Tests ; Poultry ; *Salmonella enterica ; *Salmonella enteritidis/genetics ; },
abstract = {Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.},
}
@article {pmid35385916,
year = {2022},
author = {Mallick, T and Mishra, R and Mohanty, S and Joshi, RK},
title = {Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.},
journal = {The plant pathology journal},
volume = {38},
number = {2},
pages = {102-114},
doi = {10.5423/PPJ.OA.12.2021.0190},
pmid = {35385916},
issn = {1598-2254},
support = {BT/PR23412/BPA/118/284/2017//Dept. of Biotechnology/ ; EMR/2016/005234//Science and Engineering Research Board/ ; //Dept. of Science and Technology/ ; },
abstract = {Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.},
}
@article {pmid35385325,
year = {2022},
author = {Ahmed, ASI and Sheng, MHC and Lau, KW and Wilson, SM and Wongwarawat, D and Tang, X and Ghahramanpouri, M and Nehme, A and Xu, Y and Abdipour, A and Zhang, XB and Wasnik, S and Baylink, DJ},
title = {Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.},
journal = {American journal of physiology. Cell physiology},
volume = {322},
number = {5},
pages = {C977-C990},
doi = {10.1152/ajpcell.00413.2021},
pmid = {35385325},
issn = {1522-1563},
support = {W81XWH-12-1-0023//DOD | US Army | MEDCOM | Telemedicine and Advanced Technology Research Center (TATRC)/ ; },
mesh = {Animals ; *Bone Resorption/metabolism ; Calcium/metabolism ; Calcium Channels/genetics/metabolism ; Cell Differentiation ; Mice ; Osteoclasts/metabolism ; *Osteogenesis ; RANK Ligand/metabolism ; Receptors, Calcium-Sensing/genetics/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; },
abstract = {A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.},
}
@article {pmid35385207,
year = {2022},
author = {Yang, S and Joesaar, A and Bögels, BWA and Mann, S and de Greef, TFA},
title = {Protocellular CRISPR/Cas-Based Diffusive Communication Using Transcriptional RNA Signaling.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202202436},
doi = {10.1002/anie.202202436},
pmid = {35385207},
issn = {1521-3773},
support = {101000199/ERC_/European Research Council/International ; },
abstract = {Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing.},
}
@article {pmid35384405,
year = {2022},
author = {Koch, PJ and Webb, S and Gugger, JA and Salois, MN and Koster, MI},
title = {Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.},
journal = {Current protocols},
volume = {2},
number = {4},
pages = {e408},
pmid = {35384405},
issn = {2691-1299},
support = {//National Foundation for Ectodermal Dysplasias/ ; R21EY029081/NH/NIH HHS/United States ; R21 EY029081/EY/NEI NIH HHS/United States ; R01 AR072621/AR/NIAMS NIH HHS/United States ; R01AR072621/NH/NIH HHS/United States ; },
mesh = {Cell Culture Techniques/methods ; Cell Differentiation/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Keratinocytes ; Skin ; },
abstract = {Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™ 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.},
}
@article {pmid35384352,
year = {2022},
author = {Whitley, JA and Kim, S and Lou, L and Ye, C and Alsaidan, OA and Sulejmani, E and Cai, J and Desrochers, EG and Beharry, Z and Rickman, CB and Klingeborn, M and Liu, Y and Xie, ZR and Cai, H},
title = {Encapsulating Cas9 into extracellular vesicles by protein myristoylation.},
journal = {Journal of extracellular vesicles},
volume = {11},
number = {4},
pages = {e12196},
pmid = {35384352},
issn = {2001-3078},
support = {R21 AI157831/AI/NIAID NIH HHS/United States ; U01 CA225784/CA/NCI NIH HHS/United States ; R01 EY031748/EY/NEI NIH HHS/United States ; R21 EY028671/EY/NEI NIH HHS/United States ; P30 EY005722/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Extracellular Vesicles ; Gene Editing ; Genetic Therapy ; },
abstract = {CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.},
}
@article {pmid35382990,
year = {2022},
author = {Horie, M and Yamano-Adachi, N and Kawabe, Y and Kaneoka, H and Fujita, H and Nagamori, E and Iwai, R and Sato, Y and Kanie, K and Ohta, S and Somiya, M and Ino, K},
title = {Recent advances in animal cell technologies for industrial and medical applications.},
journal = {Journal of bioscience and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiosc.2022.03.005},
pmid = {35382990},
issn = {1347-4421},
abstract = {The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.},
}
@article {pmid35380812,
year = {2022},
author = {Pan, R and Liu, J and Wang, P and Wu, D and Chen, J and Wu, Y and Li, G},
title = {Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {14},
pages = {4484-4491},
doi = {10.1021/acs.jafc.1c08262},
pmid = {35380812},
issn = {1520-5118},
mesh = {Alkalies ; Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Gold ; *Metal Nanoparticles ; Milk ; *Nucleic Acids ; Spectrum Analysis, Raman ; },
abstract = {An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64-), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.},
}
@article {pmid35378361,
year = {2022},
author = {Krueger, LA and Morris, AC},
title = {Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.},
journal = {Biochemical and biophysical research communications},
volume = {608},
number = {},
pages = {8-13},
pmid = {35378361},
issn = {1090-2104},
support = {F30 EY031545/EY/NEI NIH HHS/United States ; R01 EY021769/EY/NEI NIH HHS/United States ; TL1 TR001997/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Knock-In Techniques ; Recombinational DNA Repair ; *Zebrafish/genetics ; },
abstract = {Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.},
}
@article {pmid35377968,
year = {2022},
author = {Cheng, Y and Sretenovic, S and Zhang, Y and Pan, C and Huang, J and Qi, Y},
title = {Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2100571},
doi = {10.1002/biot.202100571},
pmid = {35377968},
issn = {1860-7314},
abstract = {CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.},
}
@article {pmid35377365,
year = {2022},
author = {Yan, Q and He, Y and Yue, Y and Jie, L and Wen, T and Zhao, Y and Zhang, M and Zhang, T},
title = {Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {181},
pages = {},
doi = {10.3791/63629},
pmid = {35377365},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Grasshoppers/genetics/metabolism ; Humans ; RNA, Guide/genetics ; Technology ; },
abstract = {The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.},
}
@article {pmid35373735,
year = {2022},
author = {Cornean, A and Gierten, J and Welz, B and Mateo, JL and Thumberger, T and Wittbrodt, J},
title = {Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35373735},
issn = {2050-084X},
support = {WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; 810172//H2020 European Research Council/ ; S/02/17//Deutsche Herzstiftung/ ; 3DMM2O//Deutsche Forschungsgemeinschaft/ ; 81X2500189//Deutsches Zentrum für Herz-Kreislaufforschung/ ; Add-on Fellowship//Joachim Herz Stiftung/ ; 3DMM2O, EXC 2082/1 Wittbrodt C3//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Adenine ; Animals ; CRISPR-Cas Systems ; Cytosine ; DNA ; *Gene Editing ; Mutation ; *Zebrafish/genetics ; },
abstract = {Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.},
}
@article {pmid35373270,
year = {2022},
author = {},
title = {A Base Editing Platform Shows Function of Cancer-Associated Variants.},
journal = {Cancer discovery},
volume = {12},
number = {4},
pages = {883},
doi = {10.1158/2159-8290.CD-RW2022-034},
pmid = {35373270},
issn = {2159-8290},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {A resource was developed to introduce and interrogate cancer-associated single-nucleotide variants.},
}
@article {pmid35373187,
year = {2022},
author = {Ravendran, S and Hernández, SS and König, S and Bak, RO},
title = {CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {793010},
pmid = {35373187},
issn = {2673-3439},
abstract = {Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.},
}
@article {pmid35371164,
year = {2022},
author = {Ali, Q and Yu, C and Hussain, A and Ali, M and Ahmar, S and Sohail, MA and Riaz, M and Ashraf, MF and Abdalmegeed, D and Wang, X and Imran, M and Manghwar, H and Zhou, L},
title = {Genome Engineering Technology for Durable Disease Resistance: Recent Progress and Future Outlooks for Sustainable Agriculture.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {860281},
pmid = {35371164},
issn = {1664-462X},
abstract = {Crop production worldwide is under pressure from multiple factors, including reductions in available arable land and sources of water, along with the emergence of new pathogens and development of resistance in pre-existing pathogens. In addition, the ever-growing world population has increased the demand for food, which is predicted to increase by more than 100% by 2050. To meet these needs, different techniques have been deployed to produce new cultivars with novel heritable mutations. Although traditional breeding continues to play a vital role in crop improvement, it typically involves long and laborious artificial planting over multiple generations. Recently, the application of innovative genome engineering techniques, particularly CRISPR-Cas9-based systems, has opened up new avenues that offer the prospects of sustainable farming in the modern agricultural industry. In addition, the emergence of novel editing systems has enabled the development of transgene-free non-genetically modified plants, which represent a suitable option for improving desired traits in a range of crop plants. To date, a number of disease-resistant crops have been produced using gene-editing tools, which can make a significant contribution to overcoming disease-related problems. Not only does this directly minimize yield losses but also reduces the reliance on pesticide application, thereby enhancing crop productivity that can meet the globally increasing demand for food. In this review, we describe recent progress in genome engineering techniques, particularly CRISPR-Cas9 systems, in development of disease-resistant crop plants. In addition, we describe the role of CRISPR-Cas9-mediated genome editing in sustainable agriculture.},
}
@article {pmid35369445,
year = {2022},
author = {Santana de Carvalho, D and Trovatti Uetanabaro, AP and Kato, RB and Aburjaile, FF and Jaiswal, AK and Profeta, R and De Oliveira Carvalho, RD and Tiwar, S and Cybelle Pinto Gomide, A and Almeida Costa, E and Kukharenko, O and Orlovska, I and Podolich, O and Reva, O and Ramos, PIP and De Carvalho Azevedo, VA and Brenig, B and Andrade, BS and de Vera, JP and Kozyrovska, NO and Barh, D and Góes-Neto, A},
title = {The Space-Exposed Kombucha Microbial Community Member Komagataeibacter oboediens Showed Only Minor Changes in Its Genome After Reactivation on Earth.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {782175},
pmid = {35369445},
issn = {1664-302X},
abstract = {Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.},
}
@article {pmid35369433,
year = {2022},
author = {Riedl, A and Fischer, J and Burgert, HG and Ruzsics, Z},
title = {Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {854690},
pmid = {35369433},
issn = {1664-302X},
abstract = {Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo. rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods.},
}
@article {pmid35368966,
year = {2022},
author = {Cai, J and Wu, D and Jin, Y and Bao, S},
title = {Effect of CMB Carrying PTX and CRISPR/Cas9 on Endometrial Cancer Naked Mouse Model.},
journal = {Journal of healthcare engineering},
volume = {2022},
number = {},
pages = {7119195},
pmid = {35368966},
issn = {2040-2309},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Endometrial Neoplasms/genetics ; Female ; Glycogen Synthase Kinase 3 ; Humans ; Mammals ; Mice ; *Paclitaxel/pharmacology/therapeutic use ; },
abstract = {Endometrial cancer, one of the most common gynecological cancers in women. Patients with advanced or recurrent disease have poor long-term outcomes. The current experiment explore the roles of cationic microbubbles (CMBs) carrying paclitaxel (PTX) and CRISPR/Cas9 plasmids on the xenotransplantation model of mice with endometrial cancer. The tumor histology, tumor cell viability, cell cycle, and invasion ability were investigated. Meanwhile, the P27, P21, GSK-3, Bcl-2 associated death promoter (Bad), mammalian target of rapamycin (mTOR), and C-erbB-2 expressions were evaluated by qRT-PCR and western blotting, respectively. CMB-PTX-CRISPR/Cas9 had an inhibitory action on the tumor growth, tumor cell viability, cell cycle, and invasion ability of the mouse xenograft model of endometrial cancer. The CMB-PTX-CRISPR/Cas9 increased the GSK-3, P21, P27, and Bad expression levels, while reduced the C-erbB-2 and mTOR expressions. CMBs loaded with both PTX and CRISPR/Cas9 plasmids may be a new combination treatment with much potential. CMB-PTX-CRISPR/Cas9 may regulate the tumor cell viability, invasion, and metastasis of endometrial cancer naked mouse model by upregulating expressions of GSK-3, P21, P27, and Bad.},
}
@article {pmid35366394,
year = {2022},
author = {Kurihara, N and Nakagawa, R and Hirano, H and Okazaki, S and Tomita, A and Kobayashi, K and Kusakizako, T and Nishizawa, T and Yamashita, K and Scott, DA and Nishimasu, H and Nureki, O},
title = {Structure of the type V-C CRISPR-Cas effector enzyme.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2022.03.006},
pmid = {35366394},
issn = {1097-4164},
abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.},
}
@article {pmid35366350,
year = {2022},
author = {Murugesan, AC and Varughese, HS},
title = {Analysis of CRISPR-Cas system and antimicrobial resistance in Staphylococcus coagulans isolates.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/lam.13713},
pmid = {35366350},
issn = {1472-765X},
support = {EMR/2016/006141//Department of Science and Technology-Science and Engineering Research Board, Government of India/ ; },
abstract = {CRISPR-Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR-Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR-Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR-Cas system. Three isolates from Antarctica seals neither contain CRISPR-Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR-Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR-Cas system, and this Type IIIA CRISPR-Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR-Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR-Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.},
}
@article {pmid35365834,
year = {2022},
author = {Poudel, R and Rodriguez, LT and Reisch, CR and Rivers, AR},
title = {GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes.},
journal = {GigaScience},
volume = {11},
number = {},
pages = {},
pmid = {35365834},
issn = {2047-217X},
support = {//U.S. Department of Agriculture/ ; 6066-21310-005-D//American Radium Society/ ; //University of Florida/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genome ; *RNA, Guide/genetics ; Software ; },
abstract = {BACKGROUND: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens.<br><br>FINDINGS: GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1-2 minutes.<br><br>CONCLUSIONS: GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.},
}
@article {pmid35364533,
year = {2022},
author = {Lin, X and Li, C and Meng, X and Yu, W and Duan, N and Wang, Z and Wu, S},
title = {CRISPR-Cas12a-mediated luminescence resonance energy transfer aptasensing platform for deoxynivalenol using gold nanoparticle-decorated Ti3C2Tx MXene as the enhanced quencher.},
journal = {Journal of hazardous materials},
volume = {433},
number = {},
pages = {128750},
doi = {10.1016/j.jhazmat.2022.128750},
pmid = {35364533},
issn = {1873-3336},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer/methods ; Gold ; Luminescence ; *Metal Nanoparticles ; Titanium ; Trichothecenes ; },
abstract = {Deoxynivalenol (DON) is a typical mycotoxin in cereals and poses tremendous threats to the ecological environment and public health. Therefore, exploiting sensitive and robust analytical methods for DON is particularly important. Here, we fabricated a CRISPR-Cas12a-mediated luminescence resonance energy transfer (LRET) aptasensor to detect DON by using single-stranded DNA modified upconversion nanoparticles (ssDNA-UCNPs) as anti-interference luminescence labels and gold nanoparticle-decorated Ti3C2Tx MXene nanosheets (MXene-Au) as enhanced quenchers. The DON aptamer can activate the trans-cleavage activity of Cas12a to indiscriminately cut nearby ssDNA-UCNPs into small fragments, which prevents ssDNA-UCNPs from adsorbing onto MXene-Au, and the upconversion luminescence (UCL) remains. Upon the binding of the aptamer with DON, the trans-cleavage activity of Cas12a was suppressed, and the ssDNA-UCNPs were not cleaved and easily adsorbed onto MXene-Au, which caused UCL quenching. Under optimized conditions, the limit of detection was determined to be 0.64 ng/mL with a linear range of 1 - 500 ng/mL. In addition, the sensor was successfully applied to detect DON in corn flour and Tai Lake water with recoveries of 96.2 - 105% and 95.2 - 104%, respectively. This platform achieves a sensitive and specific analysis of DON and greatly broadens the detection range of CRISPR-Cas sensors for non-nucleic acids hazards in the environment and food.},
}
@article {pmid35363475,
year = {2022},
author = {Malcı, K and Walls, LE and Rios-Solis, L},
title = {Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1555-1567},
pmid = {35363475},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/analysis/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.},
}
@article {pmid35362932,
year = {2022},
author = {Courtright-Lim, A},
title = {"CRISPR for Disabilities: How to Self-Regulate" or Something?.},
journal = {Journal of bioethical inquiry},
volume = {19},
number = {1},
pages = {151-161},
pmid = {35362932},
issn = {1176-7529},
mesh = {CRISPR-Cas Systems ; *Disabled Persons ; *Gene Editing ; Humans ; Stem Cell Research ; },
abstract = {The development of the CRISPR gene editing technique has been hyped as a technique that could fundamentally change scientific research and its clinical application. Unrecognized is the fact that it joins other technologies that have tried and failed under the same discourse of scientific hype. These technologies, like gene therapy and stem cell research, have moved quickly passed basic research into clinical application with dire consequences. Before hastily moving to clinical applications, it is necessary to consider basic research and determine how CRISPR/Cas systems should be applied. In the case of single gene diseases, that application is expected to have positive impacts, but as we shift to more complex diseases, the impact could be unintentionally negative. In the context of common disabilities, the level of genetic complexity may render this technology useless but potentially toxic, aggravating a social discourse that devalues those with disabilities. This paper intends to define the issues related to disability that are associated with using the CRIPSR/Cas system in basic research. It also aims to provide a decision tree to help determine whether the technology should be utilized or if alternative approaches beyond scientific research could lead to a better use of limited funding resources.},
}
@article {pmid35358830,
year = {2022},
author = {Liu, Y and Jin, Y and Chen, T and Wu, Y and Peng, X and Li, W and Wei, S and Chen, M and Zou, Q and Guo, S and Xu, J and Tang, C and Zhou, X},
title = {Generation of a homozygous ARHGAP11B knockout hiPSC line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102764},
doi = {10.1016/j.scr.2022.102764},
pmid = {35358830},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; GTPase-Activating Proteins ; Gene Editing ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {The human specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal neocortex and plays a key role in the evolutionary expansion of the neocortex. Here, we generated a homozygous ARHGAP11B knockout human induced pluripotent stem cell (hiPSC) line through CRISPR/Cas9 gene editing system. ARHGAP11B deficient cell line maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The ARHGAP11B knockout cell line can provide a new cell model for studying the evolution of human neocortex.},
}
@article {pmid35358611,
year = {2022},
author = {Bernard, BE and Landmann, E and Jeker, LT and Schumann, K},
title = {CRISPR/Cas-based Human T cell Engineering: Basic Research and Clinical Application.},
journal = {Immunology letters},
volume = {245},
number = {},
pages = {18-28},
doi = {10.1016/j.imlet.2022.03.005},
pmid = {35358611},
issn = {1879-0542},
mesh = {*CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Genetic Engineering ; Humans ; T-Lymphocytes ; },
abstract = {Engineering human T cells for the treatment of cancer, viral infections and autoimmunity has been a long-standing dream of many immunologists and hematologists. Although primary human T cells have been genetically engineered for decades, this process was challenging, time consuming and mostly limited to transgene insertions mediated by viral transduction. The absence of widely accessible tools to efficiently and precisely engineer T cells genetically in a targeted manner limited their applicability as a living drug. This fundamentally changed with the discovery of CRISPR/Cas9 and its adaptation to human T cells. CRISPR/Cas9 has made T cell engineering widely accessible and accelerated the development of engineered adoptive T cell therapies. Only 6 years after the discovery of CRISPR/Cas9 as a biotechnological tool the first CRISPR engineered T cells have been administered to patients with refractory cancers in a phase I clinical trial. Novel Cas proteins - natural and engineered ones - are rapidly emerging. These offer for instance increased flexibility, activity and/or specificity. Moreover, sophisticated protein engineering and fusions of Cas with deaminases or reverse transcriptases enable genomic DNA editing without the need for a double strand cut. Thus, the "CRISPR tool box" for experimental use as well as for novel therapeutic approaches is rapidly expanding. In this review, we will summarize the current state of CRISPR/Cas-based engineering in human T cells for basic research and its clinical applications.},
}
@article {pmid35357193,
year = {2022},
author = {Wu, T and Cao, Y and Liu, Q and Wu, X and Shang, Y and Piao, J and Li, Y and Dong, Y and Liu, D and Wang, H and Liu, J and Ding, B},
title = {Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {14},
pages = {6575-6582},
doi = {10.1021/jacs.2c01760},
pmid = {35357193},
issn = {1520-5126},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; Gene Editing ; *Nanostructures ; Ribonucleoproteins ; },
abstract = {DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.},
}
@article {pmid35355475,
year = {2022},
author = {Zhou, Z and Lü, X and Zhu, L and Zhou, J and Huang, H and Zhang, C and Liu, X},
title = {[Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {3},
pages = {1074-1085},
doi = {10.13345/j.cjb.210635},
pmid = {35355475},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.},
}
@article {pmid35354039,
year = {2022},
author = {Yi, C and Cai, C and Cheng, Z and Zhao, Y and Yang, X and Wu, Y and Wang, X and Jin, Z and Xiang, Y and Jin, M and Han, L and Zhang, A},
title = {Genome-wide CRISPR-Cas9 screening identifies the CYTH2 host gene as a potential therapeutic target of influenza viral infection.},
journal = {Cell reports},
volume = {38},
number = {13},
pages = {110559},
doi = {10.1016/j.celrep.2022.110559},
pmid = {35354039},
issn = {2211-1247},
mesh = {Antiviral Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Endosomes ; Humans ; *Influenza A virus/genetics ; *Influenza, Human/drug therapy/genetics ; },
abstract = {Host genes critical for viral infection are effective antiviral drug targets with tremendous potential due to their universal characteristics against different subtypes of viruses and minimization of drug resistance. Accordingly, we execute a genome-wide CRISPR-Cas9 screen with multiple rounds of survival selection. Enriched in this screen are several genes critical for host sialic acid biosynthesis and transportation, including the cytohesin 2 (CYTH2), tetratricopeptide repeat protein 24 (TTC24), and N-acetylneuraminate synthase (NANS), which we confirm are responsible for efficient influenza viral infection. Moreover, we reveal that CYTH2 is required for the early stage of influenza virus infection by mediating endosomal trafficking. Furthermore, CYTH2 antagonist SecinH3 blunts influenza virus infection in vivo. In summary, these data suggest that CYTH2 is an attractive target for developing host-directed antiviral drugs and therapeutics against influenza virus infection.},
}
@article {pmid35353638,
year = {2022},
author = {Rallapalli, KL and Ranzau, BL and Ganapathy, KR and Paesani, F and Komor, AC},
title = {Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {294-310},
doi = {10.1089/crispr.2021.0131},
pmid = {35353638},
issn = {2573-1602},
support = {R21 GM135736/GM/NIGMS NIH HHS/United States ; R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine/metabolism ; CRISPR-Cas Systems ; Computational Biology ; *Gene Editing ; RNA/genetics ; *RNA Editing/genetics ; },
abstract = {Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.},
}
@article {pmid35352981,
year = {2022},
author = {Li, Y and Liu, Y and Singh, J and Tangprasertchai, NS and Trivedi, R and Fang, Y and Qin, PZ},
title = {Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {341-352},
doi = {10.1089/crispr.2021.0100},
pmid = {35352981},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Endonucleases/genetics ; Gene Editing ; *RNA/chemistry/genetics ; },
abstract = {CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.},
}
@article {pmid35351985,
year = {2022},
author = {Sridhara, S and Rai, J and Whyms, C and Goswami, H and He, H and Woodside, W and Terns, MP and Li, H},
title = {Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {279},
pmid = {35351985},
issn = {2399-3642},
support = {S10 RR025080/RR/NCRR NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; S10 RR024564/RR/NCRR NIH HHS/United States ; GM099604//U.S. Department of Health &amp; Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)/ ; R01 GM099604/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphate ; Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Lactococcus lactis/genetics/metabolism ; RNA ; },
abstract = {The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.},
}
@article {pmid35351888,
year = {2022},
author = {Jia, K and Cui, YR and Huang, S and Yu, P and Lian, Z and Ma, P and Liu, J},
title = {Phage peptides mediate precision base editing with focused targeting window.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1662},
pmid = {35351888},
issn = {2041-1723},
support = {31600686//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Alleles ; Animals ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Peptides/genetics ; },
abstract = {Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.},
}
@article {pmid35351879,
year = {2022},
author = {Li, X and Zhou, L and Gao, BQ and Li, G and Wang, X and Wang, Y and Wei, J and Han, W and Wang, Z and Li, J and Gao, R and Zhu, J and Xu, W and Wu, J and Yang, B and Sun, X and Yang, L and Chen, J},
title = {Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1669},
pmid = {35351879},
issn = {2041-1723},
support = {2019YFA0802804//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018ZX10731-101-001-010//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2019YFA0802804//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFA0801401//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFC1004602//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 32070170//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31925011//National Natural Science Foundation of China (National Science Foundation of China)/ ; 91940306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31822016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21JC1404600//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Mutation ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.},
}
@article {pmid35350901,
year = {2022},
author = {Wheatley, MS and Wang, Q and Wei, W and Bottner-Parker, K and Zhao, Y and Yang, Y},
title = {Cas12a-based diagnostics for potato purple top disease complex associated with infection by 'Candidatus Phytoplasma trifolii'-related strains.},
journal = {Plant disease},
volume = {},
number = {},
pages = {},
doi = {10.1094/PDIS-09-21-2119-RE},
pmid = {35350901},
issn = {0191-2917},
abstract = {'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a trans-cleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.},
}
@article {pmid35349718,
year = {2022},
author = {Konstantakos, V and Nentidis, A and Krithara, A and Paliouras, G},
title = {CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning.},
journal = {Nucleic acids research},
volume = {50},
number = {7},
pages = {3616-3637},
pmid = {35349718},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing/methods ; *RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR-Cas9 guide design.},
}
@article {pmid35349689,
year = {2022},
author = {Zhao, D and Jiang, G and Li, J and Chen, X and Li, S and Wang, J and Zhou, Z and Pu, S and Dai, Z and Ma, Y and Bi, C and Zhang, X},
title = {Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.},
journal = {Nucleic acids research},
volume = {50},
number = {7},
pages = {4161-4170},
pmid = {35349689},
issn = {1362-4962},
mesh = {Adenine/metabolism ; Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; *RNA, Guide/genetics ; },
abstract = {CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.},
}
@article {pmid35349073,
year = {2022},
author = {Das, S and Bombaywala, S and Srivastava, S and Kapley, A and Dhodapkar, R and Dafale, NA},
title = {Genome plasticity as a paradigm of antibiotic resistance spread in ESKAPE pathogens.},
journal = {Environmental science and pollution research international},
volume = {},
number = {},
pages = {},
pmid = {35349073},
issn = {1614-7499},
abstract = {The major reason behind the spread of antibiotic resistance genes (ARGs) is persistent selective pressure in the environment encountered by bacteria. Genome plasticity plays a crucial role in dissemination of antibiotic resistance among bacterial pathogens. Mobile genetic elements harboring ARGs are reported to dodge bacterial immune system and mediate horizontal gene transfer (HGT) under selective pressure. Residual antibiotic pollutants develop selective pressures that force the bacteria to lose their defense mechanisms (CRISPR-cas) and acquire resistance. The present study targets the ESKAPE organisms (namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) causing various nosocomial infections and emerging multidrug-resistant species. The role of CRISPR-cas systems in inhibition of HGT in prokaryotes and its loss due to presence of various stressors in the environment is also focused in the study. IncF and IncH plasmids were identified in all strains of E. faecalis and K. pneumoniae, carrying Beta-lactam and fluoroquinolone resistance genes, whereas sal3, phiCTX, and SEN34 prophages harbored aminoglycoside resistance genes (aadA, aac). Various MGEs present in selected environmental niches that aid the bacterial genome plasticity and transfer of ARGs contributing to its spread are also identified.},
}
@article {pmid35348649,
year = {2022},
author = {Dong, C and Wang, X and Ma, C and Zeng, Z and Pu, DK and Liu, S and Wu, CS and Chen, S and Deng, Z and Guo, FB},
title = {Anti-CRISPRdb v2.2: an online repository of anti-CRISPR proteins including information on inhibitory mechanisms, activities and neighbors of curated anti-CRISPR proteins.},
journal = {Database : the journal of biological databases and curation},
volume = {2022},
number = {},
pages = {},
doi = {10.1093/database/baac010},
pmid = {35348649},
issn = {1758-0463},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/genetics ; },
abstract = {We previously released the Anti-CRISPRdb database hosting anti-CRISPR proteins (Acrs) and associated information. Since then, the number of known Acr families, types, structures and inhibitory activities has accumulated over time, and Acr neighbors can be used as a candidate pool for screening Acrs in further studies. Therefore, we here updated the database to include the new available information. Our newly updated database shows several improvements: (i) it comprises more entries and families because it includes both Acrs reported in the most recent literatures and Acrs obtained via performing homologous alignment; (ii) the prediction of Acr neighbors is integrated into Anti-CRISPRdb v2.2, and users can identify novel Acrs from these candidates; and (iii) this version includes experimental information on the inhibitory strength and stage for Acr-Cas/Acr-CRISPR pairs, motivating the development of tools for predicting specific inhibitory abilities. Additionally, a parameter, the rank of codon usage bias (CUBRank), was proposed and provided in the new version, which showed a positive relationship with predicted result from AcRanker; hence, it can be used as an indicator for proteins to be Acrs. CUBRank can be used to estimate the possibility of genes occurring within genome island-a hotspot hosting potential genes encoding Acrs. Based on CUBRank and Anti-CRISPRdb, we also gave the first glimpse for the emergence of Acr genes (acrs).<br><br>DATABASE URL: http://guolab.whu.edu.cn/anti-CRISPRdb.},
}
@article {pmid35347685,
year = {2022},
author = {Frey, N and Schwank, G},
title = {CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2488},
number = {},
pages = {99-111},
pmid = {35347685},
issn = {1940-6029},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Intestines ; *Organoids ; *Transforming Growth Factor beta/metabolism ; Wnt Signaling Pathway/genetics ; },
abstract = {The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.},
}
@article {pmid35347684,
year = {2022},
author = {Huang, Z and Loewer, A},
title = {Generating Somatic Knockout Cell Lines with CRISPR-Cas9 Technology to Investigate SMAD Signaling.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2488},
number = {},
pages = {81-97},
pmid = {35347684},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Signal Transduction/genetics ; Technology ; },
abstract = {Genome engineering provides a powerful tool to explore TGF-β/SMAD signaling by enabling the deletion and modification of critical components of the pathway. Over the past years, CRISPR-Cas9 technology has matured and can now be used to routinely generate knockout cell lines. Here, we describe a method to design and generate deletions of genes from the SMAD pathway in somatic human cell lines based on homologous recombination.},
}
@article {pmid35344733,
year = {2022},
author = {Rusni, S and Sassa, M and Takagi, T and Kinoshita, M and Takehana, Y and Inoue, K},
title = {Establishment of cytochrome P450 1a gene-knockout Javanese medaka, Oryzias javanicus, which distinguishes toxicity modes of the polycyclic aromatic hydrocarbons, pyrene and phenanthrene.},
journal = {Marine pollution bulletin},
volume = {178},
number = {},
pages = {113578},
doi = {10.1016/j.marpolbul.2022.113578},
pmid = {35344733},
issn = {1879-3363},
mesh = {Animals ; Cytochrome P-450 CYP1A1/genetics/metabolism ; Cytochrome P-450 Enzyme System/metabolism ; Indonesia ; *Oryzias/genetics ; *Phenanthrenes/metabolism/toxicity ; *Polycyclic Aromatic Hydrocarbons/analysis ; Pyrenes/metabolism/toxicity ; *Water Pollutants, Chemical/analysis ; },
abstract = {Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.},
}
@article {pmid35343817,
year = {2022},
author = {Brackett, NF and Davis, BW and Adli, M and Pomés, A and Chapman, MD},
title = {Evolutionary Biology and Gene Editing of Cat Allergen, Fel d 1.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {213-223},
doi = {10.1089/crispr.2021.0101},
pmid = {35343817},
issn = {2573-1602},
mesh = {*Allergens/chemistry/genetics ; Animals ; Biology ; CRISPR-Cas Systems/genetics ; Cats ; Gene Editing ; Glycoproteins/chemistry/genetics ; *Hypersensitivity/genetics/therapy ; },
abstract = {Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.},
}
@article {pmid35343224,
year = {2022},
author = {Tian, H and Niu, H and Luo, J and Yao, W and Chen, X and Wu, J and Geng, Y and Gao, W and Lei, A and Gao, Z and Tian, X and Zhao, X and Shi, H and Li, C and Hua, J},
title = {Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {13},
pages = {4030-4043},
doi = {10.1021/acs.jafc.2c00642},
pmid = {35343224},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/metabolism ; *Milk/chemistry ; *Stearoyl-CoA Desaturase/genetics/metabolism ; },
abstract = {Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.},
}
@article {pmid35343100,
year = {2022},
author = {Zheng, F and Chen, Z and Li, J and Wu, R and Zhang, B and Nie, G and Xie, Z and Zhang, H},
title = {A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {14},
pages = {e2105231},
doi = {10.1002/advs.202105231},
pmid = {35343100},
issn = {2198-3844},
support = {JCYJ20180508152903208//The Science and Technology Project of Shenzhen/ ; 2019B1515120043//Guangdong Basic and Applied Basic Research Foundation/ ; 2020A151501612//Natural Science Foundation of Guangdong Province/ ; JCYJ201904//Longhua District Science and Innovation Commission Project Grants of Shenzhen/ ; KCXFZ20201221173413038//Science and Technology Innovation Commission of Shenzhen/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Nucleic Acids ; RNA, Guide/genetics/metabolism ; Surface Plasmon Resonance ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single-guide RNA, enabling the amplification-free detection of target sequences within genomic DNA. The sensor, termed CRISPR-SPR-Chip, is used to successfully analyze recombinant plasmids with only three-base mutations with a limit of detection as low as 1.3 fM. Real-time monitoring CRISPR-SPR-Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre-amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR-empowered SPR (CRISPR-eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on-chip optic approach for clinical gene analysis.},
}
@article {pmid35341983,
year = {2022},
author = {Li, C and Chu, W and Gill, RA and Sang, S and Shi, Y and Hu, X and Yang, Y and Zaman, QU and Zhang, B},
title = {Computational tools and resources for CRISPR/Cas genome editing.},
journal = {Genomics, proteomics &amp; bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.gpb.2022.02.006},
pmid = {35341983},
issn = {2210-3244},
abstract = {The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genomic editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduced the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focused on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights on the limitations of available computational tools that surely help researchers of this field for further optimization. Lastly, we suggested a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.},
}
@article {pmid35341958,
year = {2022},
author = {Wang, S and Hu, J and Sui, C and He, G and Qu, Z and Chen, X and Wang, Y and Guo, D and Liu, X},
title = {Accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) to diagnose COVID-19, a meta-analysis.},
journal = {Microbial pathogenesis},
volume = {165},
number = {},
pages = {105498},
pmid = {35341958},
issn = {1096-1208},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; },
abstract = {OBJECTIVE: To estimate the accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) in determining coronavirus disease-19 (COVID-19).<br><br>METHODS: As of January 31, 2022, PubMed, Web of Science, Embase, Science Direct, Wiley and Springer Link were searched. Sensitivity, specificity, likelihood ratio (LR), diagnostic odds ratio (DOR) and area under the summary receiver-operating characteristic (AUC) curve were used to assess the accuracy of CRISPR.<br><br>RESULTS: According to the inclusion criteria, 5857 patients from 54 studies were included in this meta-analysis. The pooled sensitivity, specificity and AUC were 0.98, 1.00 and 1.00, respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.96, 1.00, respectively. For Cas-13, the sensitivity and specificity were 0.99 and 0.99.<br><br>CONCLUSION: This meta-analysis showed that the diagnostic performance of CRISPR is close to the gold standard, and it is expected to meet the Point of care requirements in resource poor areas.},
}
@article {pmid35340673,
year = {2022},
author = {Alduhaidhawi, AHM and AlHuchaimi, SN and Al-Mayah, TA and Al-Ouqaili, MTS and Alkafaas, SS and Muthupandian, S and Saki, M},
title = {Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater.},
journal = {Infection and drug resistance},
volume = {15},
number = {},
pages = {1143-1154},
pmid = {35340673},
issn = {1178-6973},
abstract = {Purpose: This study aimed to evaluate the presence of CRISPR-Cas system genes and their possible association with antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium species isolated from hospital wastewater (HWW) samples of several hospitals.<br><br>Methods: HWW samples (200 mL) were collected from wastewater discharged from different hospitals from October 2020 to March 2021. The isolation and identification of enterococci species were performed by standard bacteriology tests and polymerase chain reaction (PCR). Antibiotic resistance was determined using the disc diffusion. The presence of various CRISPR-Cas systems was investigated by PCR. The association of the occurrence of CRISPR-Cas systems with antibiotic resistance was analyzed with appropriate statistical tests.<br><br>Results: In total, 85 different enterococci species were isolated and identified using phenotypic methods. The results of PCR confirmed the prevalence of 50 (58.8%) E. faecalis and 35 (41.2%) E. faecium, respectively. In total, 54 (63.5%) of 85 isolates showed the presence of CRISPR-Cas loci. The incidence of CRISPR-Cas was more common in E. faecalis. CRISPR1, CRISPR2, and CRISPR3 were present in 35 (41.2%), 47 (55.3%), and 30 (35.3%) enterococci isolates, respectively. The CRISPR-Cas positive isolates showed significant lower resistance rates against vancomycin, ampicillin, chloramphenicol, erythromycin, rifampin, teicoplanin, tetracycline, imipenem, tigecycline, and trimethoprim-sulfamethoxazole in comparison with CRISPR-Cas negative isolates. The results showed that the presence of CRISPR-Cas genes was lower in multidrug-resistant (MDR) isolates (53.1%, n = 26/49) compared to the non-MDR enterococci isolates (77.8%, n = 28/36) (P = 0.023).<br><br>Conclusion: This study revealed the higher prevalence of E. faecalis than E. faecium in HWWs. Also, the lack of CRISPR-Cas genes was associated with more antibiotic resistance rates and multidrug resistance in E. faecalis and E. faecium isolates with HWW origin.},
}
@article {pmid35339825,
year = {2022},
author = {Geng, B and Wang, X and Park, KH and Lee, KE and Kim, J and Chen, P and Zhou, X and Tan, T and Yang, C and Zou, X and Janssen, PM and Cao, L and Ye, L and Wang, X and Cai, C and Zhu, H},
title = {UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway.},
journal = {Redox biology},
volume = {52},
number = {},
pages = {102295},
doi = {10.1016/j.redox.2022.102295},
pmid = {35339825},
issn = {2213-2317},
mesh = {Animals ; Escherichia coli ; *Heart Injuries ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; *Induced Pluripotent Stem Cells ; Mice ; *Myocardial Infarction/genetics/pathology ; Signal Transduction ; Ubiquitin Thiolesterase/genetics ; },
abstract = {Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.},
}
@article {pmid35339823,
year = {2022},
author = {Liu, C and Ren, L and Li, X and Fan, N and Chen, J and Zhang, D and Yang, W and Ding, S and Xu, W and Min, X},
title = {Self-electrochemiluminescence biosensor based on CRISPR/Cas12a and PdCuBP@luminol nanoemitter for highly sensitive detection of cytochrome c oxidase subunit III gene of acute kidney injury.},
journal = {Biosensors &amp; bioelectronics},
volume = {207},
number = {},
pages = {114207},
doi = {10.1016/j.bios.2022.114207},
pmid = {35339823},
issn = {1873-4235},
mesh = {*Acute Kidney Injury ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Electron Transport Complex IV ; Female ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Luminol ; Male ; },
abstract = {The cytochrome c oxidase subunit III (COX III) gene is a powerful biomarker for the early diagnosis of acute kidney injury. However, current methods for COX III gene detection are usually laborious and time-consuming, with limited sensitivity. Herein, we report a novel self-electrochemiluminescence (ECL) biosensor for highly sensitive detection of the COX III gene based on CRISPR/Cas12a and nanoemitters of luminol-loaded multicomponent metal-metalloid PdCuBP alloy mesoporous nanoclusters. The nanoemitter with excellent self-ECL in neutral media exhibited a high specific surface area for binding luminol and outstanding oxidase-like catalytic activity toward dissolved O2. Meanwhile, the CRISPR/Cas12a system, as a target-trigger, was employed to specifically recognize the COX III gene and efficiently cleave the interfacial quencher of dopamine-labeled hairpin DNA. As a result, the ECL biosensor showed superior analytical performance for COX III gene detection without exogenous coreactant. Benefiting from the high-efficiency ECL emission of the nanoemitter and Cas12a-mediated interfacial cleavage of the quencher, the developed ECL biosensor exhibited high sensitivity to COX III with a low detection limit of 0.18 pM. The established ECL biosensing method possessed excellent practical performance in urine samples. Meaningfully, the proposed strategy presents promising prospects for nucleic acid detection in the field of clinical diagnostics.},
}
@article {pmid35339702,
year = {2022},
author = {Clemmensen, SE and Kromphardt, KJK and Frandsen, RJN},
title = {Marker-free CRISPR-Cas9 based genetic engineering of the phytopathogenic fungus, Penicillium expansum.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {160},
number = {},
pages = {103689},
doi = {10.1016/j.fgb.2022.103689},
pmid = {35339702},
issn = {1096-0937},
mesh = {CRISPR-Cas Systems ; Genetic Engineering ; *Malus ; *Penicillium/genetics/metabolism ; },
abstract = {Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.},
}
@article {pmid35338974,
year = {2022},
author = {Fapohunda, FO and Qiao, S and Pan, Y and Wang, H and Liu, Y and Chen, Q and Lü, P},
title = {CRISPR Cas system: A strategic approach in detection of nucleic acids.},
journal = {Microbiological research},
volume = {259},
number = {},
pages = {127000},
doi = {10.1016/j.micres.2022.127000},
pmid = {35338974},
issn = {1618-0623},
mesh = {*CRISPR-Cas Systems ; Humans ; *Nucleic Acids ; },
abstract = {Over the decades, rapid nucleic acid detection has been difficult for scientists, especially in microbiology, biotechnology and immunology. Most technologies are finite in sensitivity, specificity or both. Early diagnosis of disease allows swift response, disease monitoring and control of the rapid spread of the disease. However, limited access to test kits, specialized laboratory equipment, and the need for highly skilled personnel has led to a detection downshift. CRISPR-based diagnostic techniques, based on clustered regularly interspaced short palindromic repeats (CRISPR), have recently altered molecular diagnosis. This modern technology is combined or paired with other methods like SHERLOCK, DETECTR, HUDSON, CDetection and so on to detect viruses, bacteria infection and other pathogenic agents, due to its precision and versatility, it can also detect diseases directly from patient samples. Rapid, sensitive, accurate and advanced molecular techniques are much needed since they soothe researchers in diagnosis and detection, and can also be employed in therapeutic treatments. In this review, detailed CRISPR Cas variants were discussed, emphasizing the use of CRISPR system as a tool in diagnostics and nucleic acids detection and the benefits of this robust tool over other amplification methods.},
}
@article {pmid35338236,
year = {2022},
author = {Stukenberg, D and Hoff, J and Faber, A and Becker, A},
title = {NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {265},
pmid = {35338236},
issn = {2399-3642},
support = {BioRoboost project (H2020-NMBP-TR-IND-2018-2020/BIOTEC-01-2018 (CSA), Project ID 210491758)//European Commission (EC)/ ; },
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing/methods ; *Vibrio/genetics ; },
abstract = {The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.},
}
@article {pmid35338140,
year = {2022},
author = {Krysler, AR and Cromwell, CR and Tu, T and Jovel, J and Hubbard, BP},
title = {Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1617},
pmid = {35338140},
issn = {2041-1723},
support = {CIHR-PS-408552//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; },
abstract = {CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.},
}
@article {pmid35337340,
year = {2022},
author = {Das, S and Bano, S and Kapse, P and Kundu, GC},
title = {CRISPR based therapeutics: a new paradigm in cancer precision medicine.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {85},
pmid = {35337340},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine.<br><br>MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo.<br><br>CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.},
}
@article {pmid35337108,
year = {2022},
author = {Park, HM and Park, Y and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H},
title = {Rethinking Protein Drug Design with Highly Accurate Structure Prediction of Anti-CRISPR Proteins.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {35337108},
issn = {1424-8247},
abstract = {Protein therapeutics play an important role in controlling the functions and activities of disease-causing proteins in modern medicine. Despite protein therapeutics having several advantages over traditional small-molecule therapeutics, further development has been hindered by drug complexity and delivery issues. However, recent progress in deep learning-based protein structure prediction approaches, such as AlphaFold2, opens new opportunities to exploit the complexity of these macro-biomolecules for highly specialised design to inhibit, regulate or even manipulate specific disease-causing proteins. Anti-CRISPR proteins are small proteins from bacteriophages that counter-defend against the prokaryotic adaptive immunity of CRISPR-Cas systems. They are unique examples of natural protein therapeutics that have been optimized by the host-parasite evolutionary arms race to inhibit a wide variety of host proteins. Here, we show that these anti-CRISPR proteins display diverse inhibition mechanisms through accurate structural prediction and functional analysis. We find that these phage-derived proteins are extremely distinct in structure, some of which have no homologues in the current protein structure domain. Furthermore, we find a novel family of anti-CRISPR proteins which are structurally similar to the recently discovered mechanism of manipulating host proteins through enzymatic activity, rather than through direct inference. Using highly accurate structure prediction, we present a wide variety of protein-manipulating strategies of anti-CRISPR proteins for future protein drug design.},
}
@article {pmid35336126,
year = {2022},
author = {de Oliveira, IMF and Godoy-Santos, F and Oyama, LB and Moreira, SM and Dias, RG and Huws, SA and Creevey, CJ and Mantovani, HC},
title = {Whole-Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen.},
journal = {Microorganisms},
volume = {10},
number = {3},
pages = {},
pmid = {35336126},
issn = {2076-2607},
support = {001//Coordenação de Aperfeicoamento de Pessoal de Nível Superior/ ; APQ-02899-18//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; //National Council for Scientific and Technological Development/ ; #CNPq//Institutos Nacionais de Ciência e Tecnologia/ ; 172629373//RCUK Newton Institutional Link Funding/ ; },
abstract = {Antimicrobial peptides (AMPs) can efficiently control different microbial pathogens and show the potential to be applied in clinical practice and livestock production. In this work, the aim was to isolate AMP-producing ruminal streptococci and to characterize their genetic features through whole-genome sequencing. We cultured 463 bacterial isolates from the rumen of Nelore bulls, 81 of which were phenotypically classified as being Streptococcaceae. Five isolates with broad-range activity were genome sequenced and confirmed as being Streptococcus lutetiensis. The genetic features linked to their antimicrobial activity or adaptation to the rumen environment were characterized through comparative genomics. The genome of S. lutetiensis UFV80 harbored a putative CRISPR-Cas9 system (Type IIA). Computational tools were used to discover novel biosynthetic clusters linked to the production of bacteriocins. All bacterial genomes harbored genetic clusters related to the biosynthesis of class I and class II bacteriocins. SDS-PAGE confirmed the results obtained in silico and demonstrated that the class II bacteriocins predicted in the genomes of three S. lutetiensis strains had identical molecular mass (5197 Da). These results demonstrate that ruminal bacteria of the Streptococcus bovis/equinus complex represent a promising source of novel antimicrobial peptides.},
}
@article {pmid35335122,
year = {2022},
author = {Jiang, Z and Abdullah, and Zhang, S and Jiang, Y and Liu, R and Xiao, Y},
title = {Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {6},
pages = {},
pmid = {35335122},
issn = {1420-3049},
support = {2018YFA0901200; 31870071; SL2020MS028//National Key R&amp;D Program of China; National Natural Science Foundation of China; Oceanic Interdisciplinary Program of Shanghai Jiao Tong University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Point Mutation ; RNA, Guide/genetics ; },
abstract = {Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.},
}
@article {pmid35333864,
year = {2022},
author = {Zhang, Q and Zhang, Y and Chai, Y},
title = {Optimization of CRISPR/LbCas12a-mediated gene editing in Arabidopsis.},
journal = {PloS one},
volume = {17},
number = {3},
pages = {e0265114},
pmid = {35333864},
issn = {1932-6203},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Promoter Regions, Genetic ; },
abstract = {CRISPR/LbCas12a system (LbCpf1) has been widely used for genome modification including plant species. However, the efficiency of CRISPR/LbCas12a varied considerably in different plant species and tissues, and the editing efficiency needs to be further improved. In this study, we tried to improve the editing efficiency of CRISPR/LbCas12a in Arabidopsis by optimizing the crRNA expression strategies and Pol II promoters. Notably, the combination of tRNA-crRNA fusion strategy and RPS5A promoter in CRISPR/LbCas12a system has highest editing efficiency, while CRISPR/LbCas12a driven by EC1f-in(crR)p had the highest ratio of homozygous &amp; bi-allelic mutants. In addition, all homozygous &amp; bi-allelic mutants can be stably inherited to the next generation and have no phenotypic separation. In this study, the editing efficiency of the CRISPR/LbCas12a system was improved by selecting the optimal crRNA expression strategies and promoter of LbCas12a in Arabidopsis, which will prove useful for optimization of CRISPR/LbCas12a methods in other plants.},
}
@article {pmid35333620,
year = {2022},
author = {Juríková, K and Sepšiová, R and Ševčovičová, A and Tomáška, Ľ and Džugasová, V},
title = {Implementing CRISPR-Cas9 Yeast Practicals into Biology Curricula.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {181-186},
doi = {10.1089/crispr.2021.0125},
pmid = {35333620},
issn = {2573-1602},
mesh = {Biology ; *CRISPR-Cas Systems/genetics ; Curriculum ; Gene Editing/methods ; Humans ; *Saccharomyces cerevisiae/genetics ; },
abstract = {CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.},
}
@article {pmid35333175,
year = {2022},
author = {Thumberger, T and Tavhelidse-Suck, T and Gutierrez-Triana, JA and Cornean, A and Medert, R and Welz, B and Freichel, M and Wittbrodt, J},
title = {Boosting targeted genome editing using the hei-tag.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35333175},
issn = {2050-084X},
support = {CRC873,project A3//Deutsche Forschungsgemeinschaft/ ; FOR2509 P10,WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; CRC1118,project S03//Deutsche Forschungsgemeinschaft/ ; GA 294354-ManISteC//H2020 European Research Council/ ; NO 810172//ERC-SyG H2020/ ; 3DMM2O, EXC 2082/1 Wittbrodt C3//Deutsche Forschungsgemeinschaft/ ; CRC873 project A3//Deutsche Forschungsgemeinschaft/ ; FOR2509 P10 WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; CRC1118 project S03//Deutsche Forschungsgemeinschaft/ ; 810172//H2020 European Research Council/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing/methods ; Mammals ; Nuclear Localization Signals ; RNA, Messenger/genetics ; },
abstract = {Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.},
}
@article {pmid35332696,
year = {2022},
author = {Aragonés, V and Aliaga, F and Pasin, F and Daròs, JA},
title = {Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2100504},
doi = {10.1002/biot.202100504},
pmid = {35332696},
issn = {1860-7314},
abstract = {Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we presentJoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci.},
}
@article {pmid35332138,
year = {2022},
author = {Peterka, M and Akrap, N and Li, S and Wimberger, S and Hsieh, PP and Degtev, D and Bestas, B and Barr, J and van de Plassche, S and Mendoza-Garcia, P and Šviković, S and Sienski, G and Firth, M and Maresca, M},
title = {Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1240},
pmid = {35332138},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA Repair ; Endonucleases/metabolism ; Gene Editing ; Mammals/genetics ; },
abstract = {Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.},
}
@article {pmid35331236,
year = {2022},
author = {Selvakumar, SC and Preethi, KA and Ross, K and Tusubira, D and Khan, MWA and Mani, P and Rao, TN and Sekar, D},
title = {CRISPR/Cas9 and next generation sequencing in the personalized treatment of Cancer.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {83},
pmid = {35331236},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; High-Throughput Nucleotide Sequencing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; },
abstract = {BACKGROUND: Cancer is caused by a combination of genetic and epigenetic abnormalities. Current cancer therapies are limited due to the complexity of their mechanism, underlining the need for alternative therapeutic approaches. Interestingly, combining the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system with next-generation sequencing (NGS) has the potential to speed up the identification, validation, and targeting of high-value targets.<br><br>MAIN TEXT: Personalized or precision medicine combines genetic information with phenotypic and environmental characteristics to produce healthcare tailored to the individual and eliminates the constraints of "one-size-fits-all" therapy. Precision medicine is now possible thanks to cancer genome sequencing. Having advantages over limited sample requirements and the recent development of biomarkers have made the use of NGS a major leap in personalized medicine. Tumor and cell-free DNA profiling using NGS, proteome and RNA analyses, and a better understanding of immunological systems, are all helping to improve cancer treatment choices. Finally, direct targeting of tumor genes in cancer cells with CRISPR/Cas9 may be achievable, allowing for eliminating genetic changes that lead to tumor growth and metastatic capability.<br><br>CONCLUSION: With NGS and CRISPR/Cas9, the goal is no longer to match the treatment for the diag