@article {pmid30883991,
year = {2019},
author = {Moses, C and Kaur, P},
title = {Applications of CRISPR systems in respiratory health: Entering a new 'red pen' era in genome editing.},
journal = {Respirology (Carlton, Vic.)},
volume = {},
number = {},
pages = {},
doi = {10.1111/resp.13527},
pmid = {30883991},
issn = {1440-1843},
abstract = {Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio-economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.},
}

@article {pmid30881132,
year = {2019},
author = {Lu, ZJ and Yu, Q and Zhou, SH and Fan, J and Shen, LF and Bao, YY and Wu, TT and Zhou, ML and Huang, YP},
title = {Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique.},
journal = {Cancer management and research},
volume = {11},
number = {},
pages = {2087-2096},
doi = {10.2147/CMAR.S183859},
pmid = {30881132},
issn = {1179-1322},
abstract = {Background: Glucose transporter (GLUT)-mediated glucose uptake is an important process in the development of laryngeal carcinoma, one of the most common malignancies of the head and neck. GLUT-1, together with HIF-1α, is also an indicator of hypoxia. Both proteins play a critical role in glucose uptake and glycolysis in laryngeal carcinoma cells under hypoxic stress. A double gene knockout model in which HIF-1α and GLUT-1 are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma.

Purpose: In this study we used the CRISPR/Cas 9 system to induce HIF-1α and GLUT-1 double gene knockout in HEp-2 cells and then used the knocked-out cells to study the role of these markers in laryngeal carcinoma, including in chemoradioresistance.

Methods: High-grade small-guide RNAs (sgRNAs) of HIF-1α and GLUT-1 were designed using an online tool and inserted into the pUC57-T7-gRNA vector. The recombinant plasmids were transfected into HEp-2 cells and positive cells were screened using the dilution method. Gene mutation and expression were determined by sequence analysis and immunoblotting.

Results: In HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the HIF-1α genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the GLUT-1 gene. Neither HIF-1α nor GLUT-1 protein was expressed in positive cells. The proliferation, migration, and invasion of HEp-2 cells were significantly decreased afterward. The possible mechanism may be that the inhibition PI3K/AKT/mTOR pathway by HIF-1α and GLUT-1 double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation.

Conclusion: Our HIF-1α and GLUT-1 double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.},
}

@article {pmid30584102,
year = {2019},
author = {Xiao, B and Yin, S and Hu, Y and Sun, M and Wei, J and Huang, Z and Wen, Y and Dai, X and Chen, H and Mu, J and Cui, L and Jiang, L},
title = {Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {1},
pages = {255-260},
doi = {10.1073/pnas.1813542116},
pmid = {30584102},
issn = {1091-6490},
support = {R01 AI116466/AI/NIAID NIH HHS/United States ; U19 AI089672/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; Erythrocytes/parasitology ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genes, Protozoan/genetics ; Histone Acetyltransferases/genetics ; Histone Deacetylases/genetics ; Humans ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/*genetics/physiology ; Recombinant Proteins ; },
abstract = {Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
*Epigenesis, Genetic
Erythrocytes/parasitology
Gene Editing/*methods
Gene Knockdown Techniques
Genes, Protozoan/genetics
Histone Acetyltransferases/genetics
Histone Deacetylases/genetics
Humans
Malaria, Falciparum/parasitology
Plasmodium falciparum/*genetics/physiology
Recombinant Proteins

@article {pmid30529264,
year = {2019},
author = {Shi, X and He, W and Guo, S and Zhang, B and Ren, S and Liu, K and Sun, T and Cui, J},
title = {RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.},
journal = {Neuroscience},
volume = {398},
number = {},
pages = {1-11},
doi = {10.1016/j.neuroscience.2018.11.052},
pmid = {30529264},
issn = {1873-7544},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Epilepsies, Myoclonic/genetics/metabolism ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques/methods ; Mice ; NAV1.1 Voltage-Gated Sodium Channel/*deficiency/genetics ; RNA, Messenger/metabolism ; Sequence Analysis, RNA ; Signal Transduction ; },
abstract = {Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.},
}

Animals
*CRISPR-Cas Systems
Cell Line
Epilepsies, Myoclonic/genetics/metabolism
*Gene Editing
Gene Expression
*Gene Knockout Techniques/methods
Mice
NAV1.1 Voltage-Gated Sodium Channel/*deficiency/genetics
RNA, Messenger/metabolism
Sequence Analysis, RNA
Signal Transduction

@article {pmid30476497,
year = {2019},
author = {Fujii, M and Clevers, H and Sato, T},
title = {Modeling Human Digestive Diseases With CRISPR-Cas9-Modified Organoids.},
journal = {Gastroenterology},
volume = {156},
number = {3},
pages = {562-576},
doi = {10.1053/j.gastro.2018.11.048},
pmid = {30476497},
issn = {1528-0012},
mesh = {CRISPR-Cas Systems/*genetics ; Colorectal Neoplasms/genetics/pathology ; Esophageal Neoplasms/genetics/pathology ; Female ; Gastrointestinal Diseases/genetics/pathology ; Gastrointestinal Neoplasms/*genetics/*pathology ; Gastrointestinal Tract/pathology ; *Genetic Predisposition to Disease ; Humans ; Male ; *Organoids ; Pancreatic Neoplasms/genetics/pathology ; Sensitivity and Specificity ; },
abstract = {Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients' tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system.},
}

CRISPR-Cas Systems/*genetics
Colorectal Neoplasms/genetics/pathology
Esophageal Neoplasms/genetics/pathology
Female
Gastrointestinal Diseases/genetics/pathology
Gastrointestinal Neoplasms/*genetics/*pathology
Gastrointestinal Tract/pathology
*Genetic Predisposition to Disease
Humans
Male
*Organoids
Pancreatic Neoplasms/genetics/pathology
Sensitivity and Specificity

@article {pmid30258161,
year = {2018},
author = {DeWeerdt, S},
title = {The genomics of brain cancer.},
journal = {Nature},
volume = {561},
number = {7724},
pages = {S54-S55},
doi = {10.1038/d41586-018-06711-8},
pmid = {30258161},
issn = {1476-4687},
mesh = {Adult ; Animals ; Brain Neoplasms/diagnosis/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Chromosome Deletion ; Chromosomes, Human, Pair 19/genetics ; Gene Editing ; *Genetic Predisposition to Disease ; Genome, Human/*genetics ; *Genomics ; Glioblastoma/diagnosis/genetics/therapy ; Glioma/diagnosis/genetics/therapy ; Humans ; Isocitrate Dehydrogenase/genetics ; Mice ; Mutation ; Oligodendroglioma/diagnosis/pathology/therapy ; *Pharmacogenomic Testing ; Precision Medicine ; Prognosis ; Survival Analysis ; Telomere Homeostasis/genetics ; Treatment Outcome ; World Health Organization ; },
}

Adult
Animals
Brain Neoplasms/diagnosis/*genetics/*therapy
CRISPR-Cas Systems/genetics
Chromosome Deletion
Chromosomes, Human, Pair 19/genetics
Gene Editing
*Genetic Predisposition to Disease
Genome, Human/*genetics
*Genomics
Glioblastoma/diagnosis/genetics/therapy
Glioma/diagnosis/genetics/therapy
Humans
Isocitrate Dehydrogenase/genetics
Mice
Mutation
Oligodendroglioma/diagnosis/pathology/therapy
*Pharmacogenomic Testing
Precision Medicine
Prognosis
Survival Analysis
Telomere Homeostasis/genetics
Treatment Outcome
World Health Organization

@article {pmid30224336,
year = {2018},
author = {Wang, H and Park, H and Liu, J and Sternberg, PW},
title = {An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3607-3616},
pmid = {30224336},
issn = {2160-1836},
support = {K99 GM126137/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R24 OD023041/OD/NIH HHS/United States ; T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/genetics ; *Gene Editing ; Mutation ; },
abstract = {Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/genetics
*Gene Editing
Mutation

@article {pmid30213867,
year = {2018},
author = {Huynh, N and Zeng, J and Liu, W and King-Jones, K},
title = {A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3593-3605},
pmid = {30213867},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics ; Drosophila/*genetics ; Drosophila Proteins/genetics ; Exocrine Glands/*metabolism ; Gene Expression Regulation ; Mixed Function Oxygenases/genetics ; },
abstract = {Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.},
}

Animals
CRISPR-Cas Systems
Cytochrome P-450 Enzyme System/genetics
Drosophila/*genetics
Drosophila Proteins/genetics
Exocrine Glands/*metabolism
Gene Expression Regulation
Mixed Function Oxygenases/genetics

@article {pmid30169977,
year = {2018},
author = {Luther, DC and Lee, YW and Nagaraj, H and Scaletti, F and Rotello, VM},
title = {Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges.},
journal = {Expert opinion on drug delivery},
volume = {15},
number = {9},
pages = {905-913},
doi = {10.1080/17425247.2018.1517746},
pmid = {30169977},
issn = {1744-7593},
support = {R01 EB022641/EB/NIBIB NIH HHS/United States ; R01 GM077173/GM/NIGMS NIH HHS/United States ; T32 GM008515/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; RNA, Messenger/genetics ; },
abstract = {INTRODUCTION: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts. Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology. Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.},
}

Animals
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/*methods
Gene Transfer Techniques
Genetic Therapy/*methods
Humans
RNA, Messenger/genetics

@article {pmid30061297,
year = {2018},
author = {Callahan, SJ and Tepan, S and Zhang, YM and Lindsay, H and Burger, A and Campbell, NR and Kim, IS and Hollmann, TJ and Studer, L and Mosimann, C and White, RM},
title = {Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).},
journal = {Disease models & mechanisms},
volume = {11},
number = {9},
pages = {},
pmid = {30061297},
issn = {1754-8411},
support = {DP2 CA186572/CA/NCI NIH HHS/United States ; F30 CA220954/CA/NCI NIH HHS/United States ; K08 AR055368/AR/NIAMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; F99 CA212436/CA/NCI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; F31 CA196305/CA/NCI NIH HHS/United States ; },
mesh = {Aging/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/pathology ; Disease Models, Animal ; Disease Progression ; *Electroporation ; Embryo, Nonmammalian/metabolism ; Gene Transfer Techniques ; Melanoma/pathology ; Plasmids/genetics ; Promoter Regions, Genetic ; *Transgenes ; Zebrafish/embryology/*genetics ; },
abstract = {Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.},
}

Aging/*genetics
Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Carcinogenesis/genetics/pathology
Disease Models, Animal
Disease Progression
*Electroporation
Embryo, Nonmammalian/metabolism
Gene Transfer Techniques
Melanoma/pathology
Plasmids/genetics
Promoter Regions, Genetic
*Transgenes
Zebrafish/embryology/*genetics

@article {pmid29981117,
year = {2018},
author = {Loyola-Vargas, VM and Avilez-Montalvo, RN},
title = {Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1815},
number = {},
pages = {131-148},
doi = {10.1007/978-1-4939-8594-4_7},
pmid = {29981117},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genome, Plant ; Plants/*genetics ; Tissue Culture Techniques/*methods ; },
abstract = {Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.},
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
*Gene Editing
*Genome, Plant
Plants/*genetics
Tissue Culture Techniques/*methods

@article {pmid29963983,
year = {2018},
author = {Rouillon, C and Athukoralage, JS and Graham, S and Grüschow, S and White, MF},
title = {Control of cyclic oligoadenylate synthesis in a type III CRISPR system.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {29963983},
issn = {2050-084X},
support = {Project grant BB/M000400/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/*biosynthesis ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics/metabolism ; Endodeoxyribonucleases/*genetics/metabolism ; Endoribonucleases/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Kinetics ; Oligoribonucleotides/*biosynthesis ; Phosphorothioate Oligonucleotides/pharmacology ; RNA Cleavage ; RNA Viruses/*genetics/metabolism ; RNA, Viral/*genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Sulfolobus solfataricus/drug effects/*genetics/immunology/metabolism ; Time Factors ; },
abstract = {The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.},
}

Adenine Nucleotides/*biosynthesis
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA-Binding Proteins/genetics/metabolism
Endodeoxyribonucleases/*genetics/metabolism
Endoribonucleases/genetics/metabolism
Escherichia coli Proteins/genetics/metabolism
Kinetics
Oligoribonucleotides/*biosynthesis
Phosphorothioate Oligonucleotides/pharmacology
RNA Cleavage
RNA Viruses/*genetics/metabolism
RNA, Viral/*genetics/metabolism
RNA-Binding Proteins/genetics/metabolism
Sulfolobus solfataricus/drug effects/*genetics/immunology/metabolism
Time Factors

@article {pmid29174564,
year = {2018},
author = {Rauch, F and Geng, Y and Lamplugh, L and Hekmatnejad, B and Gaumond, MH and Penney, J and Yamanaka, Y and Moffatt, P},
title = {Crispr-Cas9 engineered osteogenesis imperfecta type V leads to severe skeletal deformities and perinatal lethality in mice.},
journal = {Bone},
volume = {107},
number = {},
pages = {131-142},
doi = {10.1016/j.bone.2017.11.013},
pmid = {29174564},
issn = {1873-2763},
support = {MOP-137102//CIHR/Canada ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Disease Models, Animal ; Gene Editing/methods ; Gene Knock-In Techniques ; Inflammation/genetics/pathology ; Membrane Proteins/*genetics ; Mesenchymal Stem Cells/metabolism/pathology ; Mice ; Mutation ; Osteoblasts/metabolism/*pathology ; Osteogenesis/genetics ; Osteogenesis Imperfecta/*genetics/*pathology ; },
abstract = {Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and μCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Cell Differentiation/genetics
*Disease Models, Animal
Gene Editing/methods
Gene Knock-In Techniques
Inflammation/genetics/pathology
Membrane Proteins/*genetics
Mesenchymal Stem Cells/metabolism/pathology
Mice
Mutation
Osteoblasts/metabolism/*pathology
Osteogenesis/genetics
Osteogenesis Imperfecta/*genetics/*pathology

@article {pmid30878030,
year = {2018},
author = {Makarova, SS and Khromov, AV and Spechenkova, NA and Taliansky, ME and Kalinina, NO},
title = {Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.},
journal = {Biochemistry. Biokhimiia},
volume = {83},
number = {12},
pages = {1552-1562},
doi = {10.1134/S0006297918120131},
pmid = {30878030},
issn = {1608-3040},
abstract = {The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.},
}

@article {pmid30877356,
year = {2019},
author = {Zhang, YT and Jiang, JY and Shi, TQ and Sun, XM and Zhao, QY and Huang, H and Ren, LJ},
title = {Application of the CRISPR/Cas system for genome editing in microalgae.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-019-09726-x},
pmid = {30877356},
issn = {1432-0614},
support = {21878151//National Natural Science Foundation of China/ ; BK20160092//Outstanding Youth Foundation of Jiangsu Province of China/ ; 2015//Program for Innovative Research Teams in Universities of Jiangsu Province/ ; XTE1829//Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture/ ; 18KJB530007//General Program on Natural Science Research Projects of Higher Education of Jiangsu/ ; },
abstract = {Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.},
}

@article {pmid30875129,
year = {2019},
author = {Musharova, O and Sitnik, V and Vlot, M and Savitskaya, E and Datsenko, KA and Krivoy, A and Fedorov, I and Semenova, E and Brouns, SJJ and Severinov, K},
title = {Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.14237},
pmid = {30875129},
issn = {1365-2958},
abstract = {CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference, and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition. This article is protected by copyright. All rights reserved.},
}

@article {pmid30874768,
year = {2019},
author = {Atmadjaja, AN and Holby, V and Harding, AJ and Krabben, P and Smith, HK and Jenkinson, ER},
title = {CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz059},
pmid = {30874768},
issn = {1574-6968},
abstract = {The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol (ABE) process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.},
}

@article {pmid30871003,
year = {2019},
author = {Ashley, CL and Abendroth, A and McSharry, BP and Slobedman, B},
title = {Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression.},
journal = {Viruses},
volume = {11},
number = {3},
pages = {},
doi = {10.3390/v11030246},
pmid = {30871003},
issn = {1999-4915},
support = {1063738//National Health and Medical Research Council/ ; },
abstract = {The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.},
}

@article {pmid30260998,
year = {2018},
author = {Veach, RA and Wilson, MH},
title = {CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0204487},
pmid = {30260998},
issn = {1932-6203},
support = {I01 BX002190/BX/BLRD VA/United States ; I01 BX004258/BX/BLRD VA/United States ; R01 DK093660/DK/NIDDK NIH HHS/United States ; },
mesh = {Acute Kidney Injury/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cisplatin/pharmacology ; Gene Knock-In Techniques ; Gene Targeting ; Genes, Reporter ; Genetic Engineering ; Glucose/pharmacology ; Green Fluorescent Proteins/genetics ; Hepatitis A Virus Cellular Receptor 1/*genetics/*metabolism ; Homologous Recombination ; Humans ; Kidney Tubules, Proximal/cytology/drug effects/*metabolism ; Luciferases/genetics ; Up-Regulation/drug effects ; },
abstract = {We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.},
}

Acute Kidney Injury/genetics/metabolism
CRISPR-Cas Systems
Cell Line
Cisplatin/pharmacology
Gene Knock-In Techniques
Gene Targeting
Genes, Reporter
Genetic Engineering
Glucose/pharmacology
Green Fluorescent Proteins/genetics
Hepatitis A Virus Cellular Receptor 1/*genetics/*metabolism
Homologous Recombination
Humans
Kidney Tubules, Proximal/cytology/drug effects/*metabolism
Luciferases/genetics
Up-Regulation/drug effects

@article {pmid30043304,
year = {2018},
author = {Curtin, SJ},
title = {Editing the Medicago truncatula Genome: Targeted Mutagenesis Using the CRISPR-Cas9 Reagent.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1822},
number = {},
pages = {161-174},
doi = {10.1007/978-1-4939-8633-0_12},
pmid = {30043304},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Chromosome Deletion ; Chromosomes, Plant ; *Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Genetic Engineering ; *Genome, Plant ; *Genomics/methods ; Mutagenesis ; },
abstract = {Medicago truncatula is an annual plant used for studying legume biology, in particular symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi. Efforts to decipher the genetic basis of these ecologically and economically important traits are a major goal of plant and crop biology. M. truncatula is an excellent model system for this purpose, as it has several publicly available sequenced genomes, has a rapid seed-to-seed generation time, and is highly transformable. Various mutagenesis platforms such as Tnt1 retrotransposons and RNAi knockdown have been used successfully in forward and reverse genetic studies to identify and functionally characterize candidate genes. The CRISPR/Cas9 reagent is the most recent mutagenesis platform and is highly effective at generating site-directed double-stranded breaks (DSB) in M. truncatula. This protocol will demonstrate the construction of reagents using two genome engineering platforms that have successfully generated mutant plants in M. truncatula, M. sativa, and soybean systems. The reagents are easy to assemble, can be quickly retrofitted to test novel regulatory sequences for improved efficiency, and can be used for more advanced genome engineering strategies such as gene insertion or gene replacement.},
}

*CRISPR-Cas Systems
Chromosome Deletion
Chromosomes, Plant
*Gene Editing
Gene Knockout Techniques
Genes, Plant
Genetic Engineering
*Genome, Plant
*Genomics/methods
Mutagenesis

@article {pmid29475732,
year = {2018},
author = {Fehse, B and Abramowski-Mock, U},
title = {The Time Is Ripe for Somatic Genome Editing: NIH Program to Strengthen Translation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {3},
pages = {671-674},
pmid = {29475732},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Capital Financing ; *Gene Editing/methods ; *Genome ; *Genomics/methods ; Humans ; National Institutes of Health (U.S.) ; Transcription Activator-Like Effector Nucleases ; Translational Medical Research/methods/organization & administration ; United States ; },
}

Animals
CRISPR-Cas Systems
Capital Financing
*Gene Editing/methods
*Genome
*Genomics/methods
Humans
National Institutes of Health (U.S.)
Transcription Activator-Like Effector Nucleases
Translational Medical Research/methods/organization & administration
United States

@article {pmid29324392,
year = {2018},
author = {Banaszak, LG and Giudice, V and Zhao, X and Wu, Z and Gao, S and Hosokawa, K and Keyvanfar, K and Townsley, DM and Gutierrez-Rodrigues, F and Fernandez Ibanez, MDP and Kajigaya, S and Young, NS},
title = {Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.},
journal = {Blood cells, molecules & diseases},
volume = {69},
number = {},
pages = {10-22},
doi = {10.1016/j.bcmd.2017.12.002},
pmid = {29324392},
issn = {1096-0961},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Apoptosis/genetics ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Proliferation ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Damage ; *Gene Editing ; *Genomic Instability ; Humans ; K562 Cells ; Mutation ; *RNA Splicing ; Sequence Analysis, DNA ; Spliceosomes/metabolism ; },
abstract = {DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.},
}

Apoptosis/genetics
*CRISPR-Cas Systems
Cell Differentiation/genetics
Cell Line, Tumor
Cell Proliferation
DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism
DNA Damage
*Gene Editing
*Genomic Instability
Humans
K562 Cells
Mutation
*RNA Splicing
Sequence Analysis, DNA
Spliceosomes/metabolism

@article {pmid28811591,
year = {2017},
author = {Zhang, X and Li, W and Liu, C and Peng, X and Lin, J and He, S and Li, X and Han, B and Zhang, N and Wu, Y and Chen, L and Wang, L and MaYila, and Huang, J and Liu, M},
title = {Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {8149},
pmid = {28811591},
issn = {2045-2322},
mesh = {Agouti Signaling Protein/*genetics ; Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Caspase 9/metabolism ; Gene Editing ; Gene Targeting ; Genetic Loci ; Genotype ; Hair Color/*genetics ; Phenotype ; Polymorphism, Single Nucleotide ; RNA, Guide ; RNA, Messenger/genetics ; Sequence Analysis, DNA ; Sheep/*genetics ; Zygote/metabolism ; },
abstract = {Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D9 and/or D5 preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D9/D5 mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.},
}

Agouti Signaling Protein/*genetics
Amino Acid Sequence
Animals
Base Sequence
*CRISPR-Cas Systems
Caspase 9/metabolism
Gene Editing
Gene Targeting
Genetic Loci
Genotype
Hair Color/*genetics
Phenotype
Polymorphism, Single Nucleotide
RNA, Guide
RNA, Messenger/genetics
Sequence Analysis, DNA
Sheep/*genetics
Zygote/metabolism

@article {pmid30870431,
year = {2019},
author = {Thomas, M and Burgio, G and Adams, DJ and Iyer, V},
title = {Collateral damage and CRISPR genome editing.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1007994},
doi = {10.1371/journal.pgen.1007994},
pmid = {30870431},
issn = {1553-7404},
abstract = {The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.},
}

@article {pmid30867524,
year = {2019},
author = {Odamaki, T and Bottacini, F and Mitsuyama, E and Yoshida, K and Kato, K and Xiao, JZ and van Sinderen, D},
title = {Impact of a bathing tradition on shared gut microbe among Japanese families.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4380},
doi = {10.1038/s41598-019-40938-3},
pmid = {30867524},
issn = {2045-2322},
support = {FEMS-RG-2016-0103//Federation of European Microbiological Societies (FEMS)/ ; 17/IFB/5429//Science Foundation Ireland (SFI)/ ; SFI/12/RC/2273//Science Foundation Ireland (SFI)/ ; },
abstract = {Sharing of Bifidobacterium longum strains had recently been shown to occur among Japanese family members, a phenomenon that is not confined to mother-infant pairs. In the current study, we investigated if bathtub water is a possible vehicle for the exchange of strains as a consequence of a Japanese custom to share bathtub water by family members during bathing practices. A total of twenty-one subjects from five Japanese families, each consisting of parents with either 2 or 3 children, were enrolled in this study and the fecal microbiota of all participants was determined. Viable bifidobacterial strains were isolated from all bathtub water samples. A subsequent comparative genome analysis using ninety-eight strains indicated that certain strain-sets, which were isolated from feces and bathtub water, share near identical genome sequences, including CRISPR/Cas protospacers. By means of unweighted UniFrac distance analysis based on 16S rRNA gene analysis of 59 subjects from sixteen Japanese families, we showed that the fecal microbiota composition among family members that share bathtub water is significantly closer than that between family members that do not engage in this practice. Our results indicate that bathtub water represents a vehicle for the transmission of gut bacteria, and that the Japanese custom of sharing bathtub water contributes to the exchange of gut microbes, in particular bifidobacteria, among family members.},
}

@article {pmid30867223,
year = {2019},
author = {Thormann, V and Glaser, LV and Rothkegel, MC and Borschiwer, M and Bothe, M and Fuchs, A and Meijsing, SH},
title = {Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.},
journal = {Life science alliance},
volume = {2},
number = {2},
pages = {},
doi = {10.26508/lsa.201800283},
pmid = {30867223},
issn = {2575-1077},
abstract = {The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.},
}

@article {pmid30520725,
year = {2018},
author = {OhAinle, M and Helms, L and Vermeire, J and Roesch, F and Humes, D and Basom, R and Delrow, JJ and Overbaugh, J and Emerman, M},
title = {A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
doi = {10.7554/eLife.39823},
pmid = {30520725},
issn = {2050-084X},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; CFAR New Investigator Award, P30 AI027757/AI/NIAID NIH HHS/United States ; DP1 DA039543/DA/NIDA NIH HHS/United States ; P30 CA015704-43/CA/NCI NIH HHS/United States ; P30 DK056465/DK/NIDDK NIH HHS/United States ; R01 AI030927/AI/NIAID NIH HHS/United States ; CCEH Pilot Grant, DK56465/DK/NIDDK NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI30927/AI/NIAID NIH HHS/United States ; R37 AI030927/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD/genetics/immunology ; Antigens, Differentiation/genetics/immunology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/immunology ; Cell Line, Tumor ; Epithelial Cells/drug effects/*immunology/virology ; GPI-Linked Proteins/genetics/immunology ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Vectors/chemistry/immunology ; HEK293 Cells ; HIV-1/drug effects/*genetics/growth & development/immunology ; *Host-Pathogen Interactions ; Humans ; Interferon-alpha/pharmacology ; Lentivirus/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/immunology ; Nuclear Proteins/deficiency/*genetics/immunology ; Phosphotransferases (Alcohol Group Acceptor)/deficiency/*genetics/immunology ; Receptors, CCR5/genetics/immunology ; Receptors, CXCR4/genetics/immunology ; Signal Transduction ; THP-1 Cells ; Viral Tropism/genetics ; Virus Assembly/drug effects ; Virus Replication/drug effects ; },
abstract = {Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized.

Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).},
}

Antigens, CD/genetics/immunology
Antigens, Differentiation/genetics/immunology
CRISPR-Cas Systems
Carrier Proteins/genetics/immunology
Cell Line, Tumor
Epithelial Cells/drug effects/*immunology/virology
GPI-Linked Proteins/genetics/immunology
Gene Editing/*methods
Gene Expression Regulation
Genetic Vectors/chemistry/immunology
HEK293 Cells
HIV-1/drug effects/*genetics/growth & development/immunology
*Host-Pathogen Interactions
Humans
Interferon-alpha/pharmacology
Lentivirus/genetics/metabolism
Myxovirus Resistance Proteins/genetics/immunology
Nuclear Proteins/deficiency/*genetics/immunology
Phosphotransferases (Alcohol Group Acceptor)/deficiency/*genetics/immunology
Receptors, CCR5/genetics/immunology
Receptors, CXCR4/genetics/immunology
Signal Transduction
THP-1 Cells
Viral Tropism/genetics
Virus Assembly/drug effects
Virus Replication/drug effects

@article {pmid30332437,
year = {2018},
author = {Morozova, KN and Suldina, LA and Malankhanova, TB and Grigor'eva, EV and Zakian, SM and Kiseleva, E and Malakhova, AA},
title = {Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0204735},
pmid = {30332437},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Clone Cells/metabolism/ultrastructure ; Endoplasmic Reticulum/ultrastructure ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Huntingtin Protein/antagonists & inhibitors/*genetics ; Huntington Disease/*genetics/*pathology ; Lysosomes/ultrastructure ; Microscopy, Electron, Transmission ; Mitochondria/ultrastructure ; Mutant Proteins/*genetics ; Mutation ; *Trinucleotide Repeat Expansion ; },
abstract = {Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.},
}

CRISPR-Cas Systems
Clone Cells/metabolism/ultrastructure
Endoplasmic Reticulum/ultrastructure
Gene Knockout Techniques
HEK293 Cells
Humans
Huntingtin Protein/antagonists & inhibitors/*genetics
Huntington Disease/*genetics/*pathology
Lysosomes/ultrastructure
Microscopy, Electron, Transmission
Mitochondria/ultrastructure
Mutant Proteins/*genetics
Mutation
*Trinucleotide Repeat Expansion

@article {pmid30864562,
year = {2019},
author = {Veigl, SJ},
title = {A use/disuse paradigm for CRISPR-Cas systems.},
journal = {Biology & philosophy},
volume = {34},
number = {1},
pages = {13},
doi = {10.1007/s10539-018-9661-z},
pmid = {30864562},
issn = {0169-3867},
}

@article {pmid30353190,
year = {2018},
author = {Neff, EP},
title = {CRISPR-barcoding the mouse.},
journal = {Lab animal},
volume = {47},
number = {11},
pages = {309},
doi = {10.1038/s41684-018-0190-9},
pmid = {30353190},
issn = {1548-4475},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; Mutation ; },
}

Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Mice
Mutation

@article {pmid30353159,
year = {2018},
author = {Parthasarathy, S},
title = {Use the patent system to regulate gene editing.},
journal = {Nature},
volume = {562},
number = {7728},
pages = {486-488},
doi = {10.1038/d41586-018-07108-3},
pmid = {30353159},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clinical Trials as Topic/ethics/legislation & jurisprudence ; Congresses as Topic ; European Union ; Gene Editing/*ethics/*legislation & jurisprudence/statistics & numerical data ; *Government Regulation ; History, 19th Century ; History, 20th Century ; History, 21st Century ; Human Embryonic Stem Cells ; Humans ; Licensure ; Patents as Topic/history/*legislation & jurisprudence/statistics & numerical data ; Swine/genetics ; United States ; },
}

Animals
CRISPR-Cas Systems/*genetics
Clinical Trials as Topic/ethics/legislation & jurisprudence
Congresses as Topic
European Union
Gene Editing/*ethics/*legislation & jurisprudence/statistics & numerical data
*Government Regulation
History, 19th Century
History, 20th Century
History, 21st Century
Human Embryonic Stem Cells
Humans
Licensure
Patents as Topic/history/*legislation & jurisprudence/statistics & numerical data
Swine/genetics
United States

@article {pmid30087231,
year = {2018},
author = {Ibba, G and Piu, C and Uleri, E and Serra, C and Dolei, A},
title = {Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis.},
journal = {Viruses},
volume = {10},
number = {8},
pages = {},
pmid = {30087231},
issn = {1999-4915},
mesh = {Amyotrophic Lateral Sclerosis/*pathology/therapy ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; DNA-Binding Proteins/genetics ; Endogenous Retroviruses/*genetics/pathogenicity ; Gene Editing ; Gene Expression Regulation ; Humans ; Male ; NF-kappa B/genetics ; Neoplasms/*virology ; Oncogenes/*genetics ; Prostatic Neoplasms ; RNA, Viral ; Serine-Arginine Splicing Factors/genetics ; Staphylococcus aureus/enzymology ; Viral Envelope Proteins/*genetics ; },
abstract = {The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.},
}

Amyotrophic Lateral Sclerosis/*pathology/therapy
CRISPR-Cas Systems
Carcinogenesis/genetics
Cell Line, Tumor
DNA-Binding Proteins/genetics
Endogenous Retroviruses/*genetics/pathogenicity
Gene Editing
Gene Expression Regulation
Humans
Male
NF-kappa B/genetics
Neoplasms/*virology
Oncogenes/*genetics
Prostatic Neoplasms
RNA, Viral
Serine-Arginine Splicing Factors/genetics
Staphylococcus aureus/enzymology
Viral Envelope Proteins/*genetics

@article {pmid29961307,
year = {2018},
author = {Kim, YK and Nam, SA and Yang, CW},
title = {Applications of kidney organoids derived from human pluripotent stem cells.},
journal = {The Korean journal of internal medicine},
volume = {33},
number = {4},
pages = {649-659},
pmid = {29961307},
issn = {2005-6648},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Kidney ; *Kidney Diseases/therapy ; *Organoids ; *Pluripotent Stem Cells ; },
abstract = {The establishment of protocols to differentiate kidney organoids from human pluripotent stem cells provides potential applications of kidney organoids in regenerative medicine. Modeling of renal diseases, drug screening, nephrotoxicity testing of compounds, and regenerative therapy are attractive applications. Although much progress still remains to be made in the development of kidney organoids, recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated system 9 (Cas9) genome editing and three-dimensional bioprinting technologies have contributed to the application of kidney organoids in clinical fields. In this section, we review recent advances in the applications of kidney organoids to kidney disease modelling, drug screening, nephrotoxicity testing, and regenerative therapy.},
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Humans
Kidney
*Kidney Diseases/therapy
*Organoids
*Pluripotent Stem Cells

@article {pmid29906440,
year = {2018},
author = {},
title = {CRISPR Inspirations.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1560-1561},
doi = {10.1016/j.cell.2018.05.055},
pmid = {29906440},
issn = {1097-4172},
mesh = {Bacteriophages/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Haloferax volcanii/genetics/metabolism ; Immunity/genetics ; Streptococcus thermophilus/genetics ; },
}

Bacteriophages/genetics/metabolism
CRISPR-Cas Systems/*genetics
Escherichia coli/genetics/metabolism
Gene Editing
Haloferax volcanii/genetics/metabolism
Immunity/genetics
Streptococcus thermophilus/genetics

@article {pmid29906439,
year = {2018},
author = {},
title = {The Ongoing Shakeup in Organelle Biology.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1557-1559},
doi = {10.1016/j.cell.2018.05.054},
pmid = {29906439},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Organelles/*metabolism ; Protein Interaction Maps ; Proteins/genetics/metabolism ; },
abstract = {With the complexities of organelle communication and their dynamics under intense investigation, what are the new principles that are emerging, and where is the field headed? Cell's Robert Kruger recently discussed these questions with Erika Holzbaur, Jennifer Lippincott-Schwartz, and Ivan Dikic. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online.},
}

Animals
CRISPR-Cas Systems/genetics
Organelles/*metabolism
Protein Interaction Maps
Proteins/genetics/metabolism

@article {pmid29812974,
year = {2018},
author = {Luo, JJ and Bian, WP and Liu, Y and Huang, HY and Yin, Q and Yang, XJ and Pei, DS},
title = {CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {32},
number = {9},
pages = {5132-5142},
doi = {10.1096/fj.201800077RR},
pmid = {29812974},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/methods ; Genetic Therapy/methods ; Genome/*genetics ; Promoter Regions, Genetic/genetics ; Zebrafish/*genetics ; },
abstract = {Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.},
}

Animals
Animals, Genetically Modified/genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Gene Transfer Techniques
Genetic Engineering/methods
Genetic Therapy/methods
Genome/*genetics
Promoter Regions, Genetic/genetics
Zebrafish/*genetics

@article {pmid29794019,
year = {2018},
author = {Atanasov, KE and Liu, C and Erban, A and Kopka, J and Parker, JE and Alcázar, R},
title = {NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance.},
journal = {Plant physiology},
volume = {177},
number = {3},
pages = {1152-1169},
pmid = {29794019},
issn = {1532-2548},
mesh = {3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics ; Arabidopsis/genetics/*immunology/*microbiology ; Arabidopsis Proteins/*genetics/immunology ; CRISPR-Cas Systems ; Chimera ; Disease Resistance/*genetics/immunology ; Epistasis, Genetic ; Gene Expression Regulation, Plant ; *Mutation ; NLR Proteins/genetics ; Oomycetes/pathogenicity ; Plant Diseases/*genetics ; Plants, Genetically Modified ; Poland ; Proto-Oncogene Proteins c-myb/genetics ; Quantitative Trait Loci ; Self-Incompatibility in Flowering Plants/genetics/immunology ; Tobacco ; },
abstract = {Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.},
}

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics
Arabidopsis/genetics/*immunology/*microbiology
Arabidopsis Proteins/*genetics/immunology
CRISPR-Cas Systems
Chimera
Disease Resistance/*genetics/immunology
Epistasis, Genetic
Gene Expression Regulation, Plant
*Mutation
NLR Proteins/genetics
Oomycetes/pathogenicity
Plant Diseases/*genetics
Plants, Genetically Modified
Poland
Proto-Oncogene Proteins c-myb/genetics
Quantitative Trait Loci
Self-Incompatibility in Flowering Plants/genetics/immunology
Tobacco

@article {pmid29779948,
year = {2018},
author = {Koren, I and Timms, RT and Kula, T and Xu, Q and Li, MZ and Elledge, SJ},
title = {The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1622-1635.e14},
pmid = {29779948},
issn = {1097-4172},
support = {//Wellcome Trust/United Kingdom ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AG011085/AG/NIA NIH HHS/United States ; 201387/Z/16/Z//Wellcome Trust/United Kingdom ; },
mesh = {Amino Acid Motifs ; Animals ; Antigens, Neoplasm/metabolism ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Genetic Vectors/genetics/metabolism ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Leupeptins/pharmacology ; Open Reading Frames/genetics ; Peptides/metabolism ; Proteasome Endopeptidase Complex/chemistry/metabolism ; Protein Stability/drug effects ; Protein Subunits/metabolism ; Proteolysis ; Proteome/genetics/*metabolism ; Receptors, Cytokine/genetics/metabolism ; Ubiquitin-Protein Ligases/*metabolism ; },
abstract = {Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.},
}

Amino Acid Motifs
Animals
Antigens, Neoplasm/metabolism
CRISPR-Cas Systems/genetics
Computational Biology/methods
Genetic Vectors/genetics/metabolism
HEK293 Cells
Humans
Lentivirus/genetics
Leupeptins/pharmacology
Open Reading Frames/genetics
Peptides/metabolism
Proteasome Endopeptidase Complex/chemistry/metabolism
Protein Stability/drug effects
Protein Subunits/metabolism
Proteolysis
Proteome/genetics/*metabolism
Receptors, Cytokine/genetics/metabolism
Ubiquitin-Protein Ligases/*metabolism

@article {pmid29512112,
year = {2018},
author = {Howe, DK and Yeargan, M and Simpson, L and Dangoudoubiyam, S},
title = {Molecular Genetic Manipulation of Sarcocystis neurona.},
journal = {Current protocols in microbiology},
volume = {48},
number = {},
pages = {20D.2.1-20D.2.14},
doi = {10.1002/cpmc.48},
pmid = {29512112},
issn = {1934-8533},
mesh = {Animals ; CRISPR-Cas Systems ; Encephalomyelitis/parasitology/*veterinary ; *Genetic Techniques ; Horse Diseases/parasitology ; Horses ; Sarcocystis/*genetics/physiology ; Transfection/*methods ; },
abstract = {Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of S. neurona will aid in the control of EPM and neurologic disease in sea mammals, while also improving our understanding of a prominent branch on the apicomplexan phylogenetic tree. These protocols describe methods that expand the capabilities to study this prominent member of the Apicomplexa. © 2018 by John Wiley & Sons, Inc.},
}

Animals
CRISPR-Cas Systems
Encephalomyelitis/parasitology/*veterinary
*Genetic Techniques
Horse Diseases/parasitology
Horses
Sarcocystis/*genetics/physiology
Transfection/*methods

@article {pmid30176395,
year = {2018},
author = {Wu, Y and Chen, T and Liu, Y and Lv, X and Li, J and Du, G and Ledesma-Amaro, R and Liu, L},
title = {CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis.},
journal = {Metabolic engineering},
volume = {49},
number = {},
pages = {232-241},
doi = {10.1016/j.ymben.2018.08.012},
pmid = {30176395},
issn = {1096-7184},
mesh = {Acetylglucosamine/*biosynthesis/genetics ; *Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/biosynthesis/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Glucose/genetics/*metabolism ; Xylose/genetics/*metabolism ; },
abstract = {Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.},
}

Acetylglucosamine/*biosynthesis/genetics
*Bacillus subtilis/genetics/metabolism
Bacterial Proteins/biosynthesis/genetics
*CRISPR-Cas Systems
*Gene Expression Regulation, Bacterial
Glucose/genetics/*metabolism
Xylose/genetics/*metabolism

@article {pmid30126863,
year = {2018},
author = {Karlgren, M and Simoff, I and Keiser, M and Oswald, S and Artursson, P},
title = {CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition Tool Box.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {46},
number = {11},
pages = {1776-1786},
doi = {10.1124/dmd.118.082842},
pmid = {30126863},
issn = {1521-009X},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; Cytochrome P-450 Enzyme System/metabolism ; Gene Editing/methods ; Humans ; Inactivation, Metabolic/*physiology ; Pharmaceutical Preparations/*metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.},
}

Animals
CRISPR-Cas Systems/*physiology
Clustered Regularly Interspaced Short Palindromic Repeats/*physiology
Cytochrome P-450 Enzyme System/metabolism
Gene Editing/methods
Humans
Inactivation, Metabolic/*physiology
Pharmaceutical Preparations/*metabolism

@article {pmid29695624,
year = {2018},
author = {Vyas, VK and Bushkin, GG and Bernstein, DA and Getz, MA and Sewastianik, M and Barrasa, MI and Bartel, DP and Fink, GR},
title = {New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.},
journal = {mSphere},
volume = {3},
number = {2},
pages = {},
pmid = {29695624},
issn = {2379-5042},
support = {T32 GM007287/GM/NIGMS NIH HHS/United States ; R15 AI130950/AI/NIAID NIH HHS/United States ; R35 GM118135/GM/NIGMS NIH HHS/United States ; R01 GM035010/GM/NIGMS NIH HHS/United States ; F32 GM108201/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Candida albicans/genetics ; Candida glabrata/genetics ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Fungi/*genetics ; Gene Editing/*methods ; Genetic Vectors ; *Mutagenesis ; RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.},
}

*CRISPR-Cas Systems
Candida albicans/genetics
Candida glabrata/genetics
DNA Breaks, Double-Stranded
*DNA End-Joining Repair
Fungi/*genetics
Gene Editing/*methods
Genetic Vectors
*Mutagenesis
RNA, Guide/genetics
Saccharomyces cerevisiae/genetics

@article {pmid29587177,
year = {2018},
author = {O'Donnell, KA},
title = {Advances in functional genetic screening with transposons and CRISPR/Cas9 to illuminate cancer biology.},
journal = {Current opinion in genetics & development},
volume = {49},
number = {},
pages = {85-94},
doi = {10.1016/j.gde.2018.03.006},
pmid = {29587177},
issn = {1879-0380},
support = {R01 CA207763/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA Transposable Elements/*genetics ; Early Detection of Cancer ; *Genetic Testing ; Humans ; Mutagenesis, Insertional/*genetics ; Mutation/genetics ; Neoplasms/diagnosis/*genetics/pathology ; },
abstract = {Large-scale genome sequencing studies have identified a wealth of mutations in human tumors and have dramatically advanced the field of cancer genetics. However, the functional consequences of an altered gene in tumor progression cannot always be inferred from mutation status alone. This underscores the critical need for complementary methods to assign functional significance to mutated genes in cancer. Transposons are mobile genetic elements that serve as powerful tools for insertional mutagenesis. Over the last decade, investigators have employed mouse models with on-demand transposon-mediated mutagenesis to perform unbiased genetic screens to identify clinically relevant genes that participate in the pathogenesis of human cancer. Two distinct DNA transposon mutagenesis systems, Sleeping Beauty (SB) and PiggyBac (PB), have been applied extensively in vivo and more recently, in ex vivo settings. These studies have informed our understanding of the genes and pathways that drive cancer initiation, progression, and metastasis. This review highlights the latest progress on cancer gene identification for specific cancer subtypes, as well as new technological advances and incorporation of the CRISPR/Cas9 toolbox into transposon-mediated functional genetic studies.},
}

CRISPR-Cas Systems/genetics
DNA Transposable Elements/*genetics
Early Detection of Cancer
*Genetic Testing
Humans
Mutagenesis, Insertional/*genetics
Mutation/genetics
Neoplasms/diagnosis/*genetics/pathology

@article {pmid30856357,
year = {2019},
author = {Le Rhun, A and Escalera-Maurer, A and Bratovič, M and Charpentier, E},
title = {CRISPR-Cas in Streptococcus pyogenes.},
journal = {RNA biology},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/15476286.2019.1582974},
pmid = {30856357},
issn = {1555-8584},
abstract = {The discovery and characterization of the prokaryotic CRISPR-Cas immune system has led to a revolution in genome editing and engineering technologies. Despite the fact that most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of Streptococcus pyogenes, its biological importance in this organism has received little attention. Here, we provide a comprehensive overview of the current knowledge about CRISPR-Cas systems from S. pyogenes. We discuss how the interplay between CRISPR-mediated immunity and horizontal gene transfer might have modeled the evolution of this pathogen. We review the current literature about the CRISPR-Cas systems present in S. pyogenes (types I-C and II-A), and describe their distinctive biochemical and functional features. Finally, we summarize the main biotechnological applications that have arisen from the discovery of the CRISPR-Cas9 system in S. pyogenes.},
}

@article {pmid30853970,
year = {2019},
author = {Parmeciano Di Noto, G and Molina, MC and Quiroga, C},
title = {Insights Into Non-coding RNAs as Novel Antimicrobial Drugs.},
journal = {Frontiers in genetics},
volume = {10},
number = {},
pages = {57},
doi = {10.3389/fgene.2019.00057},
pmid = {30853970},
issn = {1664-8021},
abstract = {Multidrug resistant bacteria are a serious worldwide problem, especially carbapenem-resistant Enterobacteriaceae (such as Klebsiella pneumoniae and Escherichia coli), Acinetobacter baumannii and Pseudomonas aeruginosa. Since the emergence of extensive and pan-drug resistant bacteria there are few antibiotics left to treat patients, thus novel RNA-based strategies are being considered. Here, we examine the current situation of different non-coding RNAs found in bacteria as well as their function and potential application as antimicrobial agents. Furthermore, we discuss the factors that may contribute in the efficient development of RNA-based drugs, the limitations for their implementation and the use of nanocarriers for delivery.},
}

@article {pmid30853940,
year = {2019},
author = {Jaiswal, S and Singh, DK and Shukla, P},
title = {Gene Editing and Systems Biology Tools for Pesticide Bioremediation: A Review.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {87},
doi = {10.3389/fmicb.2019.00087},
pmid = {30853940},
issn = {1664-302X},
abstract = {Bioremediation is the degradation potential of microorganisms to dissimilate the complex chemical compounds from the surrounding environment. The genetics and biochemistry of biodegradation processes in datasets opened the way of systems biology. Systemic biology aid the study of interacting parts involved in the system. The significant keys of system biology are biodegradation network, computational biology, and omics approaches. Biodegradation network consists of all the databases and datasets which aid in assisting the degradation and deterioration potential of microorganisms for bioremediation processes. This review deciphers the bio-degradation network, i.e., the databases and datasets (UM-BBD, PAN, PTID, etc.) aiding in assisting the degradation and deterioration potential of microorganisms for bioremediation processes, computational biology and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation experiments. Besides, the present review also describes the gene editing tools like CRISPR Cas, TALEN, and ZFNs which can possibly make design microbe with functional gene of interest for degradation of particular recalcitrant for improved bioremediation.},
}

@article {pmid30809052,
year = {2019},
author = {Maxmen, A},
title = {Faster, better, cheaper: the rise of CRISPR in disease detection.},
journal = {Nature},
volume = {566},
number = {7745},
pages = {437},
doi = {10.1038/d41586-019-00601-3},
pmid = {30809052},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Nucleic Acids

@article {pmid30699116,
year = {2019},
author = {Leatham-Jensen, M and Uyehara, CM and Strahl, BD and Matera, AG and Duronio, RJ and McKay, DJ},
title = {Lysine 27 of replication-independent histone H3.3 is required for Polycomb target gene silencing but not for gene activation.},
journal = {PLoS genetics},
volume = {15},
number = {1},
pages = {e1007932},
doi = {10.1371/journal.pgen.1007932},
pmid = {30699116},
issn = {1553-7404},
support = {R01 GM129132/GM/NIGMS NIH HHS/United States ; R35 GM128851/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; DNA Replication/genetics ; DNA-Binding Proteins/genetics ; Drosophila melanogaster/genetics ; Epigenesis, Genetic/*genetics ; Gene Silencing ; Histone Code/genetics ; Histones/*genetics ; Humans ; Lysine/*genetics ; Methylation ; Polycomb-Group Proteins/*genetics ; Transcriptional Activation/genetics ; },
abstract = {Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.},
}

Alleles
Animals
CRISPR-Cas Systems/genetics
Chromatin/genetics
DNA Replication/genetics
DNA-Binding Proteins/genetics
Drosophila melanogaster/genetics
Epigenesis, Genetic/*genetics
Gene Silencing
Histone Code/genetics
Histones/*genetics
Humans
Lysine/*genetics
Methylation
Polycomb-Group Proteins/*genetics
Transcriptional Activation/genetics

@article {pmid30668559,
year = {2019},
author = {Zhu, YN and Wang, LZ and Li, CC and Cui, Y and Wang, M and Lin, YJ and Zhao, RP and Wang, W and Xiang, H},
title = {Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication.},
journal = {PLoS genetics},
volume = {15},
number = {1},
pages = {e1007616},
doi = {10.1371/journal.pgen.1007616},
pmid = {30668559},
issn = {1553-7404},
mesh = {Animals ; Bombyx/genetics/growth & development ; CRISPR-Cas Systems ; Domestication ; *Evolution, Molecular ; Extracellular Matrix/genetics ; Gene Knockout Techniques ; Genome, Insect/genetics ; Insect Proteins/*genetics ; Linkage Disequilibrium ; Metamorphosis, Biological/*genetics ; Phylogeny ; *Selection, Genetic ; Transcriptome/genetics ; },
abstract = {Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources.},
}

Animals
Bombyx/genetics/growth & development
CRISPR-Cas Systems
Domestication
*Evolution, Molecular
Extracellular Matrix/genetics
Gene Knockout Techniques
Genome, Insect/genetics
Insect Proteins/*genetics
Linkage Disequilibrium
Metamorphosis, Biological/*genetics
Phylogeny
*Selection, Genetic
Transcriptome/genetics

@article {pmid30082909,
year = {2019},
author = {Liu, Z and Zhang, W and Phillips, JB and Arora, R and McClellan, S and Li, J and Kim, JH and Sobol, RW and Tan, M},
title = {Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.},
journal = {Oncogene},
volume = {38},
number = {1},
pages = {88-102},
doi = {10.1038/s41388-018-0407-9},
pmid = {30082909},
issn = {1476-5594},
support = {R01 CA148629/CA/NCI NIH HHS/United States ; R01 CA149646/CA/NCI NIH HHS/United States ; R01CA149646//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/International ; CA148629//U.S. Department of Health & Human Services | NIH | Center for Scientific Review (CSR)/International ; },
mesh = {Animals ; B7 Antigens/antagonists & inhibitors/chemistry/genetics/*physiology ; Breast Neoplasms/*metabolism/pathology ; Butadienes/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Polarity ; Drug Resistance, Neoplasm/*physiology ; Enzyme Activation ; Female ; Humans ; MAP Kinase Kinase Kinases/antagonists & inhibitors/*physiology ; Mice ; Mice, Nude ; Nanog Homeobox Protein/biosynthesis/genetics ; Neoplasm Invasiveness ; Neoplasm Proteins/antagonists & inhibitors/genetics/*physiology ; Neoplastic Stem Cells/*metabolism ; Nitriles/pharmacology/therapeutic use ; Protein Domains ; Protein Interaction Mapping ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Proto-Oncogene Proteins B-raf/metabolism ; RNA Interference ; RNA, Guide/genetics ; RNA, Small Interfering/pharmacology ; Recombinant Proteins/metabolism ; SOXB1 Transcription Factors/biosynthesis/genetics ; Sequence Deletion ; Spheroids, Cellular ; Transfection ; Up-Regulation ; Vault Ribonucleoprotein Particles/*physiology ; },
abstract = {B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.},
}

Animals
B7 Antigens/antagonists & inhibitors/chemistry/genetics/*physiology
Breast Neoplasms/*metabolism/pathology
Butadienes/pharmacology/therapeutic use
CRISPR-Cas Systems
Cell Line, Tumor
Cell Polarity
Drug Resistance, Neoplasm/*physiology
Enzyme Activation
Female
Humans
MAP Kinase Kinase Kinases/antagonists & inhibitors/*physiology
Mice
Mice, Nude
Nanog Homeobox Protein/biosynthesis/genetics
Neoplasm Invasiveness
Neoplasm Proteins/antagonists & inhibitors/genetics/*physiology
Neoplastic Stem Cells/*metabolism
Nitriles/pharmacology/therapeutic use
Protein Domains
Protein Interaction Mapping
Protein Kinase Inhibitors/pharmacology/therapeutic use
Proto-Oncogene Proteins B-raf/metabolism
RNA Interference
RNA, Guide/genetics
RNA, Small Interfering/pharmacology
Recombinant Proteins/metabolism
SOXB1 Transcription Factors/biosynthesis/genetics
Sequence Deletion
Spheroids, Cellular
Transfection
Up-Regulation
Vault Ribonucleoprotein Particles/*physiology

@article {pmid29987734,
year = {2018},
author = {Allorent, G and Guglielmino, E and Giustini, C and Courtois, F},
title = {Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1829},
number = {},
pages = {367-378},
doi = {10.1007/978-1-4939-8654-5_24},
pmid = {29987734},
issn = {1940-6029},
mesh = {Biolistics/instrumentation/*methods ; *CRISPR-Cas Systems ; Cell Nucleus/genetics ; Chloroplast Proteins/*genetics ; Diatoms/*genetics ; Gene Editing ; Gene Knockout Techniques ; *Mutation ; },
abstract = {Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.},
}

Biolistics/instrumentation/*methods
*CRISPR-Cas Systems
Cell Nucleus/genetics
Chloroplast Proteins/*genetics
Diatoms/*genetics
Gene Editing
Gene Knockout Techniques
*Mutation

@article {pmid29804851,
year = {2018},
author = {Wang, J and Ji, W and Zhu, D and Wang, W and Chen, Y and Zhang, Z and Li, F},
title = {Tfap2b mutation in mice results in patent ductus arteriosus and renal malformation.},
journal = {The Journal of surgical research},
volume = {227},
number = {},
pages = {178-185},
doi = {10.1016/j.jss.2018.02.038},
pmid = {29804851},
issn = {1095-8673},
mesh = {Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Ductus Arteriosus, Patent/*genetics ; Exons/genetics ; Female ; Homozygote ; Humans ; Kidney/*abnormalities ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutagenesis ; Sequence Deletion ; Transcription Factor AP-2/*genetics ; },
abstract = {BACKGROUND: Transcription factor TFAP2B is associated with Char syndrome in humans and is characterized by patent ductus arteriosus (PDA) and facial and finger abnormalities. In a previous study, we detected a c.435_438delCCGG TFAP2B mutation in a family with PDA, and no facial dysmorphism or finger abnormalities were observed. This 4-base pair (bp) deletion in exon 2 resulted in a truncated protein of about 21 kDa in cultured cells in vitro. However, it is not clear why c.435_438delCCGG mutation carriers are present with isolated PDA instead of Char syndrome.

MATERIALS AND METHODS: We successfully established a mouse model bearing Tfap2b c.435_438delCCGG mutation using CRISPR/Cas9 technology. The mutant mice were phenotyped using histological analysis, and the development of ductus smooth muscles in mutant mice was examined by immunohistochemistry.

RESULTS: The c.435_438delCCGG homozygous mutant mice were characterized by delayed closure of the ductus arteriosus (DA) and renal malformation. Furthermore, the c.435_438delCCGG mutation might result in PDA by affecting the development of ductus arterious smooth muscle cells.

CONCLUSIONS: Using the c.435_438delCCGG homozygous mice, we verified the nature of the c.435_438delCCGG mutation and established a new and useful animal model to explore the function of Tfap2b and the mechanisms of PDA and renal formation. These findings may be useful for the development of therapies for those rare disorder.},
}

Animals
Base Sequence/genetics
CRISPR-Cas Systems/genetics
Disease Models, Animal
Ductus Arteriosus, Patent/*genetics
Exons/genetics
Female
Homozygote
Humans
Kidney/*abnormalities
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Mutagenesis
Sequence Deletion
Transcription Factor AP-2/*genetics

@article {pmid29446996,
year = {2018},
author = {Yang, Y and Wang, Q and Li, Q and Men, K and He, Z and Deng, H and Ji, W and Wei, Y},
title = {Recent Advances in Therapeutic Genome Editing in China.},
journal = {Human gene therapy},
volume = {29},
number = {2},
pages = {136-145},
doi = {10.1089/hum.2017.210},
pmid = {29446996},
issn = {1557-7422},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Gene Editing/*trends ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*trends ; Genome, Human ; Humans ; },
abstract = {Editing of the genome to correct disease-causing mutations is a promising approach for the treatment of human diseases. Recent advances in the development of programmable nuclease-based genome editing tools have substantially improved the ability to make precise changes in the human genome. Genome editing technologies are already being used to correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional gene therapies. Chinese scientists have made remarkable breakthroughs in the field of therapeutic genome editing, particularly with the first clinical trial involving the clustered regularly interspaced short palindromic repeats-caspase 9 system that began in China. Herein, current progress toward developing programmable nuclease-based gene therapies is introduced, as well as future prospects and challenges in China.},
}

CRISPR-Cas Systems/*genetics
China
Gene Editing/*trends
Genetic Diseases, Inborn/genetics/*therapy
Genetic Therapy/*trends
Genome, Human
Humans

@article {pmid29338433,
year = {2018},
author = {He, ZY and Zhang, YG and Yang, YH and Ma, CC and Wang, P and Du, W and Li, L and Xiang, R and Song, XR and Zhao, X and Yao, SH and Wei, YQ},
title = {In Vivo Ovarian Cancer Gene Therapy Using CRISPR-Cas9.},
journal = {Human gene therapy},
volume = {29},
number = {2},
pages = {223-233},
doi = {10.1089/hum.2017.209},
pmid = {29338433},
issn = {1557-7422},
mesh = {CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; DNA (Cytosine-5-)-Methyltransferase 1/*genetics/therapeutic use ; Drug Resistance, Neoplasm/genetics ; Female ; Folate Receptor 1/genetics/therapeutic use ; Gene Editing ; Gene Expression Regulation, Neoplastic ; *Gene Transfer Techniques ; *Genetic Therapy ; Humans ; Liposomes/administration & dosage/chemistry ; Ovarian Neoplasms/drug therapy/*genetics/pathology ; Paclitaxel/administration & dosage/adverse effects ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) genome editing technology holds great promise for the field of human gene therapy. However, a lack of safe and effective delivery systems restricts its biomedical application. Here, a folate receptor-targeted liposome (F-LP) was used to deliver CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA targeting the ovarian cancer-related DNA methyltransferase 1 (DNMT1) gene (gDNMT1). F-LP efficiently bound the gDNMT1 plasmid and formed a stable complex (F-LP/gDNMT1) that was safe for injection. F-LP/gDNMT1 effectively mutated endogenous DNMT1 in vitro, and then expressed the Cas9 endonuclease and downregulated DNMT1 in vivo. The tumor growth of both paclitaxel-sensitive and -resistant ovarian cancers were inhibited by F-LP/gDNMT1, which shows fewer adverse effects than paclitaxel injection. Therefore, CRISPR-Cas9-targeted DNMT1 manipulation may be a potential therapeutic regimen for ovarian cancer, and lipid-mediated delivery systems represent promising delivery vectors of CRISPR-Cas9 technology for precise genome editing therapeutics.},
}

CRISPR-Cas Systems/genetics
Cell Proliferation/drug effects
DNA (Cytosine-5-)-Methyltransferase 1/*genetics/therapeutic use
Drug Resistance, Neoplasm/genetics
Female
Folate Receptor 1/genetics/therapeutic use
Gene Editing
Gene Expression Regulation, Neoplastic
*Gene Transfer Techniques
*Genetic Therapy
Humans
Liposomes/administration & dosage/chemistry
Ovarian Neoplasms/drug therapy/*genetics/pathology
Paclitaxel/administration & dosage/adverse effects

@article {pmid29120617,
year = {2017},
author = {Ma, Y and Wang, M and Li, W and Zhang, Z and Zhang, X and Wu, G and Tan, T and Cui, Z and Zhang, XE},
title = {Live Visualization of HIV-1 Proviral DNA Using a Dual-Color-Labeled CRISPR System.},
journal = {Analytical chemistry},
volume = {89},
number = {23},
pages = {12896-12901},
doi = {10.1021/acs.analchem.7b03584},
pmid = {29120617},
issn = {1520-6882},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Color ; DNA, Viral/*genetics ; HIV Infections/*diagnostic imaging ; HIV-1/*genetics ; Humans ; Proviruses/*genetics ; Quantum Dots/chemistry ; },
abstract = {HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mapped based on the colocalized signals of QDs in living cells. Compared to the existing zinc finger proteins and TALENs, the CRISPR system is much easier to operate and more efficient in imaging of internal genomic loci. Therefore, the proposed method could be not only a powerful tool for imaging proviral HIV-1, but also a versatile platform to image single genomic loci in living cells.},
}

CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Color
DNA, Viral/*genetics
HIV Infections/*diagnostic imaging
HIV-1/*genetics
Humans
Proviruses/*genetics
Quantum Dots/chemistry

@article {pmid30722791,
year = {2019},
author = {Gonçalves, E and Behan, FM and Louzada, S and Arnol, D and Stronach, EA and Yang, F and Yusa, K and Stegle, O and Iorio, F and Garnett, MJ},
title = {Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {27},
doi = {10.1186/s13059-019-1637-z},
pmid = {30722791},
issn = {1474-760X},
support = {//Wellcome Trust/United Kingdom ; C44943/A22536//Cancer Research UK/United Kingdom ; 206194//Wellcome Trust/United Kingdom ; 098051//Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Genomic Structural Variation ; Genomics/*methods ; Humans ; Neoplasms/genetics ; Ploidies ; Software ; *Whole Genome Sequencing ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed.

RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects.

CONCLUSION: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.},
}

*CRISPR-Cas Systems
Cell Line, Tumor
*Genomic Structural Variation
Genomics/*methods
Humans
Neoplasms/genetics
Ploidies
Software
*Whole Genome Sequencing

@article {pmid30717767,
year = {2019},
author = {Teng, F and Li, J and Cui, T and Xu, K and Guo, L and Gao, Q and Feng, G and Chen, C and Han, D and Zhou, Q and Li, W},
title = {Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {15},
doi = {10.1186/s13059-019-1620-8},
pmid = {30717767},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Mice ; },
abstract = {CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements. Here, we harness six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that remarkably increase the targeting range in the genomes. Moreover, we identify optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a.},
}

Animals
*CRISPR-Associated Proteins
*CRISPR-Cas Systems
Gene Editing/*methods
Humans
Mice

@article {pmid30678704,
year = {2019},
author = {Zhu, S and Cao, Z and Liu, Z and He, Y and Wang, Y and Yuan, P and Li, W and Tian, F and Bao, Y and Wei, W},
title = {Guide RNAs with embedded barcodes boost CRISPR-pooled screens.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {20},
doi = {10.1186/s13059-019-1628-0},
pmid = {30678704},
issn = {1474-760X},
mesh = {Bacterial Proteins ; Bacterial Toxins ; *CRISPR-Cas Systems ; Gene Targeting ; Genomics/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; *RNA, Guide ; },
abstract = {We report a new method using re-designed guide RNAs with internal barcodes (iBARs) embedded in their loop regions. Our iBAR approach outperforms the conventional method by producing screening results with much lower false-positive and false-negative rates especially with a high multiplicity of infection (MOI). Importantly, the iBAR approach reduces the starting cells at high MOI significantly with greatly improved efficiency and accuracy compared with the canonical CRISPR screens at a low MOI. This new system is particularly useful when the source of cells is limited or when it is difficult to control viral infection for in vivo screening.},
}

Bacterial Proteins
Bacterial Toxins
*CRISPR-Cas Systems
Gene Targeting
Genomics/*methods
HEK293 Cells
HeLa Cells
Humans
*RNA, Guide

@article {pmid30588718,
year = {2019},
author = {Jo, N and Sogabe, Y and Yamada, Y and Ukai, T and Kagawa, H and Mitsunaga, K and Woltjen, K and Yamada, Y},
title = {Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling.},
journal = {Cancer science},
volume = {110},
number = {3},
pages = {926-938},
doi = {10.1111/cas.13924},
pmid = {30588718},
issn = {1349-7006},
support = {JP16H06276//Japan Society for the Promotion of Science/ ; //Naito Foundation/ ; //Takeda Science Foundation/ ; JP18cm0106203//Japan Agency for Medical Research and Development/ ; JP18gm1110004//Japan Agency for Medical Research and Development/ ; },
mesh = {Adenoma/*genetics/pathology ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Gene Editing/methods ; Intestinal Neoplasms/*genetics/pathology ; Kidney Neoplasms/*genetics/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mutation/genetics ; RNA, Guide/*genetics ; Wilms Tumor/*genetics/pathology ; },
abstract = {The emergence of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has dramatically advanced how we manipulate the genome. Regarding in vivo experiments, Cas9-transgenic animals could provide efficient and complex genome editing. However, this potential has not been fully realized partly due to a lack of convenient platforms and limited examples of successful disease modeling. Here, we devised two doxycycline (Dox)-inducible Cas9 platforms that efficiently enable conditional genome editing at multiple loci in vitro and in vivo. In these platforms, we took advantage of a site-specific multi-segment cloning strategy for rapid and easy integration of multiple single guide (sg)RNAs. We found that a platform containing rtTA at the Rosa26 locus and TRE-Cas9 together with multiple sgRNAs at the Col1a1 locus showed higher efficiency of inducible insertions and deletions (indels) with minimal leaky editing. Using this platform, we succeeded to model Wilms' tumor and the progression of intestinal adenomas with multiple mutations including an activating mutation with a large genomic deletion. Collectively, the established platform should make complicated disease modeling in the mouse easily attainable, extending the range of in vivo experiments in various biological fields including cancer research.},
}

Adenoma/*genetics/pathology
Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Female
Gene Editing/methods
Intestinal Neoplasms/*genetics/pathology
Kidney Neoplasms/*genetics/pathology
Mice
Mice, Inbred C57BL
Mice, Inbred ICR
Mutation/genetics
RNA, Guide/*genetics
Wilms Tumor/*genetics/pathology

@article {pmid29987710,
year = {2018},
author = {Naim, F and Dugdale, B and Kleidon, J and Brinin, A and Shand, K and Waterhouse, P and Dale, J},
title = {Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9.},
journal = {Transgenic research},
volume = {27},
number = {5},
pages = {451-460},
pmid = {29987710},
issn = {1573-9368},
mesh = {Alleles ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Musa/*genetics ; Oxidoreductases/*genetics ; Plant Proteins/genetics ; Plants, Genetically Modified ; RNA, Guide ; },
abstract = {Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar "Williams". Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1-105 nt) or deletions (1-55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.},
}

Alleles
*CRISPR-Cas Systems
Gene Editing/*methods
Musa/*genetics
Oxidoreductases/*genetics
Plant Proteins/genetics
Plants, Genetically Modified
RNA, Guide

@article {pmid29522859,
year = {2018},
author = {Kherraf, ZE and Conne, B and Amiri-Yekta, A and Kent, MC and Coutton, C and Escoffier, J and Nef, S and Arnoult, C and Ray, PF},
title = {Creation of knock out and knock in mice by CRISPR/Cas9 to validate candidate genes for human male infertility, interest, difficulties and feasibility.},
journal = {Molecular and cellular endocrinology},
volume = {468},
number = {},
pages = {70-80},
doi = {10.1016/j.mce.2018.03.002},
pmid = {29522859},
issn = {1872-8057},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Female ; Gene Knock-In Techniques/*methods ; *Genetic Association Studies ; Humans ; Infertility, Male/*genetics ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation/genetics ; Nucleotide Motifs/genetics ; },
abstract = {High throughput sequencing (HTS) and CRISPR/Cas9 are two recent technologies that are currently revolutionizing biological and clinical research. Both techniques are complementary as HTS permits to identify new genetic variants and genes involved in various pathologies and CRISPR/Cas9 permits to create animals or cell models to validate the effect of the identified variants, to characterize the pathogeny of the identified variants and the function of the genes of interest and ultimately to provide ways of correcting the molecular defects. We analyzed a cohort of 78 infertile men presenting with multiple morphological anomalies of the sperm flagella (MMAF), a severe form of male infertility. Using whole exome sequencing (WES), homozygous mutations in autosomal candidate genes were identified in 63% of the tested subjects. We decided to produce by CRISPR/cas9 four knock-out (KO) and one knock-in (KI) mouse lines to confirm these results and to increase our understanding of the physiopathology associated with these genetic variations. Overall 31% of the live pups obtained presented a mutational event in one of the targeted regions. All identified events were insertions or deletions localized near the PAM sequence. Surprisingly we observed a high rate of germline mosaicism as 30% of the F1 displayed a different mutation than the parental event characterized on somatic tissue (tail), indicating that CRISPR/Cas9 mutational events kept happening several cell divisions after the injection. Overall, we created mouse models for 5 distinct loci and in each case homozygous animals could be obtained in approximately 6 months. These results demonstrate that the combined use of WES and CRISPR/Cas9 is an efficient and timely strategy to identify and validate mutations responsible for infertility phenotypes in human.},
}

Animals
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Female
Gene Knock-In Techniques/*methods
*Genetic Association Studies
Humans
Infertility, Male/*genetics
Male
Mice, Inbred C57BL
Mice, Knockout
Mutation/genetics
Nucleotide Motifs/genetics

@article {pmid29367726,
year = {2018},
author = {Sakuma, T and Yamamoto, T},
title = {Genome editing for dissecting and curing human genetic diseases.},
journal = {Journal of human genetics},
volume = {63},
number = {2},
pages = {105},
doi = {10.1038/s10038-017-0380-0},
pmid = {29367726},
issn = {1435-232X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Diseases, Inborn/*therapy ; *Genetic Therapy ; *Genome, Human ; Humans ; },
}

*CRISPR-Cas Systems
*Gene Editing
Genetic Diseases, Inborn/*therapy
*Genetic Therapy
*Genome, Human
Humans

@article {pmid29325827,
year = {2018},
author = {Nødvig, CS and Hoof, JB and Kogle, ME and Jarczynska, ZD and Lehmbeck, J and Klitgaard, DK and Mortensen, UH},
title = {Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli.},
journal = {Fungal genetics and biology : FG & B},
volume = {115},
number = {},
pages = {78-89},
doi = {10.1016/j.fgb.2018.01.004},
pmid = {29325827},
issn = {1096-0937},
mesh = {Aspergillus/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Breaks, Double-Stranded ; Gene Deletion ; Gene Editing/*methods ; Gene Targeting ; Genetic Vectors ; Oligonucleotides/genetics ; RNA, Transfer/genetics ; },
abstract = {CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.},
}

Aspergillus/*genetics
CRISPR-Cas Systems/*genetics
*DNA Breaks, Double-Stranded
Gene Deletion
Gene Editing/*methods
Gene Targeting
Genetic Vectors
Oligonucleotides/genetics
RNA, Transfer/genetics

@article {pmid28203699,
year = {2018},
author = {Yan, J and Chuai, G and Zhou, C and Zhu, C and Yang, J and Zhang, C and Gu, F and Xu, H and Wei, J and Liu, Q},
title = {Benchmarking CRISPR on-target sgRNA design.},
journal = {Briefings in bioinformatics},
volume = {19},
number = {4},
pages = {721-724},
doi = {10.1093/bib/bbx001},
pmid = {28203699},
issn = {1477-4054},
mesh = {Benchmarking/*methods ; *CRISPR-Cas Systems ; Computer Simulation ; *Gene Editing ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based gene editing has been widely implemented in various cell types and organisms. A major challenge in the effective application of the CRISPR system is the need to design highly efficient single-guide RNA (sgRNA) with minimal off-target cleavage. Several tools are available for sgRNA design, while limited tools were compared. In our opinion, benchmarking the performance of the available tools and indicating their applicable scenarios are important issues. Moreover, whether the reported sgRNA design rules are reproducible across different sgRNA libraries, cell types and organisms remains unclear. In our study, a systematic and unbiased benchmark of the sgRNA predicting efficacy was performed on nine representative on-target design tools, based on six benchmark data sets covering five different cell types. The benchmark study presented here provides novel quantitative insights into the available CRISPR tools.},
}

Benchmarking/*methods
*CRISPR-Cas Systems
Computer Simulation
*Gene Editing
Humans
RNA, Guide/*genetics

@article {pmid28132023,
year = {2017},
author = {Lu, XJ and Sun, HM and Xu, Y and Yu, X and Gu, B},
title = {The applications and advances of CRISPR-Cas9 in medical research.},
journal = {Briefings in functional genomics},
volume = {16},
number = {1},
pages = {1-3},
doi = {10.1093/bfgp/elw036},
pmid = {28132023},
issn = {2041-2657},
mesh = {*Biomedical Research ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Genetic Therapy ; Humans ; Models, Biological ; },
}

*Biomedical Research
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Genetic Therapy
Humans
Models, Biological

@article {pmid30537986,
year = {2018},
author = {Chen, N and Zhao, G and Yan, X and Lv, Z and Yin, H and Zhang, S and Song, W and Li, X and Li, L and Du, Z and Jia, L and Zhou, L and Li, W and Hoffman, AR and Hu, JF and Cui, J},
title = {A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {218},
doi = {10.1186/s13059-018-1594-y},
pmid = {30537986},
issn = {1474-760X},
support = {I01 BX002905/BX/BLRD VA/United States ; BX002905/VA/VA/United States ; },
mesh = {Breast Neoplasms/*metabolism ; CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism ; DNA Methylation ; *Gene Expression Regulation, Neoplastic ; Humans ; Mixed Function Oxygenases/metabolism ; Neoplasm Metastasis ; Proto-Oncogene Protein c-fli-1/*metabolism ; Proto-Oncogene Proteins/metabolism ; },
abstract = {BACKGROUND: Friend leukemia virus integration 1 (FLI1), an ETS transcription factor family member, acts as an oncogenic driver in hematological malignancies and promotes tumor growth in solid tumors. However, little is known about the mechanisms underlying the activation of this proto-oncogene in tumors.

RESULTS: Immunohistochemical staining showed that FLI1 is aberrantly overexpressed in advanced stage and metastatic breast cancers. Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. Notably, FECR1 utilizes a positive feedback mechanism to activate FLI1 by inducing DNA hypomethylation in CpG islands of the promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1, a demethylase that is actively involved in DNA demethylation. FECR1 also binds to and downregulates in trans DNMT1, a methyltransferase that is essential for the maintenance of DNA methylation.

CONCLUSIONS: These data suggest that FECR1 circular RNA acts as an upstream regulator to control breast cancer tumor growth by coordinating the regulation of DNA methylating and demethylating enzymes. Thus, FLI1 drives tumor metastasis not only through the canonical oncoprotein pathway, but also by using epigenetic mechanisms mediated by its exonic circular RNA.},
}

Breast Neoplasms/*metabolism
CRISPR-Cas Systems
DNA (Cytosine-5-)-Methyltransferase 1/metabolism
DNA Methylation
*Gene Expression Regulation, Neoplastic
Humans
Mixed Function Oxygenases/metabolism
Neoplasm Metastasis
Proto-Oncogene Protein c-fli-1/*metabolism
Proto-Oncogene Proteins/metabolism

@article {pmid30501614,
year = {2018},
author = {Zhang, Y and Massel, K and Godwin, ID and Gao, C},
title = {Applications and potential of genome editing in crop improvement.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {210},
doi = {10.1186/s13059-018-1586-y},
pmid = {30501614},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Plant Breeding ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism's genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.},
}

CRISPR-Cas Systems
Crops, Agricultural/*genetics
*Gene Editing
*Plant Breeding
Transcription Activator-Like Effector Nucleases
Zinc Finger Nucleases

@article {pmid30477585,
year = {2018},
author = {Chong, ZS and Ohnishi, S and Yusa, K and Wright, GJ},
title = {Pooled extracellular receptor-ligand interaction screening using CRISPR activation.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {205},
pmid = {30477585},
issn = {1474-760X},
support = {206194//Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Genomics/*methods ; Humans ; Ligands ; Receptors, Cell Surface/*analysis ; },
abstract = {Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale.},
}

CRISPR-Cas Systems
Genomics/*methods
Humans
Ligands
Receptors, Cell Surface/*analysis

@article {pmid30442181,
year = {2018},
author = {Ji, X and Si, X and Zhang, Y and Zhang, H and Zhang, F and Gao, C},
title = {Conferring DNA virus resistance with high specificity in plants using virus-inducible genome-editing system.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {197},
pmid = {30442181},
issn = {1474-760X},
mesh = {Arabidopsis ; *CRISPR-Cas Systems ; *Disease Resistance ; *Geminiviridae ; Gene Editing/*methods ; Tobacco ; },
abstract = {The CRISPR/Cas9 system has recently been engineered to confer resistance to geminiviruses in plants. However, we show here that the usefulness of this antiviral strategy is undermined by off-target effects identified by deep sequencing in Arabidopsis. We construct two virus-inducible CRISPR/Cas9 vectors that efficiently inhibit beet severe curly top virus (BSCTV) accumulation in both transient assays (Nicotiana benthamiana) and transgenic lines (Arabidopsis). Deep sequencing detects no off-target effect in candidate sites of the transgenic Arabidopsis. This kind of virus-inducible genome-editing system should be widely applicable for generating virus-resistant plants without off-target costs.},
}

Arabidopsis
*CRISPR-Cas Systems
*Disease Resistance
*Geminiviridae
Gene Editing/*methods
Tobacco

@article {pmid29625148,
year = {2018},
author = {Wang, Q and Liu, S and Liu, Z and Ke, Z and Li, C and Yu, X and Chen, S and Guo, D},
title = {Genome scale screening identification of SaCas9/gRNAs for targeting HIV-1 provirus and suppression of HIV-1 infection.},
journal = {Virus research},
volume = {250},
number = {},
pages = {21-30},
doi = {10.1016/j.virusres.2018.04.002},
pmid = {29625148},
issn = {1872-7492},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Genetic Vectors ; *Genome, Viral ; HIV Infections/therapy ; HIV-1/*genetics ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Proviruses/*genetics ; *RNA, Guide ; Staphylococcus aureus/genetics ; Virion/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing approach has been widely used in anti-HIV-1 gene therapy research. However, the major challenges facing the therapeutic application of CRISPR/Cas9 are the precise genome cleavage efficacy and efficient delivery of Cas9/gRNA specifically to the HIV-infected cells. Recently, a small size Cas9 from Staphylococcus aureus (SaCas9) has shown promise in genome editing in eukaryotic cells, suggesting a potential usage in blocking HIV-1 infection by targeting the HIV-1 genome. Here, we designed 43 guide RNAs (gRNAs) against the HIV-1 genome, thereby identifying 8 gRNAs that efficiently and specifically disrupt the target DNA by SaCas9. In addition, we found the selected gRNAs induce SaCas9 to disrupt the latent HIV-1 provirus and suppress HIV-1 proviral reactivation in latently infected Jurkat C11 cells. We further confirmed that the dual or triple gRNAs in an all-in-one lentiviral vector could reduce viral production in TZM-bl cells as well as in Jurkat T cells. Moreover, we did not detect any off-target cleavages in the predicted sites, suggesting that through all-in-one lentiviral vector-mediated HIV-1 genome editing, the selected SaCas9/gRNAs can provide an alternative and flexible strategy for anti-HIV gene therapy.},
}

*CRISPR-Cas Systems
Gene Editing
Genetic Therapy
Genetic Vectors
*Genome, Viral
HIV Infections/therapy
HIV-1/*genetics
Humans
Jurkat Cells
Lentivirus/genetics
Proviruses/*genetics
*RNA, Guide
Staphylococcus aureus/genetics
Virion/genetics

@article {pmid30840895,
year = {2019},
author = {Mogila, I and Kazlauskiene, M and Valinskyte, S and Tamulaitiene, G and Tamulaitis, G and Siksnys, V},
title = {Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits.},
journal = {Cell reports},
volume = {26},
number = {10},
pages = {2753-2765.e4},
doi = {10.1016/j.celrep.2019.02.029},
pmid = {30840895},
issn = {2211-1247},
abstract = {The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function. We show that the Cas10-Csm4 subcomplex binds the 5'-handle of crRNA and triggers Csm3 oligomerization to form a padlock for crRNA binding. We demonstrate that Csm5 plays a key role in target RNA binding while Csm2 ensures RNA cleavage at multiple sites by Csm3. Finally, guided by deletion analysis, we engineered a minimal Csm complex containing only the Csm3, Csm4, and Cas10 subunits and crRNA and demonstrated that it retains all three catalytic activities, thus paving the way for practical applications.},
}

@article {pmid30838976,
year = {2019},
author = {Wegner, M and Diehl, V and Bittl, V and de Bruyn, R and Wiechmann, S and Matthess, Y and Hebel, M and Hayes, MG and Schaubeck, S and Benner, C and Heinz, S and Bremm, A and Dikic, I and Ernst, A and Kaulich, M},
title = {Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.42549},
pmid = {30838976},
issn = {2050-084X},
support = {IIIL5-518/17.004//Hessisches Ministerium für Wissenschaft und Kunst/ ; EXC115/2//Deutsche Forschungsgemeinschaft/ ; IIIL5-519/03/03.001//Hessisches Ministerium für Wissenschaft und Kunst/ ; EXC147/2//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Current technologies to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology to efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents that uncouples sequence diversity from sequence distribution. We demonstrate fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed at generating the up-to-date largest gRNA library to simultaneously interrogate the coding and noncoding human genome and identify genes, predicted promoter flanking regions, transcription factor and CTCF binding sites linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.},
}

@article {pmid30837341,
year = {2019},
author = {Nasko, DJ and Ferrell, BD and Moore, RM and Bhavsar, JD and Polson, SW and Wommack, KE},
title = {CRISPR Spacers Indicate Preferential Matching of Specific Virioplankton Genes.},
journal = {mBio},
volume = {10},
number = {2},
pages = {},
doi = {10.1128/mBio.02651-18},
pmid = {30837341},
issn = {2150-7511},
abstract = {Viral infection exerts selection pressure on marine microbes, as virus-induced cell lysis causes 20 to 50% of cell mortality, resulting in fluxes of biomass into oceanic dissolved organic matter. Archaeal and bacterial populations can defend against viral infection using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system, which relies on specific matching between a spacer sequence and a viral gene. If a CRISPR spacer match to any gene within a viral genome is equally effective in preventing lysis, no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness, certain viral genes may demonstrate a greater frequency of CRISPR spacer matches. Indeed, homology search analyses of bacterioplankton CRISPR spacer sequences against virioplankton sequences revealed preferential matching of replication proteins, nucleic acid binding proteins, and viral structural proteins. Positive selection pressure for effective viral defense is one parsimonious explanation for these observations. CRISPR spacers from virioplankton metagenomes preferentially matched methyltransferase and phage integrase genes within virioplankton sequences. These virioplankton CRISPR spacers may assist infected host cells in defending against competing phage. Analyses also revealed that half of the spacer-matched viral genes were unknown, some genes matched several spacers, and some spacers matched multiple genes, a many-to-many relationship. Thus, CRISPR spacer matching may be an evolutionary algorithm, agnostically identifying those genes under stringent selection pressure for sustaining viral infection and lysis. Investigating this subset of viral genes could reveal those genetic mechanisms essential to virus-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes.IMPORTANCE The CRISPR-Cas system is one means by which bacterial and archaeal populations defend against viral infection which causes 20 to 50% of cell mortality in the ocean. We tested the hypothesis that certain viral genes are preferentially targeted for the initial attack of the CRISPR-Cas system on a viral genome. Using CASC, a pipeline for CRISPR spacer discovery, and metagenome data from oceanic microbes and viruses, we found a clear subset of viral genes with high match frequencies to CRISPR spacers. Moreover, we observed a many-to-many relationship of spacers and viral genes. These high-match viral genes were involved in nucleotide metabolism, DNA methylation, and viral structure. It is possible that CRISPR spacer matching is an evolutionary algorithm pointing to those viral genes most important to sustaining infection and lysis. Studying these genes may advance the understanding of virus-host interactions in nature and provide new technologies for leveraging CRISPR-Cas systems in beneficial microbes.},
}

@article {pmid30613384,
year = {2018},
author = {Conboy, I and Murthy, N and Etienne, J and Robinson, Z},
title = {Making gene editing a therapeutic reality.},
journal = {F1000Research},
volume = {7},
number = {},
pages = {},
doi = {10.12688/f1000research.16106.1},
pmid = {30613384},
issn = {2046-1402},
support = {R01 EB023776/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Muscular Dystrophy, Duchenne/genetics ; Myotonic Dystrophy/genetics ; },
abstract = {This review discusses current bottlenecks in making CRISPR-Cas9-mediated genome editing a therapeutic reality and it outlines recent strategies that aim to overcome these hurdles as well as the scope of current clinical trials that pioneer the medical translation of CRISPR-Cas9. Additionally, this review outlines the specifics of disease-modifying gene editing in recessive versus dominant genetic diseases with the focus on genetic myopathies that are exemplified by Duchenne muscular dystrophy and myotonic dystrophies.},
}

Animals
CRISPR-Cas Systems/genetics
Gene Editing/*methods
Genetic Therapy/*methods
Humans
Muscular Dystrophy, Duchenne/genetics
Myotonic Dystrophy/genetics

@article {pmid30365569,
year = {2018},
author = {Zhao, Q and Busch, B and Jiménez-Soto, LF and Ishikawa-Ankerhold, H and Massberg, S and Terradot, L and Fischer, W and Haas, R},
title = {Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation.},
journal = {PLoS pathogens},
volume = {14},
number = {10},
pages = {e1007359},
pmid = {30365569},
issn = {1553-7374},
mesh = {Antigens, Bacterial/genetics/*metabolism ; Antigens, CD/genetics/*metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion Molecules/antagonists & inhibitors/genetics/*metabolism ; GPI-Linked Proteins/antagonists & inhibitors/genetics/metabolism ; Humans ; Integrins/antagonists & inhibitors/genetics/*metabolism ; Protein Transport ; Stomach Neoplasms/genetics/*metabolism/pathology ; Tumor Cells, Cultured ; },
abstract = {Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and β1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp β-lactamase reporter system clearly show that neither β1 integrin heterodimers (α1β1, α2β1 or α5β1), nor any other αβ integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe.},
}

Antigens, Bacterial/genetics/*metabolism
Antigens, CD/genetics/*metabolism
Bacterial Proteins/genetics/*metabolism
CRISPR-Cas Systems
Cell Adhesion Molecules/antagonists & inhibitors/genetics/*metabolism
GPI-Linked Proteins/antagonists & inhibitors/genetics/metabolism
Humans
Integrins/antagonists & inhibitors/genetics/*metabolism
Protein Transport
Stomach Neoplasms/genetics/*metabolism/pathology
Tumor Cells, Cultured

@article {pmid29844277,
year = {2018},
author = {Chen, YC and Sheng, J and Trang, P and Liu, F},
title = {Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections.},
journal = {Viruses},
volume = {10},
number = {6},
pages = {},
pmid = {29844277},
issn = {1999-4915},
support = {R01 AI041927/AI/NIAID NIH HHS/United States ; R01 AI091536/AI/NIAID NIH HHS/United States ; R01 DE023935/DE/NIDCR NIH HHS/United States ; R01 DE025462/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cytomegalovirus/genetics ; Gene Editing ; *Genome, Viral ; Herpesviridae/*genetics ; Herpesviridae Infections/prevention & control/*therapy ; Herpesvirus 4, Human/genetics ; Herpesvirus 8, Human/genetics ; Humans ; Mice ; Mutagenesis ; Simplexvirus/genetics ; Virus Latency ; },
abstract = {The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein⁻Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections.},
}

Animals
*CRISPR-Cas Systems
Cytomegalovirus/genetics
Gene Editing
*Genome, Viral
Herpesviridae/*genetics
Herpesviridae Infections/prevention & control/*therapy
Herpesvirus 4, Human/genetics
Herpesvirus 8, Human/genetics
Humans
Mice
Mutagenesis
Simplexvirus/genetics
Virus Latency

@article {pmid29794970,
year = {2018},
author = {Zhang, Y and Tang, N and Sadigh, Y and Baigent, S and Shen, Z and Nair, V and Yao, Y},
title = {Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function.},
journal = {Viruses},
volume = {10},
number = {6},
pages = {},
pmid = {29794970},
issn = {1999-4915},
support = {BB/P016472/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antigens, Viral/genetics ; *CRISPR-Cas Systems ; Chickens/immunology/virology ; Gene Deletion ; Gene Editing/*methods ; *Genome, Viral ; Herpesvirus 2, Gallid/*genetics/*pathogenicity ; Marek Disease/prevention & control ; Oncogene Proteins, Viral/genetics ; Phosphoproteins/genetics ; Poultry Diseases/prevention & control/virology ; Virulence/genetics ; },
abstract = {Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek's disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.},
}

Animals
Antigens, Viral/genetics
*CRISPR-Cas Systems
Chickens/immunology/virology
Gene Deletion
Gene Editing/*methods
*Genome, Viral
Herpesvirus 2, Gallid/*genetics/*pathogenicity
Marek Disease/prevention & control
Oncogene Proteins, Viral/genetics
Phosphoproteins/genetics
Poultry Diseases/prevention & control/virology
Virulence/genetics

@article {pmid29734705,
year = {2018},
author = {Schilling, T and Dietrich, S and Hoppert, M and Hertel, R},
title = {A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages.},
journal = {Viruses},
volume = {10},
number = {5},
pages = {},
pmid = {29734705},
issn = {1999-4915},
mesh = {Bacillus Phages/*genetics ; Bacillus subtilis/*virology ; *CRISPR-Cas Systems ; *Genetic Engineering ; Mutagenesis ; Prophages/genetics ; Reproducibility of Results ; *Software ; },
abstract = {Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus. This toolkit comprises the application CutSPR, a bioinformatic tool for rapid primer design, and facilitates the cloning of specific CRISPR-Cas9-based mutagenesis plasmids. The employment of the prophage-free and super-competent B. subtilis TS01 strain enables an easy and fast introduction of specific constructs for in vivo phage mutagenesis. Clean gene deletions and a functional clean gene insertion into the genome of the model phage vB_BsuP-Goe1 served as proof of concept and demonstrate reliability and high efficiency. The here presented toolkit allows comprehensive investigation of the diverse phage genetic pool, a better understanding of phage biology, and safe phage applications.},
}

Bacillus Phages/*genetics
Bacillus subtilis/*virology
*CRISPR-Cas Systems
*Genetic Engineering
Mutagenesis
Prophages/genetics
Reproducibility of Results
*Software

@article {pmid29684735,
year = {2018},
author = {McDougall, WM and Perreira, JM and Reynolds, EC and Brass, AL},
title = {CRISPR genetic screens to discover host-virus interactions.},
journal = {Current opinion in virology},
volume = {29},
number = {},
pages = {87-100},
doi = {10.1016/j.coviro.2018.03.007},
pmid = {29684735},
issn = {1879-6265},
support = {HHSN272200900041C/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Flavivirus/genetics/metabolism ; *Genetic Testing ; *Host-Pathogen Interactions ; Humans ; Receptors, Virus/metabolism ; Virus Diseases/*virology ; Viruses/*genetics ; Workflow ; },
abstract = {Viruses impose an immense burden on human health. With the goal of treating and preventing viral infections, researchers have carried out genetic screens to improve our understanding of viral dependencies and identify potential anti-viral strategies. The emergence of CRISPR genetic screening tools has facilitated this effort by enabling host-virus screens to be undertaken in a more versatile and fidelitous manner than previously possible. Here we review the growing number of CRISPR screens which continue to increase our understanding of host-virus interactions.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Flavivirus/genetics/metabolism
*Genetic Testing
*Host-Pathogen Interactions
Humans
Receptors, Virus/metabolism
Virus Diseases/*virology
Viruses/*genetics
Workflow

@article {pmid30835493,
year = {2019},
author = {Chen, K and Wang, Y and Zhang, R and Zhang, H and Gao, C},
title = {CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture.},
journal = {Annual review of plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-arplant-050718-100049},
pmid = {30835493},
issn = {1545-2123},
abstract = {Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genome editing with crop breeding. We summarize the applications of genome editing for trait improvement, development of fine-tuning gene regulation, strategies for breeding virus resistance, and the use of high-throughput mutant libraries. We outline future perspectives for genome editing in plant synthetic biology and domestication, advances in delivery systems, editing specificity, homology-directed repair, and gene drives. Finally, we discuss the challenges and opportunities for precision plant breeding and its bright future in agriculture. Expected final online publication date for the Annual Review of Plant Biology Volume 70 is April 29, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}

@article {pmid30834930,
year = {2019},
author = {van Sluijs, L and van Houte, S and van der Oost, J and Brouns, SJJ and Buckling, A and Westra, ER},
title = {Addiction systems antagonize bacterial adaptive immunity.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz047},
pmid = {30834930},
issn = {1574-6968},
abstract = {CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-antitoxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.},
}

@article {pmid30833733,
year = {2019},
author = {Fu, BXH and Smith, JD and Fuchs, RT and Mabuchi, M and Curcuru, J and Robb, GB and Fire, AZ},
title = {Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41564-019-0382-0},
pmid = {30833733},
issn = {2058-5276},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.},
}

@article {pmid30204788,
year = {2018},
author = {Vernon, SW and Goodchild, J and Baines, RA},
title = {The VAChTY49N mutation provides insecticide-resistance but perturbs evoked cholinergic neurotransmission in Drosophila.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0203852},
pmid = {30204788},
issn = {1932-6203},
support = {//Wellcome Trust/United Kingdom ; BB/L027690/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; 087742/Z/08/Z//Wellcome Trust/United Kingdom ; },
mesh = {Acetylcholine/*metabolism ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Drosophila ; Drosophila Proteins/*genetics/metabolism ; Insecticide Resistance/*genetics/physiology ; Insecticides/pharmacology ; Larva ; Motor Activity/physiology ; Patch-Clamp Techniques ; *Point Mutation ; Synapses/metabolism ; Synaptic Transmission/*genetics/physiology ; Vesicular Acetylcholine Transport Proteins/*genetics/metabolism ; },
abstract = {Global agriculture and the control of insect disease vectors have developed with a heavy reliance on insecticides. The increasing incidence of resistance, for virtually all insecticides, threatens both food supply and effective control of insect borne disease. CASPP ((5-chloro-1'-[(E)-3-(4-chlorophenyl)allyl]spiro[indoline-3,4'-piperidine]-1-yl}-
(2-chloro-4-pyridyl)methanone)) compounds are a potential new class of neuroactive insecticide specifically targeting the Vesicular Acetylcholine Transporter (VAChT). Resistance to CASPP, under laboratory conditions, has been reported following either up-regulation of wildtype VAChT expression or the presence of a specific point mutation (VAChTY49N). However, the underlying mechanism of CASPP-resistance, together with the consequence to insect viability of achieving resistance, is unknown. In this study, we use electrophysiological characterisation of cholinergic release at Drosophila larval interneuron→motoneuron synapses to investigate the physiological implications of these two identified modes of CASPP resistance. We show that both VAChT up-regulation or the expression of VAChTY49N increases miniature (mini) release frequency. Mini frequency appears deterministic of CASPP activity. However, maintenance of SV release is not indicative of resistance in all cases. This is evidenced through expression of syntaxin or complexin mutants (sytx3-61/cpxSH1) that show similarly high mini release frequency but are not resistant to CASPP. The VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles. This observation suggests a functional trade-off for this point mutation, which is not seen when wildtype VAChT is up-regulated.},
}

Acetylcholine/*metabolism
Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Drosophila
Drosophila Proteins/*genetics/metabolism
Insecticide Resistance/*genetics/physiology
Insecticides/pharmacology
Larva
Motor Activity/physiology
Patch-Clamp Techniques
*Point Mutation
Synapses/metabolism
Synaptic Transmission/*genetics/physiology
Vesicular Acetylcholine Transport Proteins/*genetics/metabolism

@article {pmid29860898,
year = {2018},
author = {Li, P and Marino, MP and Zou, J and Argaw, T and Morreale, MT and Iaffaldano, BJ and Reiser, J},
title = {Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.},
journal = {Human gene therapy methods},
volume = {29},
number = {3},
pages = {135-145},
pmid = {29860898},
issn = {1946-6544},
mesh = {CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics/metabolism ; Gene Editing/*methods ; Gene Targeting/*methods/standards ; HEK293 Cells ; HeLa Cells ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Lentivirus/genetics/metabolism ; Viral Proteins/*genetics/metabolism ; },
abstract = {The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells. The Rep 78 and ZFN-based approaches were also compared in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors. The targeting efficiencies involving the Rep 78 protein were similar to those involving the AAVS1-specific ZFN, while the targeting specificity for the Rep 78 protein was lower compared to that of the ZFN. It is anticipated that the Rep 78 nickase-based targeting approach may ultimately contribute to the reduction of risks associated with other genome editing approaches involving DNA double-strand breaks.},
}

CRISPR-Cas Systems
DNA-Binding Proteins/*genetics/metabolism
Gene Editing/*methods
Gene Targeting/*methods/standards
HEK293 Cells
HeLa Cells
Humans
Induced Pluripotent Stem Cells/metabolism
Lentivirus/genetics/metabolism
Viral Proteins/*genetics/metabolism

@article {pmid29540833,
year = {2018},
author = {Acosta, J and Wang, W and Feldser, DM},
title = {Off and back-on again: a tumor suppressor's tale.},
journal = {Oncogene},
volume = {37},
number = {23},
pages = {3058-3069},
pmid = {29540833},
issn = {1476-5594},
support = {P30 CA016520/CA/NCI NIH HHS/United States ; R00 CA158581/CA/NCI NIH HHS/United States ; R01 CA193602/CA/NCI NIH HHS/United States ; R21 CA205340/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Floxuridine/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; *Genes, Tumor Suppressor ; Genetic Engineering/*methods ; Genetic Vectors ; Humans ; Mice ; Mice, Transgenic ; Neoplasms, Experimental/*genetics ; RNA Interference ; Recombinases/genetics ; },
abstract = {Tumor suppressor genes play critical roles orchestrating anti-cancer programs that are both context dependent and mechanistically diverse. Beyond canonical tumor suppressive programs that control cell division, cell death, and genome stability, unexpected tumor suppressor gene activities that regulate metabolism, immune surveillance, the epigenetic landscape, and others have recently emerged. This diversity underscores the important roles these genes play in maintaining cellular homeostasis to suppress cancer initiation and progression, but also highlights a tremendous challenge in discerning precise context-specific programs of tumor suppression controlled by a given tumor suppressor. Fortunately, the rapid sophistication of genetically engineered mouse models of cancer has begun to shed light on these context-dependent tumor suppressor activities. By using techniques that not only toggle "off" tumor suppressor genes in nascent tumors, but also facilitate the timely restoration of gene function "back-on again" in disease specific contexts, precise mechanisms of tumor suppression can be revealed in an unbiased manner. This review discusses the development and implementation of genetic systems designed to toggle tumor suppressor genes off and back-on again and their potential to uncover the tumor suppressor's tale.},
}

Alleles
Animals
CRISPR-Cas Systems
Floxuridine/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
*Genes, Tumor Suppressor
Genetic Engineering/*methods
Genetic Vectors
Humans
Mice
Mice, Transgenic
Neoplasms, Experimental/*genetics
RNA Interference
Recombinases/genetics

@article {pmid28808289,
year = {2017},
author = {Lombardi, L and Turner, SA and Zhao, F and Butler, G},
title = {Gene editing in clinical isolates of Candida parapsilosis using CRISPR/Cas9.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {8051},
pmid = {28808289},
issn = {2045-2322},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Candida parapsilosis/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA End-Joining Repair/genetics ; Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Candida parapsilosis is one of the most common causes of candidiasis, particularly in the very young and the very old. Studies of gene function are limited by the lack of a sexual cycle, the diploid genome, and a paucity of molecular tools. We describe here the development of a plasmid-based CRISPR-Cas9 system for gene editing in C. parapsilosis. A major advantage of the system is that it can be used in any genetic background, which we showed by editing genes in 20 different isolates. Gene editing is carried out in a single transformation step. The CAS9 gene is expressed only when the plasmid is present, and it can be removed easily from transformed strains. There is theoretically no limit to the number of genes that can be edited in any strain. Gene editing is increased by homology-directed repair in the presence of a repair template. Editing by non-homologous end joining (NHEJ) also occurs in some genetic backgrounds. Finally, we used the system to introduce unique tags at edited sites.},
}

Bacterial Proteins/*genetics
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
Candida parapsilosis/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA End-Joining Repair/genetics
Gene Editing/methods
Plasmids/genetics

@article {pmid30828340,
year = {2019},
author = {Muñoz, IV and Sarrocco, S and Malfatti, L and Baroncelli, R and Vannacci, G},
title = {CRISPR-Cas for Fungal Genome Editing: A New Tool for the Management of Plant Diseases.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {135},
doi = {10.3389/fpls.2019.00135},
pmid = {30828340},
issn = {1664-462X},
}

@article {pmid30715580,
year = {2019},
author = {Pérez, L and Soto, E and Farré, G and Juanos, J and Villorbina, G and Bassie, L and Medina, V and Serrato, AJ and Sahrawy, M and Rojas, JA and Romagosa, I and Muñoz, P and Zhu, C and Christou, P},
title = {CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.},
journal = {Plant cell reports},
volume = {38},
number = {3},
pages = {417-433},
doi = {10.1007/s00299-019-02388-z},
pmid = {30715580},
issn = {1432-203X},
support = {BIO2014-54426//Ministerio de Economía y Competitividad/ ; IJCI- 2014-19528//Ministerio de Ciencia e Innovación/ ; BIO2014-54441-P//Universitat de Lleida/ ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Endosperm/metabolism ; Mutation/genetics ; Oryza/genetics/*metabolism ; Starch Synthase/genetics/*metabolism ; Waxes/metabolism ; },
abstract = {KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.},
}

Alleles
CRISPR-Cas Systems/genetics
Endosperm/metabolism
Mutation/genetics
Oryza/genetics/*metabolism
Starch Synthase/genetics/*metabolism
Waxes/metabolism

@article {pmid30540754,
year = {2018},
author = {Ramanagoudr-Bhojappa, R and Carrington, B and Ramaswami, M and Bishop, K and Robbins, GM and Jones, M and Harper, U and Frederickson, SC and Kimble, DC and Sood, R and Chandrasekharappa, SC},
title = {Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.},
journal = {PLoS genetics},
volume = {14},
number = {12},
pages = {e1007821},
doi = {10.1371/journal.pgen.1007821},
pmid = {30540754},
issn = {1553-7404},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Damage ; Fanconi Anemia/*genetics/physiopathology ; Female ; Fertility/genetics/physiology ; Frameshift Mutation ; Gene Knockout Techniques ; Humans ; Male ; Phenotype ; RNA Splicing/genetics ; Sex Determination Processes/genetics/physiology ; Sexual Development/genetics/physiology ; Zebrafish/*genetics/growth & development/physiology ; Zebrafish Proteins/genetics/physiology ; },
abstract = {Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.},
}

Animals
CRISPR-Cas Systems
DNA Damage
Fanconi Anemia/*genetics/physiopathology
Female
Fertility/genetics/physiology
Frameshift Mutation
Gene Knockout Techniques
Humans
Male
Phenotype
RNA Splicing/genetics
Sex Determination Processes/genetics/physiology
Sexual Development/genetics/physiology
Zebrafish/*genetics/growth & development/physiology
Zebrafish Proteins/genetics/physiology

@article {pmid30254120,
year = {2018},
author = {Cernak, P and Estrela, R and Poddar, S and Skerker, JM and Cheng, YF and Carlson, AK and Chen, B and Glynn, VM and Furlan, M and Ryan, OW and Donnelly, MK and Arkin, AP and Taylor, JW and Cate, JHD},
title = {Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.},
journal = {mBio},
volume = {9},
number = {5},
pages = {},
pmid = {30254120},
issn = {2150-7511},
mesh = {Biotechnology ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genes, Mating Type, Fungal/genetics ; *Genetic Engineering ; Kluyveromyces/*genetics/metabolism ; Lipid Metabolism ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/*methods ; Temperature ; Thermotolerance ; },
abstract = {Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.},
}

Biotechnology
CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
*Gene Editing
Genes, Mating Type, Fungal/genetics
*Genetic Engineering
Kluyveromyces/*genetics/metabolism
Lipid Metabolism
Saccharomyces cerevisiae/genetics
Synthetic Biology/*methods
Temperature
Thermotolerance

@article {pmid30135210,
year = {2018},
author = {Wright, FA and Bonzerato, CG and Sliter, DA and Wojcikiewicz, RJH},
title = {The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {40},
pages = {15706-15714},
pmid = {30135210},
issn = {1083-351X},
support = {R01 DK107944/DK/NIDDK NIH HHS/United States ; R01 GM121621/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; *Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cell Line, Transformed ; Gene Editing ; Gonadotrophs/cytology/metabolism ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/*genetics/metabolism ; Membrane Proteins/deficiency/*genetics ; Mice ; Mutation ; Nerve Tissue Proteins/*genetics/metabolism ; Phosphatidylinositol Phosphates/*metabolism ; Protein Binding ; Proteolysis ; Sequence Alignment ; Sequence Homology, Amino Acid ; Spastic Paraplegia, Hereditary/genetics/metabolism/pathology ; Ubiquitination ; },
abstract = {The erlin1/2 complex is a ∼2-MDa endoplasmic reticulum membrane-located ensemble of the ∼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia. Here, we used gene editing to ablate erlin1 or erlin2 expression to better define their individual roles in the cell and examined the functional effects of a spastic paraplegia-linked mutation to erlin2 (threonine to isoleucine at position 65; T65I). Our results revealed that erlin2 is the dominant player in mediating the interaction between the erlin1/2 complex and IP3Rs and that the T65I mutation dramatically inhibits this interaction and the ability of the erlin1/2 complex to promote IP3R ubiquitination and degradation. Remarkably, we also discovered that the erlin1/2 complex specifically binds to phosphatidylinositol 3-phosphate, that erlin2 binds this phospholipid much more strongly than does erlin1, that the binding is inhibited by T65I mutation of erlin2, and that multiple determinants within the erlin2 polypeptide comprise the phosphatidylinositol 3-phosphate-binding site. Overall, these results indicate that erlin2 is the primary mediator of the cellular roles of the erlin1/2 complex and that disease-linked mutations of erlin2 can affect both IP3R processing and lipid binding.},
}

Amino Acid Sequence
*Amino Acid Substitution
Animals
CRISPR-Cas Systems
Cell Line, Transformed
Gene Editing
Gonadotrophs/cytology/metabolism
Humans
Inositol 1,4,5-Trisphosphate Receptors/*genetics/metabolism
Membrane Proteins/deficiency/*genetics
Mice
Mutation
Nerve Tissue Proteins/*genetics/metabolism
Phosphatidylinositol Phosphates/*metabolism
Protein Binding
Proteolysis
Sequence Alignment
Sequence Homology, Amino Acid
Spastic Paraplegia, Hereditary/genetics/metabolism/pathology
Ubiquitination

@article {pmid30126845,
year = {2018},
author = {Cai, X and Zhang, D and Wang, J and Liu, X and Ouyang, G and Xiao, W},
title = {Deletion of the fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {40},
pages = {15370-15380},
pmid = {30126845},
issn = {1083-351X},
mesh = {Adaptation, Physiological/*genetics ; Animals ; Apoptosis/genetics ; Brain/metabolism/physiopathology ; CREB-Binding Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Embryo, Nonmammalian ; Epithelial Cells/metabolism/pathology ; Gene Deletion ; Gene Editing/methods ; Gene Expression Regulation, Developmental ; Hypoxia/*genetics/metabolism/physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Mixed Function Oxygenases/deficiency/*genetics ; Oxygen Consumption/genetics ; Protein Subunits/genetics/metabolism ; Signal Transduction ; Zebrafish/*genetics/growth & development/metabolism ; p300-CBP Transcription Factors/genetics/metabolism ; },
abstract = {Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.},
}

Adaptation, Physiological/*genetics
Animals
Apoptosis/genetics
Brain/metabolism/physiopathology
CREB-Binding Protein/genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Embryo, Nonmammalian
Epithelial Cells/metabolism/pathology
Gene Deletion
Gene Editing/methods
Gene Expression Regulation, Developmental
Hypoxia/*genetics/metabolism/physiopathology
Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism
Mixed Function Oxygenases/deficiency/*genetics
Oxygen Consumption/genetics
Protein Subunits/genetics/metabolism
Signal Transduction
Zebrafish/*genetics/growth & development/metabolism
p300-CBP Transcription Factors/genetics/metabolism

@article {pmid30082317,
year = {2018},
author = {Rodríguez-Castañeda, F and Lemma, RB and Cuervo, I and Bengtsen, M and Moen, LM and Ledsaak, M and Eskeland, R and Gabrielsen, OS},
title = {The SUMO protease SENP1 and the chromatin remodeler CHD3 interact and jointly affect chromatin accessibility and gene expression.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {40},
pages = {15439-15454},
pmid = {30082317},
issn = {1083-351X},
mesh = {Animals ; CCCTC-Binding Factor/genetics/metabolism ; COS Cells ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cercopithecus aethiops ; Chromatin/*chemistry/metabolism/ultrastructure ; *Chromatin Assembly and Disassembly ; Cloning, Molecular ; Cysteine Endopeptidases/genetics/*metabolism ; DNA Helicases/genetics/*metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing/methods ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; HEK293 Cells ; Humans ; K562 Cells ; Leukocytes/metabolism/pathology ; Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics/*metabolism ; Protein Binding ; *Protein Processing, Post-Translational ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {The small ubiquitin-like modifier (SUMO) post-translationally modifies lysine residues of transcription factors and co-regulators and thereby contributes to an important layer of control of the activities of these transcriptional regulators. Likewise, deSUMOylation of these factors by the sentrin-specific proteases (SENPs) also plays a role in gene regulation, but whether SENPs functionally interact with other regulatory factors that control gene expression is unclear. In the present work, we focused on SENP1, specifically, on its role in activation of gene expression investigated through analysis of the SENP1 interactome, which revealed that SENP1 physically interacts with the chromatin remodeler chromodomain helicase DNA-binding protein 3 (CHD3). Using several additional methods, including GST pulldown and co-immunoprecipitation assays, we validated and mapped this interaction, and using CRISPR-Cas9-generated CHD3- and SENP1-KO cells (in the haploid HAP1 cell line), we investigated whether these two proteins are functionally linked in regulating chromatin remodeling and gene expression. Genome-wide ATAC-Seq analysis of the CHD3- and SENP1-KO cells revealed a large degree of overlap in differential chromatin openness between these two mutant cell lines. Moreover, motif analysis and comparison with ChIP-Seq profiles in K562 cells pointed to an association of CHD3 and SENP1 with CCCTC-binding factor (CTCF) and SUMOylated chromatin-associated factors. Lastly, genome-wide RNA-Seq also indicated that these two proteins co-regulate the expression of several genes. We propose that the functional link between chromatin remodeling by CHD3 and deSUMOylation by SENP1 uncovered here provides another level of control of gene expression.},
}

Animals
CCCTC-Binding Factor/genetics/metabolism
COS Cells
CRISPR-Cas Systems
Cell Line, Tumor
Cercopithecus aethiops
Chromatin/*chemistry/metabolism/ultrastructure
*Chromatin Assembly and Disassembly
Cloning, Molecular
Cysteine Endopeptidases/genetics/*metabolism
DNA Helicases/genetics/*metabolism
Escherichia coli/genetics/metabolism
Gene Editing/methods
Gene Expression
Genetic Vectors/chemistry/metabolism
HEK293 Cells
Humans
K562 Cells
Leukocytes/metabolism/pathology
Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics/*metabolism
Protein Binding
*Protein Processing, Post-Translational
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid30069047,
year = {2018},
author = {Luo, J and Sun, X and Cormack, BP and Boeke, JD},
title = {Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast.},
journal = {Nature},
volume = {560},
number = {7718},
pages = {392-396},
doi = {10.1038/s41586-018-0374-x},
pmid = {30069047},
issn = {1476-4687},
support = {MCB-1616111//National Science Foundation/International ; },
mesh = {Artificial Gene Fusion/*methods ; CRISPR-Cas Systems/genetics ; Chromosomes, Fungal/*genetics ; Crosses, Genetic ; *Gene Editing ; *Karyotype ; Microbial Viability/*genetics ; Reproduction/genetics ; Saccharomyces cerevisiae/*genetics ; Spores, Fungal/genetics/physiology ; },
abstract = {Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere-telomere fusions and genome duplication events3-5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7-10, but CRISPR-Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR-Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.},
}

Artificial Gene Fusion/*methods
CRISPR-Cas Systems/genetics
Chromosomes, Fungal/*genetics
Crosses, Genetic
*Gene Editing
*Karyotype
Microbial Viability/*genetics
Reproduction/genetics
Saccharomyces cerevisiae/*genetics
Spores, Fungal/genetics/physiology

@article {pmid30043639,
year = {2018},
author = {Albayrak, G and Konac, E and Ugras Dikmen, A and Bilen, CY},
title = {FOXA1 knock-out via CRISPR/Cas9 altered Casp-9, Bax, CCND1, CDK4, and fibronectin expressions in LNCaP cells.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {243},
number = {12},
pages = {990-994},
pmid = {30043639},
issn = {1535-3699},
mesh = {Apoptosis ; CRISPR-Cas Systems ; Caspase 9/metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1/metabolism ; Cyclin-Dependent Kinase 4/metabolism ; Epithelial-Mesenchymal Transition ; Fibronectins/metabolism ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Hepatocyte Nuclear Factor 3-alpha/*genetics ; Humans ; Male ; Mutation ; Prostatic Neoplasms/*metabolism/pathology ; Protein Binding ; Protein Interaction Mapping ; Signal Transduction ; bcl-2-Associated X Protein/metabolism ; },
abstract = {Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P < 0.05, fold change: ∼40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, β-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment.},
}

Apoptosis
CRISPR-Cas Systems
Caspase 9/metabolism
Cell Cycle
Cell Line, Tumor
Cell Proliferation
Cyclin D1/metabolism
Cyclin-Dependent Kinase 4/metabolism
Epithelial-Mesenchymal Transition
Fibronectins/metabolism
*Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Hepatocyte Nuclear Factor 3-alpha/*genetics
Humans
Male
Mutation
Prostatic Neoplasms/*metabolism/pathology
Protein Binding
Protein Interaction Mapping
Signal Transduction
bcl-2-Associated X Protein/metabolism

@article {pmid29925955,
year = {2018},
author = {Phelan, JD and Young, RM and Webster, DE and Roulland, S and Wright, GW and Kasbekar, M and Shaffer, AL and Ceribelli, M and Wang, JQ and Schmitz, R and Nakagawa, M and Bachy, E and Huang, DW and Ji, Y and Chen, L and Yang, Y and Zhao, H and Yu, X and Xu, W and Palisoc, MM and Valadez, RR and Davies-Hill, T and Wilson, WH and Chan, WC and Jaffe, ES and Gascoyne, RD and Campo, E and Rosenwald, A and Ott, G and Delabie, J and Rimsza, LM and Rodriguez, FJ and Estephan, F and Holdhoff, M and Kruhlak, MJ and Hewitt, SM and Thomas, CJ and Pittaluga, S and Oellerich, T and Staudt, LM},
title = {A multiprotein supercomplex controlling oncogenic signalling in lymphoma.},
journal = {Nature},
volume = {560},
number = {7718},
pages = {387-391},
pmid = {29925955},
issn = {1476-4687},
support = {U01 CA157581/CA/NCI NIH HHS/United States ; Z01 BC011008-01//NULL/International ; },
mesh = {Animals ; Biopsy ; CRISPR-Cas Systems/genetics ; *Carcinogenesis/genetics ; Drug Design ; Female ; Humans ; Lymphoma, Large B-Cell, Diffuse/genetics/*metabolism/*pathology ; Mice ; Multiprotein Complexes/chemistry/*metabolism ; Mutation ; Myeloid Differentiation Factor 88/genetics/metabolism ; NF-kappa B/metabolism ; Proteomics ; Pyrazoles/pharmacology/therapeutic use ; Pyrimidines/pharmacology/therapeutic use ; Receptors, Antigen, B-Cell/antagonists & inhibitors/genetics/metabolism ; *Signal Transduction/drug effects/genetics ; TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Toll-Like Receptor 9/genetics/metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCβ to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.},
}

Animals
Biopsy
CRISPR-Cas Systems/genetics
*Carcinogenesis/genetics
Drug Design
Female
Humans
Lymphoma, Large B-Cell, Diffuse/genetics/*metabolism/*pathology
Mice
Multiprotein Complexes/chemistry/*metabolism
Mutation
Myeloid Differentiation Factor 88/genetics/metabolism
NF-kappa B/metabolism
Proteomics
Pyrazoles/pharmacology/therapeutic use
Pyrimidines/pharmacology/therapeutic use
Receptors, Antigen, B-Cell/antagonists & inhibitors/genetics/metabolism
*Signal Transduction/drug effects/genetics
TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism
Toll-Like Receptor 9/genetics/metabolism
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid29859265,
year = {2018},
author = {Shrestha, A and Khan, A and Dey, N},
title = {cis-trans Engineering: Advances and Perspectives on Customized Transcriptional Regulation in Plants.},
journal = {Molecular plant},
volume = {11},
number = {7},
pages = {886-898},
doi = {10.1016/j.molp.2018.05.008},
pmid = {29859265},
issn = {1752-9867},
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Genetic Engineering/*methods ; Plant Proteins/*genetics/metabolism ; *Plants, Genetically Modified ; Promoter Regions, Genetic ; Transcription Factors/metabolism ; Transcription, Genetic ; },
abstract = {Coordinated transcriptional control employing synthetic promoters and transcription factors (TFs) can be used to achieve customized regulation of gene expression in planta. Synthetic promoter technology has yielded a series of promoters with modified cis-regulatory elements that provide useful tools for efficient modulation of gene expression. In addition, the use of zinc fingers (ZFs), transcription activator-like effectors (TALEs), and catalytically inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) has made it feasible to engineer TFs that can produce targeted gene expression regulation; these approaches are particularly effective when artificial TFs are coupled with transcriptional activators or repressors. This review focuses on strategies used to engineer both promoters and TFs in the context of targeted transcriptional regulation. We also discuss the creation of synthetic inducible platforms, which can be used to impart stress tolerance to plants. We propose that combinatorial "cis-trans engineering" using a CRISPR-dCas9-based bipartite module could be used to regulate the expression of multiple target genes. This approach provides an attractive tool for introduction of specific qualitative traits into plants, thus enhancing their overall environmental adaptability.},
}

*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
Genetic Engineering/*methods
Plant Proteins/*genetics/metabolism
*Plants, Genetically Modified
Promoter Regions, Genetic
Transcription Factors/metabolism
Transcription, Genetic