@article {pmid38732215,
year = {2024},
author = {Rizk, R and Devost, D and Pétrin, D and Hébert, TE},
title = {KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.},
journal = {International journal of molecular sciences},
volume = {25},
number = {9},
pages = {},
pmid = {38732215},
issn = {1422-0067},
support = {PJT-156298/CAPMC/CIHR/Canada ; },
mesh = {Humans ; HEK293 Cells ; *Cell Proliferation/genetics ; *Potassium Channels/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protein Isoforms/genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; },
abstract = {We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gβ1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gβ1. Our work demonstrates a unique relationship between KCTD proteins and Gβ1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.},
}

Humans
HEK293 Cells
*Cell Proliferation/genetics
*Potassium Channels/metabolism/genetics
CRISPR-Cas Systems
Gene Knockout Techniques
Protein Isoforms/genetics/metabolism
Gene Editing
Gene Expression Regulation

@article {pmid38732145,
year = {2024},
author = {Martínez, M and Rizzuto, I and Molina, R},
title = {Knowing Our Enemy in the Antimicrobial Resistance Era: Dissecting the Molecular Basis of Bacterial Defense Systems.},
journal = {International journal of molecular sciences},
volume = {25},
number = {9},
pages = {},
pmid = {38732145},
issn = {1422-0067},
support = {PID2022-136750NB-I00//Agencia Estatal de Investigación/ ; RYC2021-030916-I//Agencia Estatal de Investigación/ ; },
mesh = {*Bacteria/genetics ; *Bacteriophages/physiology/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; Humans ; Bacterial Infections/immunology/microbiology ; },
abstract = {Bacteria and their phage adversaries are engaged in an ongoing arms race, resulting in the development of a broad antiphage arsenal and corresponding viral countermeasures. In recent years, the identification and utilization of CRISPR-Cas systems have driven a renewed interest in discovering and characterizing antiphage mechanisms, revealing a richer diversity than initially anticipated. Currently, these defense systems can be categorized based on the bacteria's strategy associated with the infection cycle stage. Thus, bacterial defense systems can degrade the invading genetic material, trigger an abortive infection, or inhibit genome replication. Understanding the molecular mechanisms of processes related to bacterial immunity has significant implications for phage-based therapies and the development of new biotechnological tools. This review aims to comprehensively cover these processes, with a focus on the most recent discoveries.},
}

*Bacteria/genetics
*Bacteriophages/physiology/genetics
*CRISPR-Cas Systems
Drug Resistance, Bacterial/genetics
Humans
Bacterial Infections/immunology/microbiology

@article {pmid38732031,
year = {2024},
author = {Liu, W and Wang, W and Wang, Z and Fan, X and Li, W and Huang, Y and Yang, X and Tang, Z},
title = {CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.},
journal = {International journal of molecular sciences},
volume = {25},
number = {9},
pages = {},
pmid = {38732031},
issn = {1422-0067},
mesh = {*Muscle Development/genetics ; *Peptide Elongation Factor 1/genetics/metabolism ; Animals ; Mice ; *Cell Proliferation/genetics ; *Cell Differentiation/genetics ; *Myoblasts/metabolism/cytology ; CRISPR-Cas Systems ; Cell Line ; MicroRNAs/genetics/metabolism ; Humans ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.},
}

*Muscle Development/genetics
*Peptide Elongation Factor 1/genetics/metabolism
Animals
Mice
*Cell Proliferation/genetics
*Cell Differentiation/genetics
*Myoblasts/metabolism/cytology
CRISPR-Cas Systems
Cell Line
MicroRNAs/genetics/metabolism
Humans
RNA-Binding Proteins/metabolism/genetics

@article {pmid38731837,
year = {2024},
author = {Melnikova, L and Molodina, V and Georgiev, P and Golovnin, A},
title = {Development of a New Model System to Study Long-Distance Interactions Supported by Architectural Proteins.},
journal = {International journal of molecular sciences},
volume = {25},
number = {9},
pages = {},
pmid = {38731837},
issn = {1422-0067},
support = {22-24-00719//Russian Science Foundation/ ; },
mesh = {*Drosophila Proteins/metabolism/genetics ; Animals ; *Promoter Regions, Genetic ; *Enhancer Elements, Genetic ; Drosophila melanogaster/genetics/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/metabolism/genetics ; Chromatin/metabolism/genetics ; Insulator Elements/genetics ; Binding Sites ; Protein Binding ; Nuclear Proteins/metabolism/genetics ; Gene Editing/methods ; Repressor Proteins/metabolism/genetics ; Microtubule-Associated Proteins ; },
abstract = {Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.},
}

*Drosophila Proteins/metabolism/genetics
Animals
*Promoter Regions, Genetic
*Enhancer Elements, Genetic
Drosophila melanogaster/genetics/metabolism
CRISPR-Cas Systems
DNA-Binding Proteins/metabolism/genetics
Chromatin/metabolism/genetics
Insulator Elements/genetics
Binding Sites
Protein Binding
Nuclear Proteins/metabolism/genetics
Gene Editing/methods
Repressor Proteins/metabolism/genetics
Microtubule-Associated Proteins

@article {pmid38730523,
year = {2024},
author = {Lee, KE and Xu, Y and Geng, B and Kim, J and Kellon, N and He, Z and Zhang, Z and Li, D and Gouchoe, DA and Zhu, H},
title = {Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.},
journal = {Biotechnology journal},
volume = {19},
number = {5},
pages = {e2300676},
pmid = {38730523},
issn = {1860-7314},
support = {R01 EY032583/EY/NEI NIH HHS/United States ; R01 EY030621/EY/NEI NIH HHS/United States ; R01 AR067766/AR/NIAMS NIH HHS/United States ; R01 HL153876/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Leptin/genetics/metabolism ; *Gene Editing/methods ; Mice ; Muscle, Skeletal/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; },
abstract = {Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.},
}

Animals
*Receptors, Leptin/genetics/metabolism
*Gene Editing/methods
Mice
Muscle, Skeletal/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
CRISPR-Cas Systems/genetics
Mice, Inbred C57BL

@article {pmid38730242,
year = {2024},
author = {Bibert, S and Quinodoz, M and Perriot, S and Krebs, FS and Jan, M and Malta, RC and Collinet, E and Canales, M and Mathias, A and Faignart, N and Roulet-Perez, E and Meylan, P and Brouillet, R and Opota, O and Lozano-Calderon, L and Fellmann, F and Guex, N and Zoete, V and Asner, S and Rivolta, C and Du Pasquier, R and Bochud, PY},
title = {Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3969},
pmid = {38730242},
issn = {2041-1723},
support = {33IC30_179636//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 314730_192616//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Humans ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Female ; *Encephalitis, Herpes Simplex/genetics ; Infant ; *Mutation ; *Herpesvirus 1, Human/genetics ; Induced Pluripotent Stem Cells/metabolism ; Toll-Like Receptor 3/genetics/metabolism ; Ubiquitination ; Neurons/metabolism ; Neural Stem Cells/metabolism/virology ; CRISPR-Cas Systems ; },
abstract = {Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.},
}

Humans
*Ubiquitin-Protein Ligases/genetics/metabolism
Female
*Encephalitis, Herpes Simplex/genetics
Infant
*Mutation
*Herpesvirus 1, Human/genetics
Induced Pluripotent Stem Cells/metabolism
Toll-Like Receptor 3/genetics/metabolism
Ubiquitination
Neurons/metabolism
Neural Stem Cells/metabolism/virology
CRISPR-Cas Systems

@article {pmid38729924,
year = {2024},
author = {Berjis, A and Muthumani, D and Aguilar, OA and Pomp, O and Johnson, O and Finck, AV and Engel, NW and Chen, L and Plachta, N and Scholler, J and Lanier, LL and June, CH and Sheppard, NC},
title = {Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3937},
pmid = {38729924},
issn = {2041-1723},
mesh = {*Granzymes/metabolism ; *Interleukin-15/metabolism ; *Killer Cells, Natural/immunology/metabolism ; *Apoptosis ; Humans ; *Interleukin-18/metabolism ; Animals ; *Cryopreservation/methods ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; },
abstract = {Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.},
}

*Granzymes/metabolism
*Interleukin-15/metabolism
*Killer Cells, Natural/immunology/metabolism
*Apoptosis
Humans
*Interleukin-18/metabolism
Animals
*Cryopreservation/methods
Mice
Cell Line, Tumor
CRISPR-Cas Systems

@article {pmid38729496,
year = {2024},
author = {Karimi, MR and Jariani, P and Yang, JL and Naghavi, MR},
title = {A comprehensive review of the molecular and genetic mechanisms underlying gum and resin synthesis in Ferula species.},
journal = {International journal of biological macromolecules},
volume = {269},
number = {Pt 2},
pages = {132168},
doi = {10.1016/j.ijbiomac.2024.132168},
pmid = {38729496},
issn = {1879-0003},
abstract = {Ferula spp. are plants that produce oleo-gum-resins (OGRs), which are plant exudates with various colors. These OGRs have various industrial applications in pharmacology, perfumery, and food. The main constituents of these OGRs are terpenoids, a diverse group of organic compounds with different structures and functions. The biosynthesis of OGRs in Ferula spp., particularly galbanum, holds considerable economic and ecological importance. However, the molecular and genetic underpinnings of this biosynthetic pathway remain largely enigmatic. This review provides an overview of the current state of knowledge on the biosynthesis of OGRs in Ferula spp., highlighting the major enzymes, genes, and pathways involved in the synthesis of different terpenoid classes, such as monoterpenes, sesquiterpenes, and triterpenes. It also examines the potential of using omics techniques, such as transcriptomics and metabolomics, and genome editing tools, such as CRISPR/Cas, to increase the yield and quality of Ferula OGRs, as well as to create novel bioactive compounds with enhanced properties. Moreover, this review addresses the current challenges and opportunities of applying gene editing in Ferula spp., and suggests some directions for future research and development.},
}

@article {pmid38729048,
year = {2024},
author = {Tan, C and Xie, G and Wu, S and Song, C and Zhang, J and Yi, X and Wang, J and Tang, H},
title = {Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.},
journal = {Biosensors & bioelectronics},
volume = {258},
number = {},
pages = {116373},
doi = {10.1016/j.bios.2024.116373},
pmid = {38729048},
issn = {1873-4235},
abstract = {Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.},
}

@article {pmid38662916,
year = {2024},
author = {Nemudraia, A and Nemudryi, A and Wiedenheft, B},
title = {Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells.},
journal = {Science (New York, N.Y.)},
volume = {384},
number = {6697},
pages = {808-814},
doi = {10.1126/science.adk5518},
pmid = {38662916},
issn = {1095-9203},
support = {K99 AI171893/AI/NIAID NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Repair ; HEK293 Cells ; RNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism ; },
abstract = {Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
DNA Repair
HEK293 Cells
RNA/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases/metabolism

@article {pmid38658794,
year = {2024},
author = {Ryu, J and Barkal, S and Yu, T and Jankowiak, M and Zhou, Y and Francoeur, M and Phan, QV and Li, Z and Tognon, M and Brown, L and Love, MI and Bhat, V and Lettre, G and Ascher, DB and Cassa, CA and Sherwood, RI and Pinello, L},
title = {Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.},
journal = {Nature genetics},
volume = {56},
number = {5},
pages = {925-937},
pmid = {38658794},
issn = {1546-1718},
support = {R01 HL164409/HL/NHLBI NIH HHS/United States ; UM1 HG012010/HG/NHGRI NIH HHS/United States ; UM1HG012010//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01HL164409//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R35HG010717-01//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01HG010372//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01HL164409//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01HG010372//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; UM1HG012010//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01HL164409//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01GM143249//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; GNT1174405//Department of Health | National Health and Medical Research Council (NHMRC)/ ; GNT1174405//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Phenotype ; *Genotype ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Bayes Theorem ; Receptors, LDL/genetics ; HEK293 Cells ; },
abstract = {CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization.},
}

Humans
*Gene Editing/methods
*Phenotype
*Genotype
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Bayes Theorem
Receptors, LDL/genetics
HEK293 Cells

@article {pmid38641083,
year = {2024},
author = {Vora, DS and Bhandari, SM and Sundar, D},
title = {DNA shape features improve prediction of CRISPR/Cas9 activity.},
journal = {Methods (San Diego, Calif.)},
volume = {226},
number = {},
pages = {120-126},
doi = {10.1016/j.ymeth.2024.04.012},
pmid = {38641083},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; *Gene Editing/methods ; *Neural Networks, Computer ; *Machine Learning ; Nucleic Acid Conformation ; Humans ; Bayes Theorem ; Epigenesis, Genetic ; },
abstract = {The CRISPR/Cas9 genome editing technology has transformed basic and translational research in biology and medicine. However, the advances are hindered by off-target effects and a paucity in the knowledge of the mechanism of the Cas9 protein. Machine learning models have been proposed for the prediction of Cas9 activity at unintended sites, yet feature engineering plays a major role in the outcome of the predictors. This study evaluates the improvement in the performance of similar predictors upon inclusion of epigenetic and DNA shape feature groups in the conventionally used sequence-based Cas9 target and off-target datasets. The approach involved the utilization of neural networks trained on a diverse range of parameters, allowing us to systematically assess the performance increase for the meticulously designed datasets- (i) sequence only, (ii) sequence and epigenetic features, and (iii) sequence, epigenetic and DNA shape feature datasets. The addition of DNA shape information significantly improved predictive performance, evaluated by Akaike and Bayesian information criteria. The evaluation of individual feature importance by permutation and LIME-based methods also indicates that not only sequence features like mismatches and nucleotide composition, but also base pairing parameters like opening and stretch, that are indicative of distortion in the DNA-RNA hybrid in the presence of mismatches, influence model outcomes.},
}

*CRISPR-Cas Systems/genetics
*DNA/genetics/chemistry
*Gene Editing/methods
*Neural Networks, Computer
*Machine Learning
Nucleic Acid Conformation
Humans
Bayes Theorem
Epigenesis, Genetic

@article {pmid38605144,
year = {2024},
author = {Chidley, C and Darnell, AM and Gaudio, BL and Lien, EC and Barbeau, AM and Vander Heiden, MG and Sorger, PK},
title = {A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.},
journal = {Nature cell biology},
volume = {26},
number = {5},
pages = {825-838},
pmid = {38605144},
issn = {1476-4679},
support = {U54 CA225088/CA/NCI NIH HHS/United States ; P30-CA014051//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R35-CA242379//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; U54-CA225088//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Tumor Microenvironment ; *Cell Proliferation ; Animals ; CRISPR-Cas Systems ; Nutrients/metabolism ; Cell Line, Tumor ; Biological Transport ; Glucose/metabolism ; Amino Acids/metabolism ; Serotonin/metabolism ; Amino Acid Transport Systems/metabolism/genetics ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.},
}

Humans
*Tumor Microenvironment
*Cell Proliferation
Animals
CRISPR-Cas Systems
Nutrients/metabolism
Cell Line, Tumor
Biological Transport
Glucose/metabolism
Amino Acids/metabolism
Serotonin/metabolism
Amino Acid Transport Systems/metabolism/genetics
Mice
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38160731,
year = {2024},
author = {Kislitsin, VY and Chulkin, AM and Dotsenko, AS and Sinelnikov, IG and Sinitsyn, AP and Rozhkova, AM},
title = {The role of intracellular β-glucosidase in cellulolytic response induction in filamentous fungus Penicillium verruculosum.},
journal = {Research in microbiology},
volume = {175},
number = {4},
pages = {104178},
doi = {10.1016/j.resmic.2023.104178},
pmid = {38160731},
issn = {1769-7123},
mesh = {*Penicillium/genetics/enzymology ; *beta-Glucosidase/metabolism/genetics ; Gene Expression Regulation, Fungal ; Fungal Proteins/genetics/metabolism ; Cellulose/metabolism ; Cellobiose/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Neurospora crassa/genetics/enzymology ; Cellulose 1,4-beta-Cellobiosidase/metabolism/genetics ; Gene Editing ; },
abstract = {In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular β-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.},
}

*Penicillium/genetics/enzymology
*beta-Glucosidase/metabolism/genetics
Gene Expression Regulation, Fungal
Fungal Proteins/genetics/metabolism
Cellulose/metabolism
Cellobiose/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Neurospora crassa/genetics/enzymology
Cellulose 1,4-beta-Cellobiosidase/metabolism/genetics
Gene Editing

@article {pmid37952706,
year = {2024},
author = {Zhang, Y and Zhang, Z and Chen, Y and Tan, X and Liu, Y and Tian, Z and Wang, J and Zhang, X and Zhang, D},
title = {Protein kinase A regulatory subunit is required for normal growth, zoosporogenesis, and pathogenicity in Phytophthora sojae.},
journal = {Research in microbiology},
volume = {175},
number = {4},
pages = {104152},
doi = {10.1016/j.resmic.2023.104152},
pmid = {37952706},
issn = {1769-7123},
mesh = {*Phytophthora/genetics/pathogenicity/growth & development/enzymology ; *Glycine max/microbiology ; *Plant Diseases/microbiology ; *Cyclic AMP-Dependent Protein Kinases/metabolism/genetics ; Virulence ; *Hyphae/growth & development/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Signal Transduction ; Spores/growth & development/genetics ; Plant Leaves/microbiology ; Gene Editing ; },
abstract = {Phytophthora sojae, one of the most devastating Oomycete pathogens, causes severe diseases that lead to economic loss in the soybean industry. The production of zoospores play a crucial role during the development of Phytophthora disease. In this work, CRISPR/Cas9 genome editing technology were used to obtain protein kinase A regulatory subunit (PsPkaR) knockout mutants. The role of PsPkaR in the production of zoospores and pathogenicity of P. sojae was analyzed. The overall findings indicate that PsPkaR is involved in regulating the growth process of P. sojae, primarily affecting the hyphal morphology and growth rate. Additionally, PsPkaR participates in the regulation of the release process of zoospores. Specifically, knocking-out PsPkaR resulted in incomplete cytoplasmic differentiation and uneven protoplast division, leading to abnormal release of zoospores. Furthermore, when the PsPkaR knockout mutants were inoculated on soybean leaves, the pathogenicity was significantly reduced compared to that of the wild-type and control strains. These findings of this study provide important clues and evidence regarding the role of the cAMP-PKA signaling pathway in the interaction between P. sojae and its host. This work contributes to a better understanding of the pathogenic mechanism of P. sojae and the development of corresponding prevention and control strategies.},
}

*Phytophthora/genetics/pathogenicity/growth & development/enzymology
*Glycine max/microbiology
*Plant Diseases/microbiology
*Cyclic AMP-Dependent Protein Kinases/metabolism/genetics
Virulence
*Hyphae/growth & development/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Signal Transduction
Spores/growth & development/genetics
Plant Leaves/microbiology
Gene Editing

@article {pmid37386294,
year = {2024},
author = {Xiang, G and Li, Y and Sun, J and Huo, Y and Cao, S and Cao, Y and Guo, Y and Yang, L and Cai, Y and Zhang, YE and Wang, H},
title = {Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors.},
journal = {Nature biotechnology},
volume = {42},
number = {5},
pages = {745-757},
pmid = {37386294},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; Humans ; *DNA Transposable Elements/genetics ; Escherichia coli/genetics ; Evolution, Molecular ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; },
abstract = {As the evolutionary ancestor of Cas12 nuclease, the transposon (IS200/IS605)-encoded TnpB proteins act as compact RNA-guided DNA endonucleases. To explore their evolutionary diversity and potential as genome editors, we screened TnpBs from 64 annotated IS605 members and identified 25 active in Escherichia coli, of which three are active in human cells. Further characterization of these 25 TnpBs enables prediction of the transposon-associated motif (TAM) and the right-end element RNA (reRNA) directly from genomic sequences. We established a framework for annotating TnpB systems in prokaryotic genomes and applied it to identify 14 additional candidates. Among these, ISAam1 (369 amino acids (aa)) and ISYmu1 (382 aa) TnpBs demonstrated robust editing activity across dozens of genomic loci in human cells. Both RNA-guided genome editors demonstrated similar editing efficiency as SaCas9 (1,053 aa) while being substantially smaller. The enormous diversity of TnpBs holds potential for the discovery of additional valuable genome editors.},
}

*Gene Editing/methods
Humans
*DNA Transposable Elements/genetics
Escherichia coli/genetics
Evolution, Molecular
CRISPR-Cas Systems/genetics
Endonucleases/genetics/metabolism

@article {pmid38727638,
year = {2024},
author = {Severi, AA and Akbari, B},
title = {CRISPR-Cas9 delivery strategies and applications: Review and update.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {3},
pages = {e23598},
doi = {10.1002/dvg.23598},
pmid = {38727638},
issn = {1526-968X},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Neoplasms/genetics/therapy ; Animals ; Genetic Therapy/methods ; Gene Transfer Techniques ; },
abstract = {Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Neoplasms/genetics/therapy
Animals
Genetic Therapy/methods
Gene Transfer Techniques

@article {pmid38727241,
year = {2024},
author = {Wünschiers, R and Leidenfrost, RM and Holtorf, H and Dittrich, B and Dürr, T and Braun, J},
title = {CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.},
journal = {Journal of microbiology & biology education},
volume = {},
number = {},
pages = {e0018723},
doi = {10.1128/jmbe.00187-23},
pmid = {38727241},
issn = {1935-7877},
abstract = {Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.},
}

@article {pmid38726868,
year = {2024},
author = {Kim, GE and Park, HH},
title = {AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae357},
pmid = {38726868},
issn = {1362-4962},
support = {NRF-2021R1A2C3003331//National Research Foundation of Korea/ ; },
abstract = {CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages.},
}

@article {pmid38726521,
year = {2023},
author = {Wang, QJ},
title = {[Genetics and hearing loss－from gene function to gene therapy].},
journal = {Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery},
volume = {58},
number = {Z1},
pages = {41-51},
doi = {10.3760/cma.j.cn115330-20231209-00281},
pmid = {38726521},
issn = {1673-0860},
support = {82350005, 82222016, 82271189, 82271171//National Natural Science Foundation of China/ ; },
mesh = {*Genetic Therapy/methods ; Humans ; *Hearing Loss/genetics/therapy ; Deafness/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing ; },
}

*Genetic Therapy/methods
Humans
*Hearing Loss/genetics/therapy
Deafness/genetics/therapy
CRISPR-Cas Systems
Gene Editing

@article {pmid38725755,
year = {2024},
author = {Asif, M and Khan, WJ and Aslam, S and Aslam, A and Chowdhury, MA},
title = {The Use of CRISPR-Cas9 Genetic Technology in Cardiovascular Disease: A Comprehensive Review of Current Progress and Future Prospective.},
journal = {Cureus},
volume = {16},
number = {4},
pages = {e57869},
pmid = {38725755},
issn = {2168-8184},
abstract = {Over the last century, there have been major landmark developments in the field of medicine, enabling us to control and cure various diseases on a larger scale. A few of these include the discovery of antibiotics, the development of vaccines, and the origin of organ and tissue transplants. The continued quest for innovation in microbiology and medicine has helped humankind save millions of lives and decrease morbidity at the global level. Genetic medicine has grown significantly in the last two decades and appears to be the next frontier of curative therapies for chronic diseases. One important landmark in genetic medicine is the development of CRISPR (clustered, regularly interspaced short palindromic repeats) technology. In this article, we describe the basic structure and function of the CRISPR-Cas9 system, which, simply put, consists of an RNA part and a protein. It works as a molecular scissor that can perform targeted cuts followed by repairs in and around the genes of interest to attain favorable translational outcomes. We focused on summarizing recent studies using CRISPR-Cas9 technology in diagnosing and treating cardiovascular disease. These studies are primarily experimental and limited to animal models. However, their results are promising enough to anticipate that this technology will undoubtedly be available in clinical medicine in the coming years. CRISPR-Cas9-mediated gene editing has been used to study and potentially treat congenital heart disease, hyperlipidemias, arrhythmogenic cardiomyopathies, and the prevention of ischemia-reperfusion injury. Despite the current progress, we recognize the several challenges this technology faces, including funding for research, improving precision and reproducible results for human subjects, and establishing protocols for ethical compliance so that it is acceptable to the scientific community and the general public.},
}

@article {pmid38725495,
year = {2024},
author = {Wu, Y and Jin, R and Chang, Y and Liu, M},
title = {A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.},
journal = {Chemical science},
volume = {15},
number = {18},
pages = {6934-6942},
pmid = {38725495},
issn = {2041-6520},
abstract = {A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.},
}

@article {pmid38725064,
year = {2024},
author = {Yan, M and Yu, Z},
title = {Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits.},
journal = {Microbiome},
volume = {12},
number = {1},
pages = {82},
pmid = {38725064},
issn = {2049-2618},
support = {2021-67015-33393//USDA National Institute of Food and Agriculture/ ; 2021-67015-33393//USDA National Institute of Food and Agriculture/ ; },
mesh = {*Rumen/microbiology/virology ; Animals ; *Viruses/classification/genetics ; *Metagenome ; Gastrointestinal Microbiome ; Virome ; Ruminants/microbiology/virology ; Methane/metabolism ; Animal Feed ; Bacteria/classification/genetics ; },
abstract = {BACKGROUND: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined.

RESULTS: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions.

CONCLUSIONS: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.},
}

*Rumen/microbiology/virology
Animals
*Viruses/classification/genetics
*Metagenome
Gastrointestinal Microbiome
Virome
Ruminants/microbiology/virology
Methane/metabolism
Animal Feed
Bacteria/classification/genetics

@article {pmid38724931,
year = {2024},
author = {Zhan, T and Li, X and Liu, J and Ye, C},
title = {CRISPR-based gene expression platform for precise regulation of bladder cancer.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {66},
pmid = {38724931},
issn = {1689-1392},
mesh = {*Urinary Bladder Neoplasms/genetics/therapy/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Gene Editing/methods ; beta Catenin/metabolism/genetics ; NF-kappa B/metabolism/genetics ; Gene Expression/genetics ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and β-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular β-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.},
}

*Urinary Bladder Neoplasms/genetics/therapy/metabolism
Humans
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
*Gene Editing/methods
beta Catenin/metabolism/genetics
NF-kappa B/metabolism/genetics
Gene Expression/genetics
Gene Expression Regulation, Neoplastic
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid38724663,
year = {2024},
author = {},
title = {CRISPR therapy restores some vision to people with blindness.},
journal = {Nature},
volume = {629},
number = {8012},
pages = {507},
pmid = {38724663},
issn = {1476-4687},
mesh = {Humans ; *Genetic Therapy/trends/methods ; *Blindness/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}

Humans
*Genetic Therapy/trends/methods
*Blindness/therapy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing
Animals
Mice
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid38724644,
year = {2024},
author = {Lim, JJ and Murata, Y and Yuri, S and Kitamuro, K and Kawai, T and Isotani, A},
title = {Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {10636},
pmid = {38724644},
issn = {2045-2322},
support = {22K06067//Japan Society for the Promotion of Science/ ; 21K19267//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; Rats ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Forkhead Transcription Factors/genetics/metabolism ; Thymus Gland/metabolism ; Models, Animal ; Blastocyst/metabolism ; },
abstract = {Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNA[ms] system that induces cell death with a single-guide RNA (sgRNA[ms]) targeting multiple sites in the genome. The Cas9-sgRNA[ms] system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNA[ms] at the Rosa26 locus (Foxn1[Cas9]; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1[Cas9]; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNA[ms] system because Rosa26_ms could be maintained as homozygous.},
}

Animals
*CRISPR-Cas Systems
Mice
Rats
*RNA, Guide, CRISPR-Cas Systems/genetics
*Forkhead Transcription Factors/genetics/metabolism
Thymus Gland/metabolism
Models, Animal
Blastocyst/metabolism

@article {pmid38666524,
year = {2024},
author = {Xu, J and Zhou, T and Xue, D and Sui, Z and Yang, H and Yuan, X and Wang, Q},
title = {Integrating recombinase polymerase amplification with CRISPR/Cas9-initiated nicking-rolling circle amplification in Staphylococcus aureus assay.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {40},
pages = {5314-5317},
doi = {10.1039/d4cc00238e},
pmid = {38666524},
issn = {1364-548X},
mesh = {*Staphylococcus aureus/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques ; *Recombinases/metabolism/genetics ; },
abstract = {We integrate recombinase polymerase amplification (RPA) with CRISPR/Cas9-initiated nicking rolling circle amplification (CRISPR/Cas9-nRCA) for detecting Staphylococcus aureus. This approach utilizes a unique dimeric G-triplex structure, demonstrating firstly enhanced ThT fluorescence for target detection. The proof-of-concept study introduces a new avenue for integrating isothermal amplifications with CRISPR/Cas9 in the fields of pathogen detection and disease diagnosis.},
}

*Staphylococcus aureus/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques
*Recombinases/metabolism/genetics

@article {pmid38615930,
year = {2024},
author = {Liang, JH and Wang, WT and Wang, R and Gao, R and Du, KX and Duan, ZW and Zhang, XY and Li, Y and Wu, JZ and Yin, H and Shen, HR and Wang, L and Li, JY and Guo, JR and Xu, W},
title = {PRMT5 activates lipid metabolic reprogramming via MYC contributing to the growth and survival of mantle cell lymphoma.},
journal = {Cancer letters},
volume = {591},
number = {},
pages = {216877},
doi = {10.1016/j.canlet.2024.216877},
pmid = {38615930},
issn = {1872-7980},
mesh = {*Lymphoma, Mantle-Cell/genetics/metabolism/pathology ; Humans ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Lipid Metabolism ; *Cell Proliferation ; *Fatty Acid Synthase, Type I/metabolism/genetics ; Cell Line, Tumor ; Sterol Regulatory Element Binding Protein 1/metabolism/genetics ; Sterol Regulatory Element Binding Protein 2/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Animals ; Mice ; Male ; Prognosis ; Female ; Cholesterol/metabolism ; CRISPR-Cas Systems ; Metabolic Reprogramming ; },
abstract = {Mantle cell lymphoma (MCL) is an incurable and aggressive subtype of non-Hodgkin B-cell lymphoma. Increased lipid uptake, storage, and lipogenesis occur in a variety of cancers and contribute to rapid tumor growth. However, no data has been explored for the roles of lipid metabolism reprogramming in MCL. Here, we identified aberrant lipid metabolism reprogramming and PRMT5 as a key regulator of cholesterol and fatty acid metabolism reprogramming in MCL patients. High PRMT5 expression predicts adverse outcome prognosis in 105 patients with MCL and GEO database (GSE93291). PRMT5 deficiency resulted in proliferation defects and cell death by CRISPR/Cas9 editing. Moreover, PRMT5 inhibitors including SH3765 and EPZ015666 worked through blocking SREBP1/2 and FASN expression in MCL. Furthermore, PRMT5 was significantly associated with MYC expression in 105 MCL samples and the GEO database (GSE93291). CRISPR MYC knockout indicated PRMT5 can promote MCL outgrowth by inducing SREBP1/2 and FASN expression through the MYC pathway.},
}

*Lymphoma, Mantle-Cell/genetics/metabolism/pathology
Humans
*Protein-Arginine N-Methyltransferases/genetics/metabolism
*Proto-Oncogene Proteins c-myc/metabolism/genetics
*Lipid Metabolism
*Cell Proliferation
*Fatty Acid Synthase, Type I/metabolism/genetics
Cell Line, Tumor
Sterol Regulatory Element Binding Protein 1/metabolism/genetics
Sterol Regulatory Element Binding Protein 2/metabolism/genetics
Gene Expression Regulation, Neoplastic
Animals
Mice
Male
Prognosis
Female
Cholesterol/metabolism
CRISPR-Cas Systems
Metabolic Reprogramming

@article {pmid38608339,
year = {2024},
author = {Chen, Z and Hu, J and Dai, J and Zhou, C and Hua, Y and Hua, X and Zhao, Y},
title = {Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.},
journal = {Microbiological research},
volume = {284},
number = {},
pages = {127713},
doi = {10.1016/j.micres.2024.127713},
pmid = {38608339},
issn = {1618-0623},
mesh = {*Deinococcus/genetics ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems ; Genome, Bacterial ; DNA Breaks, Double-Stranded ; Homologous Recombination ; Bacterial Proteins/genetics/metabolism ; Plasmids/genetics ; Mutagenesis ; Genomic Instability ; Clustered Regularly Interspaced Short Palindromic Repeats ; Rec A Recombinases/genetics/metabolism ; DNA Damage ; },
abstract = {Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.},
}

*Deinococcus/genetics
*Gene Editing/methods
*DNA Repair/genetics
*CRISPR-Cas Systems
Genome, Bacterial
DNA Breaks, Double-Stranded
Homologous Recombination
Bacterial Proteins/genetics/metabolism
Plasmids/genetics
Mutagenesis
Genomic Instability
Clustered Regularly Interspaced Short Palindromic Repeats
Rec A Recombinases/genetics/metabolism
DNA Damage

@article {pmid38591870,
year = {2024},
author = {Negroni, YL and Doro, I and Tamborrino, A and Luzzi, I and Fortunato, S and Hensel, G and Khosravi, S and Maretto, L and Stevanato, P and Lo Schiavo, F and de Pinto, MC and Krupinska, K and Zottini, M},
title = {The Arabidopsis Mitochondrial Nucleoid-Associated Protein WHIRLY2 Is Required for a Proper Response to Salt Stress.},
journal = {Plant & cell physiology},
volume = {65},
number = {4},
pages = {576-589},
doi = {10.1093/pcp/pcae025},
pmid = {38591870},
issn = {1471-9053},
support = {2017FBS8YN PRIN-2017//Italian Ministry/ ; },
mesh = {*Arabidopsis/genetics/physiology/metabolism/drug effects ; *Arabidopsis Proteins/metabolism/genetics ; *Salt Stress/genetics ; *Mitochondria/metabolism/drug effects ; *DNA, Mitochondrial/genetics/metabolism ; Mitochondrial Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.},
}

*Arabidopsis/genetics/physiology/metabolism/drug effects
*Arabidopsis Proteins/metabolism/genetics
*Salt Stress/genetics
*Mitochondria/metabolism/drug effects
*DNA, Mitochondrial/genetics/metabolism
Mitochondrial Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid38579711,
year = {2024},
author = {Haideri, T and Lin, J and Bao, X and Lian, XL},
title = {MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {19},
number = {5},
pages = {744-757},
doi = {10.1016/j.stemcr.2024.03.005},
pmid = {38579711},
issn = {2213-6711},
support = {R21 EB026035/EB/NIBIB NIH HHS/United States ; R37 CA265926/CA/NCI NIH HHS/United States ; R56 DK133147/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *Pluripotent Stem Cells/metabolism/cytology ; *Cell Lineage/genetics ; *Gene Knock-In Techniques/methods ; Genes, Reporter ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing/methods ; RNA, Messenger/genetics/metabolism ; },
abstract = {Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.},
}

Humans
*Pluripotent Stem Cells/metabolism/cytology
*Cell Lineage/genetics
*Gene Knock-In Techniques/methods
Genes, Reporter
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line
Gene Editing/methods
RNA, Messenger/genetics/metabolism

@article {pmid38574388,
year = {2024},
author = {Homanics, GE and Park, JE and Bailey, L and Schaeffer, DJ and Schaeffer, L and He, J and Li, S and Zhang, T and Haber, A and Spruce, C and Greenwood, A and Murai, T and Schultz, L and Mongeau, L and Ha, SK and Oluoch, J and Stein, B and Choi, SH and Huhe, H and Thathiah, A and Strick, PL and Carter, GW and Silva, AC and Sukoff Rizzo, SJ},
title = {Early molecular events of autosomal-dominant Alzheimer's disease in marmosets with PSEN1 mutations.},
journal = {Alzheimer's & dementia : the journal of the Alzheimer's Association},
volume = {20},
number = {5},
pages = {3455-3471},
doi = {10.1002/alz.13806},
pmid = {38574388},
issn = {1552-5279},
support = {/NH/NIH HHS/United States ; U19AG074866/AG/NIA NIH HHS/United States ; /NH/NIH HHS/United States ; IPA 2019 No. 16//University of Pittsburgh Medical Center/ ; },
mesh = {Animals ; *Presenilin-1/genetics ; *Alzheimer Disease/genetics ; *Callithrix ; Male ; Female ; Brain/pathology/metabolism ; Amyloid beta-Peptides/metabolism ; Disease Models, Animal ; Point Mutation/genetics ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Mutation/genetics ; Humans ; },
abstract = {INTRODUCTION: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan.

METHODS: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures.

RESULTS: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood.

DISCUSSION: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression.

HIGHLIGHTS: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.},
}

Animals
*Presenilin-1/genetics
*Alzheimer Disease/genetics
*Callithrix
Male
Female
Brain/pathology/metabolism
Amyloid beta-Peptides/metabolism
Disease Models, Animal
Point Mutation/genetics
Animals, Genetically Modified
CRISPR-Cas Systems
Gene Knock-In Techniques
Mutation/genetics
Humans

@article {pmid38558112,
year = {2024},
author = {Kumar, K and Basak, R and Rai, A and Mukhopadhyay, A},
title = {GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.},
journal = {Molecular microbiology},
volume = {121},
number = {5},
pages = {1063-1078},
doi = {10.1111/mmi.15255},
pmid = {38558112},
issn = {1365-2958},
support = {2018BLZ8469//Indian Institute of Technology, New Delhi, India/ ; //J.C. Bose Fellowship/ ; CRG/2021/000099//Science and Engineering Research Board, Government of India/ ; SR/S2/JCB-24/2009//Science and Engineering board, India/ ; },
mesh = {*Virulence Factors/metabolism/genetics ; *Protozoan Proteins/metabolism/genetics ; *Metalloendopeptidases/metabolism/genetics ; Golgi Apparatus/metabolism ; Endoplasmic Reticulum/metabolism ; Macrophages/parasitology/metabolism ; Animals ; Leishmania/metabolism/genetics ; Protein Transport ; CRISPR-Cas Systems ; Golgi Matrix Proteins/metabolism/genetics ; },
abstract = {Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.},
}

*Virulence Factors/metabolism/genetics
*Protozoan Proteins/metabolism/genetics
*Metalloendopeptidases/metabolism/genetics
Golgi Apparatus/metabolism
Endoplasmic Reticulum/metabolism
Macrophages/parasitology/metabolism
Animals
Leishmania/metabolism/genetics
Protein Transport
CRISPR-Cas Systems
Golgi Matrix Proteins/metabolism/genetics

@article {pmid38411275,
year = {2024},
author = {Furusawa, T and Cavero, R and Liu, Y and Li, H and Xu, X and Andresson, T and Reinhold, W and White, O and Boufraqech, M and Meyer, TJ and Hartmann, O and Diefenbacher, ME and Pommier, Y and Weyemi, U},
title = {Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.},
journal = {Molecular carcinogenesis},
volume = {63},
number = {6},
pages = {1024-1037},
doi = {10.1002/mc.23705},
pmid = {38411275},
issn = {1098-2744},
support = {/CA/NCI NIH HHS/United States ; //NCI center for cancer research/ ; /CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; *Drug Resistance, Neoplasm/genetics ; *Monocarboxylic Acid Transporters/genetics/metabolism ; Cell Line, Tumor ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects/genetics ; },
abstract = {Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clustered regularly interspaced short palindromic repeats knockout screen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer (NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylic acid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*Lung Neoplasms/genetics/drug therapy/metabolism/pathology
*Drug Resistance, Neoplasm/genetics
*Monocarboxylic Acid Transporters/genetics/metabolism
Cell Line, Tumor
Mitochondrial Membrane Transport Proteins/genetics/metabolism
Phthalazines/pharmacology
Piperazines/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Mitochondria/metabolism/drug effects/genetics

@article {pmid38243597,
year = {2024},
author = {Zhang, C and Tang, Y and Tang, S and Chen, L and Li, T and Yuan, H and Xu, Y and Zhou, Y and Zhang, S and Wang, J and Wen, H and Jiang, W and Pang, Y and Deng, X and Cao, X and Zhou, J and Song, X and Liu, Q},
title = {An inducible CRISPR activation tool for accelerating plant regeneration.},
journal = {Plant communications},
volume = {5},
number = {5},
pages = {100823},
doi = {10.1016/j.xplc.2024.100823},
pmid = {38243597},
issn = {2590-3462},
mesh = {*Regeneration/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; },
abstract = {The inducible CRISPR activation (CRISPR-a) system offers unparalleled precision and versatility for regulating endogenous genes, making it highly sought after in plant research. In this study, we developed a chemically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system. We systematically compared different induction strategies and achieved high efficiency in target gene activation. We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration. Further experiments showed that induced activation of these morphogenic genes can accelerate regeneration and improve regeneration efficiency in both eudicot and monocot plants, including alfalfa, woodland strawberry, and sheepgrass. Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.},
}

*Regeneration/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Plants, Genetically Modified/genetics
Gene Expression Regulation, Plant

@article {pmid38113380,
year = {2024},
author = {Arimura, SI and Nakazato, I},
title = {Genome Editing of Plant Mitochondrial and Chloroplast Genomes.},
journal = {Plant & cell physiology},
volume = {65},
number = {4},
pages = {477-483},
doi = {10.1093/pcp/pcad162},
pmid = {38113380},
issn = {1471-9053},
support = {20H00417, Core-to-Core, 22J20237//Japan Society for the Promotion of Science/ ; JPMJPR12B2 (PRESTO) and JPMJTR22UG (ASTEP)//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing/methods ; *Genome, Chloroplast/genetics ; *Genome, Plant/genetics ; *Genome, Mitochondrial/genetics ; CRISPR-Cas Systems ; Plants/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Chloroplasts/genetics ; },
abstract = {Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.},
}

*Gene Editing/methods
*Genome, Chloroplast/genetics
*Genome, Plant/genetics
*Genome, Mitochondrial/genetics
CRISPR-Cas Systems
Plants/genetics
Transcription Activator-Like Effector Nucleases/genetics/metabolism
Chloroplasts/genetics

@article {pmid38723038,
year = {2024},
author = {Kadry, MO and Abdel-Megeed, RM},
title = {CRISPR-Cas9 genome and long non-coding RNAs as a novel diagnostic index for prostate cancer therapy via liposomal-coated compounds.},
journal = {PloS one},
volume = {19},
number = {5},
pages = {e0302264},
pmid = {38723038},
issn = {1932-6203},
mesh = {Male ; *Prostatic Neoplasms/genetics/drug therapy/pathology/diagnosis ; Animals ; *Liposomes ; Rats ; *RNA, Long Noncoding/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; },
abstract = {CRISPR/Cas9 is a recently discovered genomic editing technique that altered scientist's sight in studying genes function. Cas9 is controlled via guide (g) RNAs, which match the DNA targeted in cleavage to modify the respective gene. The development in prostate cancer (PC) modeling directed not only to novel resources for recognizing the signaling pathways overriding prostate cell carcinoma, but it has also created a vast reservoir for complementary tools to examine therapies counteracting this type of cancer. Various cultured somatic rat models for prostate cancer have been developed that nearly mimic human prostate cancer. Nano-medicine can passively target cancer cells via increasing bioavailability and conjugation via specific legend, contributing to reduced systemic side-effects and increased efficacy. This article highlights liposomal loaded Nano-medicine as a potential treatment for prostate cancer and clarifies the CRISPR/Cas9 variation accompanied with prostate cancer. PC is induced experimentally in western rat model via ethinyl estradiol for 4 weeks and SC. dose of 3, 2'- dimethyl-4-aminobiphenyl estradiol (DAE) (50mg/kg) followed by treatment via targeted liposomal-coated compounds such as liposomal dexamethasone (DXM), liposomal doxorubicin (DOX) and liposomal Turmeric (TUR) (3mg/kg IP) for four weeks in a comparative study to their non-targeted analogue dexamethasone, doxorubicin and Turmeric. 3, 2'- dimethyl-4-aminobiphenylestradiol elicit prostate cancer in western rats within 5 months. Simultaneous supplementations with these liposomal compounds influence on prostate cancer; tumor markers were investigated via prostate-specific antigen (PSA), Nitric oxide (NOX) and CRISPR/Cas9 gene editing. Several long non-coding RNAs were reported to be deregulated in prostate cell carcinoma, including MALAT1. On the other hand, gene expression of apoptotic biomarkers focal adhesion kinase (AKT-1), phosphatidylinistol kinase (PI3K) and glycogen synthase kinase-3 (GSK-3) was also investigated and further confirming these results via histopathological examination. Liposomal loaded dexamethasone; doxorubicin and Turmeric can be considered as promising therapeutic agents for prostate cancer via modulating CRISPR/Cas9 gene editing and long non coding gene MALAT1.},
}

Male
*Prostatic Neoplasms/genetics/drug therapy/pathology/diagnosis
Animals
*Liposomes
Rats
*RNA, Long Noncoding/genetics
*CRISPR-Cas Systems/genetics
Humans
Gene Editing/methods

@article {pmid38722107,
year = {2024},
author = {Fazelzadeh Haghighi, M and Jafari Khamirani, H and Fallahi, J and Monfared, AA and Ashrafi Dehkordi, K and Tabei, SMB},
title = {Novel insight into FCSK-congenital disorder of glycosylation through a CRISPR-generated cell model.},
journal = {Molecular genetics & genomic medicine},
volume = {12},
number = {5},
pages = {e2445},
pmid = {38722107},
issn = {2324-9269},
support = {3834//Shahrekord University of Medical Sciences/ ; },
mesh = {*Congenital Disorders of Glycosylation/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems ; Fucose/metabolism ; Glycosylation ; Receptors, Notch/metabolism/genetics ; Phosphotransferases (Alcohol Group Acceptor) ; },
abstract = {BACKGROUND: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency.

METHODS: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses.

RESULTS: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation.

CONCLUSION: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.},
}

*Congenital Disorders of Glycosylation/genetics/pathology/metabolism
Humans
*CRISPR-Cas Systems
Fucose/metabolism
Glycosylation
Receptors, Notch/metabolism/genetics
Phosphotransferases (Alcohol Group Acceptor)

@article {pmid38722096,
year = {2024},
author = {Panara, V and Yu, H and Peng, D and Staxäng, K and Hodik, M and Filipek-Gorniok, B and Kazenwadel, J and Skoczylas, R and Mason, E and Allalou, A and Harvey, NL and Haitina, T and Hogan, BM and Koltowska, K},
title = {Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.},
journal = {Development (Cambridge, England)},
volume = {151},
number = {9},
pages = {},
doi = {10.1242/dev.202525},
pmid = {38722096},
issn = {1477-9129},
support = {2017.0144//Knut och Alice Wallenbergs Stiftelse/ ; M13/17//Ragnar Söderbergs stiftelse/ ; VR-MH-2016-01437//Vetenskapsrådet/ ; //Kjell och Märta Beijers Stiftelse/ ; 1146706//National Health and Medical Research Council/ ; },
mesh = {Animals ; *Homeodomain Proteins/metabolism/genetics ; *Zebrafish/genetics/embryology ; *Tumor Suppressor Proteins/metabolism/genetics ; *Gene Expression Regulation, Developmental ; *Enhancer Elements, Genetic/genetics ; *Lymphatic Vessels/metabolism/embryology ; *Zebrafish Proteins/metabolism/genetics ; *Endothelial Cells/metabolism ; Lymphangiogenesis/genetics ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Mice ; },
abstract = {During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.},
}

Animals
*Homeodomain Proteins/metabolism/genetics
*Zebrafish/genetics/embryology
*Tumor Suppressor Proteins/metabolism/genetics
*Gene Expression Regulation, Developmental
*Enhancer Elements, Genetic/genetics
*Lymphatic Vessels/metabolism/embryology
*Zebrafish Proteins/metabolism/genetics
*Endothelial Cells/metabolism
Lymphangiogenesis/genetics
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics
Mice

@article {pmid38695755,
year = {2024},
author = {Pian, H and Wang, H and Wang, H and Li, Z},
title = {Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria.},
journal = {Analytical chemistry},
volume = {96},
number = {19},
pages = {7524-7531},
doi = {10.1021/acs.analchem.4c00230},
pmid = {38695755},
issn = {1520-6882},
mesh = {*Malaria/diagnosis/parasitology ; *Point-of-Care Systems ; Humans ; *CRISPR-Cas Systems/genetics ; Plasmodium/genetics/isolation & purification ; Transcription, Genetic ; },
abstract = {Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts.},
}

*Malaria/diagnosis/parasitology
*Point-of-Care Systems
Humans
*CRISPR-Cas Systems/genetics
Plasmodium/genetics/isolation & purification
Transcription, Genetic

@article {pmid38684052,
year = {2024},
author = {Blanluet, C and Kuo, CJ and Bhattacharya, A and Santiago, JG},
title = {Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping.},
journal = {Analytical chemistry},
volume = {96},
number = {19},
pages = {7444-7451},
doi = {10.1021/acs.analchem.3c05657},
pmid = {38684052},
issn = {1520-6882},
mesh = {Kinetics ; Humans ; *CRISPR-Cas Systems/genetics ; Lung Neoplasms/genetics ; Genotyping Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genotype ; },
abstract = {Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.},
}

Kinetics
Humans
*CRISPR-Cas Systems/genetics
Lung Neoplasms/genetics
Genotyping Techniques/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genotype

@article {pmid38625092,
year = {2024},
author = {Zhao, S and Liu, J and Zuo, Z},
title = {Secondary Conformational Checkpoint in CRISPR-Cas9.},
journal = {Journal of chemical theory and computation},
volume = {20},
number = {9},
pages = {3440-3448},
doi = {10.1021/acs.jctc.4c00120},
pmid = {38625092},
issn = {1549-9626},
mesh = {*CRISPR-Cas Systems ; *Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Protein Conformation ; Gene Editing ; Mutation ; DNA/chemistry/metabolism ; },
abstract = {A specific checkpoint between target DNA binding and cleavage primarily governs the precision of Cas9 gene editing. Although various CRISPR-Cas9 variants have been developed to improve DNA cleavage accuracy, we still lack a comprehensive understanding of how they work at the molecular level. Herein, we have focused on studying the late-stage conformational transitions of Cas9 and an evolved Cas9 mutant (evoCas9) that start from the precleavage state. Our submilliseconds of dynamic simulations reveal that the presence of base mismatches leads the HNH nuclease domain of Cas9 to alter its principal functional modes of motion, thereby impairing its conformational activation. This observation suggests the existence of a secondary conformational checkpoint that fine-tunes the final DNA cleavage activation. Remarkably, evoCas9 is prone to deviating from the normal activation pathway with base mismatches. This is characterized by a noticeable shift in the positioning of the HNH domain and a significantly perturbed allosteric communication network within the enzyme. Therefore, the mutations evolved in evoCas9 also reinforce the secondary checkpoint in addition to the previously identified primary checkpoint, collectively ensuring this variant's high gene-editing accuracy. This mechanism should also apply to other Cas9-guide RNA variants with enhanced fidelity.},
}

*CRISPR-Cas Systems
*Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
Protein Conformation
Gene Editing
Mutation
DNA/chemistry/metabolism

@article {pmid38177342,
year = {2024},
author = {Eghbalsaied, S and Lawler, C and Petersen, B and Hajiyev, RA and Bischoff, SR and Frankenberg, S},
title = {CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.},
journal = {Gene therapy},
volume = {31},
number = {5-6},
pages = {209-223},
pmid = {38177342},
issn = {1476-5462},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Genome, Mitochondrial ; Mitochondrial Diseases/therapy/genetics/metabolism ; DNA, Mitochondrial/genetics ; Mitochondria/genetics/metabolism ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; },
abstract = {Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
*Genome, Mitochondrial
Mitochondrial Diseases/therapy/genetics/metabolism
DNA, Mitochondrial/genetics
Mitochondria/genetics/metabolism
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Therapy/methods

@article {pmid37587230,
year = {2024},
author = {Burdo, TH and Chen, C and Kaminski, R and Sariyer, IK and Mancuso, P and Donadoni, M and Smith, MD and Sariyer, R and Caocci, M and Liao, S and Liu, H and Huo, W and Zhao, H and Misamore, J and Lewis, MG and Simonyan, V and Thompson, EE and Xu, EY and Cradick, TJ and Gordon, J and Khalili, K},
title = {Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.},
journal = {Gene therapy},
volume = {31},
number = {5-6},
pages = {224-233},
pmid = {37587230},
issn = {1476-5462},
mesh = {Animals ; *Simian Immunodeficiency Virus/genetics/drug effects ; *Macaca mulatta ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Simian Acquired Immunodeficiency Syndrome/virology/therapy ; Tissue Distribution ; Genetic Vectors/genetics/administration & dosage/pharmacokinetics ; Genetic Therapy/methods ; Dependovirus/genetics ; },
abstract = {In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 10[12], 1.4 × 10[13] and 1.4 × 10[14] genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.},
}

Animals
*Simian Immunodeficiency Virus/genetics/drug effects
*Macaca mulatta
*Gene Editing/methods
*CRISPR-Cas Systems
*Simian Acquired Immunodeficiency Syndrome/virology/therapy
Tissue Distribution
Genetic Vectors/genetics/administration & dosage/pharmacokinetics
Genetic Therapy/methods
Dependovirus/genetics

@article {pmid38719574,
year = {2024},
author = {Li, M and Ren, X and Xu, M and Dong, S and Li, X and Chen, X and Wang, C and Yang, F},
title = {Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.},
journal = {Biotechnology journal},
volume = {19},
number = {5},
pages = {e2400178},
doi = {10.1002/biot.202400178},
pmid = {38719574},
issn = {1860-7314},
mesh = {*Bacillus subtilis/genetics/enzymology/metabolism ; *Isomaltose/metabolism/analogs & derivatives ; *Glucosyltransferases/genetics/metabolism ; *Sucrose/metabolism ; *Recombinant Proteins/genetics/metabolism ; Metabolic Engineering/methods ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL[-1] in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL[-1] during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.},
}

*Bacillus subtilis/genetics/enzymology/metabolism
*Isomaltose/metabolism/analogs & derivatives
*Glucosyltransferases/genetics/metabolism
*Sucrose/metabolism
*Recombinant Proteins/genetics/metabolism
Metabolic Engineering/methods
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems/genetics
Bacterial Proteins/genetics/metabolism

@article {pmid38719418,
year = {2024},
author = {Wei, J and Zhang, J and Wang, W and Zhou, H and Ma, H and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {Precision miRNA profiling: Electrochemiluminescence powered by CRISPR-Cas13a and hybridization chain reaction.},
journal = {Analytica chimica acta},
volume = {1307},
number = {},
pages = {342641},
doi = {10.1016/j.aca.2024.342641},
pmid = {38719418},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Nucleic Acid Hybridization ; *CRISPR-Cas Systems/genetics ; *Luminescent Measurements ; *Electrochemical Techniques/methods ; Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.},
}

*MicroRNAs/analysis/genetics
Humans
*Nucleic Acid Hybridization
*CRISPR-Cas Systems/genetics
*Luminescent Measurements
*Electrochemical Techniques/methods
Biosensing Techniques/methods
Limit of Detection

@article {pmid38718125,
year = {2024},
author = {Wang, Z and Wang, H and Lin, S and Angers, S and Sargent, EH and Kelley, SO},
title = {Phenotypic targeting using magnetic nanoparticles for rapid characterization of cellular proliferation regulators.},
journal = {Science advances},
volume = {10},
number = {19},
pages = {eadj1468},
pmid = {38718125},
issn = {2375-2548},
mesh = {Humans ; *Cell Proliferation/drug effects ; *Magnetite Nanoparticles/chemistry ; Cell Line, Tumor ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.},
}

Humans
*Cell Proliferation/drug effects
*Magnetite Nanoparticles/chemistry
Cell Line, Tumor
Phenotype
CRISPR-Cas Systems

@article {pmid38717950,
year = {2024},
author = {Mesaki, K and Yamamoto, H and Juvet, S and Yeung, J and Guan, Z and Akhter, A and Yao, Y and Dickie, C and Mangat, H and Wang, A and Wilson, GW and Mariscal, A and Hu, J and Davidson, AR and Kleinstiver, BP and Cypel, M and Liu, M and Keshavjee, S},
title = {CRISPR-Cas genome editing in ex-vivo human lungs to rewire the translational path of genome-targeting therapeutics.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2023.223},
pmid = {38717950},
issn = {1557-7422},
abstract = {The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex-vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients However, assessing genome-targeting interventions in a clinically-relevant manner prior to clinical trials remains a major challenge due to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating pre-implantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene IL-10. We combined this approach with adenoviral vector (AdV)-mediated IL-10 delivery to create favorable kinetics for early (immediate post-implantation) graft immunomodulation. Using ex-vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex-vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.},
}

@article {pmid38677125,
year = {2024},
author = {Liu, LL and Yin, YQ and Ma, KX and Xing, JC and Ren, XX and Huang, JY and Liao, M and Qi, WB and Huang, LH},
title = {Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.},
journal = {Veterinary microbiology},
volume = {293},
number = {},
pages = {110099},
doi = {10.1016/j.vetmic.2024.110099},
pmid = {38677125},
issn = {1873-2542},
mesh = {*Virus Replication/genetics ; *Encephalitis Virus, Japanese/genetics/physiology ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Animals ; Host-Pathogen Interactions/genetics ; Encephalitis, Japanese/virology ; Cell Line ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.},
}

*Virus Replication/genetics
*Encephalitis Virus, Japanese/genetics/physiology
Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
Animals
Host-Pathogen Interactions/genetics
Encephalitis, Japanese/virology
Cell Line
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38621604,
year = {2024},
author = {Wang, Y and Li, K and Shen, W and Huang, X and Wu, L},
title = {Point-of-care testing of methamphetamine and cocaine utilizing wearable sensors.},
journal = {Analytical biochemistry},
volume = {691},
number = {},
pages = {115526},
doi = {10.1016/j.ab.2024.115526},
pmid = {38621604},
issn = {1096-0309},
mesh = {*Cocaine/analysis ; *Methamphetamine/analysis ; *Wearable Electronic Devices ; Humans ; *Point-of-Care Testing ; Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; Substance Abuse Detection/methods ; },
abstract = {The imperative for the point-of-care testing of methamphetamine and cocaine in drug abuse prevention necessitates innovative solutions. To address this need, we have introduced a multi-channel wearable sensor harnessing CRISPR/Cas12a system. A CRISPR/Cas12a based system, integrated with aptamers specific to methamphetamine and cocaine, has been engineered. These aptamers function as signal-mediated intermediaries, converting methamphetamine and cocaine into nucleic acid signals, subsequently generating single-stranded DNA to activate the Cas12 protein. Additionally, we have integrated a microfluidic system and magnetic separation technology into the CRISPR system, enabling rapid and precise detection of cocaine and methamphetamine. The proposed sensing platform demonstrated exceptional sensitivity, achieving a detection limit as low as 0.1 ng/mL. This sensor is expected to be used for on-site drug detection in the future.},
}

*Cocaine/analysis
*Methamphetamine/analysis
*Wearable Electronic Devices
Humans
*Point-of-Care Testing
Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
Substance Abuse Detection/methods

@article {pmid38579481,
year = {2024},
author = {Zhang, D and Jiang, S and Xia, N and Zhang, J and Liu, A and Deng, D and Zhang, C and Sun, Y and Chen, N and Kang, X and Pan, Z and Zheng, W and Zhu, J},
title = {Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L.},
journal = {Veterinary microbiology},
volume = {293},
number = {},
pages = {110073},
doi = {10.1016/j.vetmic.2024.110073},
pmid = {38579481},
issn = {1873-2542},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; Animals ; Swine ; *African Swine Fever/virology/diagnosis ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods/veterinary ; Viral Structural Proteins/genetics ; },
abstract = {African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.},
}

*African Swine Fever Virus/genetics/isolation & purification
Animals
Swine
*African Swine Fever/virology/diagnosis
*CRISPR-Cas Systems
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods/veterinary
Viral Structural Proteins/genetics

@article {pmid38528117,
year = {2024},
author = {Hetzler, Z and Marinakos, SM and Lott, N and Mohammad, N and Lass-Napiorkowska, A and Kolbe, J and Turrentine, L and Fields, D and Overton, L and Marie, H and Hucknall, A and Rammo, O and George, H and Wei, Q},
title = {Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a.},
journal = {Gene therapy},
volume = {31},
number = {5-6},
pages = {304-313},
pmid = {38528117},
issn = {1476-5462},
mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems ; Humans ; *Genome, Viral ; Genetic Vectors/genetics ; HEK293 Cells ; },
abstract = {Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).},
}

*Dependovirus/genetics
*CRISPR-Cas Systems
Humans
*Genome, Viral
Genetic Vectors/genetics
HEK293 Cells

@article {pmid38497898,
year = {2024},
author = {Simões, S and Lino, M and Barrera, A and Rebelo, C and Tomatis, F and Vilaça, A and Breunig, C and Neuner, A and Peça, J and González, R and Carvalho, A and Stricker, S and Ferreira, L},
title = {Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {21},
pages = {e202401004},
doi = {10.1002/anie.202401004},
pmid = {38497898},
issn = {1521-3773},
support = {SFRH/BPD/105327/2014//Fundação para a Ciência e a Tecnologia/ ; POCI-01-0145-FEDER-016682//Fundação para a Ciência e a Tecnologia/ ; EXPL/BIA-CEL/0989/2021//Fundação para a Ciência e a Tecnologia/ ; 952266//European Commission/ ; 764958//European Commission/ ; 101129812//European Innovation Council/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Infrared Rays ; Animals ; *Brain/metabolism ; Mice ; Ribonucleoproteins/metabolism/chemistry/genetics ; Nanoparticles/chemistry ; Humans ; },
abstract = {The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Infrared Rays
Animals
*Brain/metabolism
Mice
Ribonucleoproteins/metabolism/chemistry/genetics
Nanoparticles/chemistry
Humans

@article {pmid38344908,
year = {2024},
author = {Hernandes, N and Qi, XM and Bhide, S and Brown, C and Camm, BJ and Baxter, SW and Robin, C},
title = {Acetylcholine esterase of Drosophila melanogaster: a laboratory model to explore insecticide susceptibility gene drives.},
journal = {Pest management science},
volume = {80},
number = {6},
pages = {2950-2964},
doi = {10.1002/ps.8003},
pmid = {38344908},
issn = {1526-4998},
support = {DP190102512//Australian Research Council/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/drug effects ; *Acetylcholinesterase/genetics/metabolism ; *Insecticide Resistance/genetics ; *Gene Drive Technology ; *Insecticides/pharmacology ; CRISPR-Cas Systems ; Drosophila Proteins/genetics ; },
abstract = {BACKGROUND: One of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses.

RESULTS: We have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygous, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of gene drive resistance (GDR): rather than being mediated by the conventional non-homologous end-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas.

CONCLUSIONS: Insecticide susceptibility gene drives could be useful tools to control pest insects however problems with particularities of target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as a 'safety-switch' to prevent the unwanted spread of gene drives. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

Animals
*Drosophila melanogaster/genetics/drug effects
*Acetylcholinesterase/genetics/metabolism
*Insecticide Resistance/genetics
*Gene Drive Technology
*Insecticides/pharmacology
CRISPR-Cas Systems
Drosophila Proteins/genetics

@article {pmid37736806,
year = {2024},
author = {Alayoubi, AM and Khawaji, ZY and Mohammed, MA and Mercier, FE},
title = {CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia.},
journal = {Annals of hematology},
volume = {103},
number = {6},
pages = {1805-1817},
pmid = {37736806},
issn = {1432-0584},
mesh = {Humans ; *CRISPR-Cas Systems ; *Hemophilia A/therapy/genetics ; *Genetic Therapy/methods ; *Hematologic Neoplasms/therapy/genetics ; Gene Editing/methods ; Animals ; Hemoglobinopathies/therapy/genetics ; beta-Thalassemia/therapy/genetics ; Immunotherapy, Adoptive ; },
abstract = {Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).},
}

Humans
*CRISPR-Cas Systems
*Hemophilia A/therapy/genetics
*Genetic Therapy/methods
*Hematologic Neoplasms/therapy/genetics
Gene Editing/methods
Animals
Hemoglobinopathies/therapy/genetics
beta-Thalassemia/therapy/genetics
Immunotherapy, Adoptive

@article {pmid37269979,
year = {2023},
author = {Chen, X and Zhao, D and Hou, X and Li, J and Pu, S and Fei, J and Li, S and Zhou, Z and Bi, C and Zhang, X},
title = {Genomic allele-specific base editing with imperfect gRNA.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {50},
number = {10},
pages = {799-802},
doi = {10.1016/j.jgg.2023.05.010},
pmid = {37269979},
issn = {1673-8527},
mesh = {*Gene Editing ; *Alleles ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Genomics/methods ; },
}

*Gene Editing
*Alleles
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
Humans
Genomics/methods

@article {pmid38717584,
year = {2024},
author = {Yin, W and Li, L and Yang, Y and Yang, Y and Liang, R and Ma, L and Dai, J and Mao, G and Ma, Y},
title = {Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.4c00432},
pmid = {38717584},
issn = {2379-3694},
abstract = {Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 10[7] fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 μL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 10[4] fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.},
}

@article {pmid38575054,
year = {2024},
author = {Bhattacharya, S and Agarwal, A and Muniyappa, K},
title = {Deciphering the Substrate Specificity Reveals that CRISPR-Cas12a Is a Bifunctional Enzyme with Both Endo- and Exonuclease Activities.},
journal = {Journal of molecular biology},
volume = {436},
number = {10},
pages = {168550},
doi = {10.1016/j.jmb.2024.168550},
pmid = {38575054},
issn = {1089-8638},
mesh = {Substrate Specificity ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Acidaminococcus/enzymology/genetics ; Endodeoxyribonucleases/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics/chemistry ; Exonucleases/metabolism/genetics ; DNA, Cruciform/metabolism/genetics ; DNA/metabolism/genetics ; },
abstract = {The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.},
}

Substrate Specificity
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Acidaminococcus/enzymology/genetics
Endodeoxyribonucleases/metabolism/chemistry/genetics
Bacterial Proteins/metabolism/genetics/chemistry
Exonucleases/metabolism/genetics
DNA, Cruciform/metabolism/genetics
DNA/metabolism/genetics

@article {pmid38716572,
year = {2024},
author = {Fang, T and Chen, G},
title = {Non-viral vector-based genome editing for cancer immunotherapy.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4bm00286e},
pmid = {38716572},
issn = {2047-4849},
abstract = {Despite the exciting promise of cancer immunotherapy in the clinic, immune checkpoint blockade therapy and T cell-based therapies are often associated with low response rates, intrinsic and adaptive immune resistance, and systemic side effects. CRISPR-Cas-based genome editing appears to be an effective strategy to overcome these unmet clinical needs. As a safer delivery platform for the CRISPR-Cas system, non-viral nanoformulations have been recently explored to target tumor cells and immune cells, aiming to improve cancer immunotherapy on a gene level. In this review, we summarized the efforts of non-viral vector-based CRISPR-Cas-mediated genome editing in tumor cells and immune cells for cancer immunotherapy. Their design rationale and specific applications were highlighted.},
}

@article {pmid38715208,
year = {2024},
author = {Sandor, R and Wagh, SG and Kelterborn, S and Großkinsky, DK and Novak, O and Olsen, N and Paul, B and Petřík, I and Wu, S and Hegemann, P and Strnad, M and Červený, J and Roitsch, T},
title = {Cytokinin-deficient Chlamydomonas reinhardtii CRISPR-Cas9 mutants show reduced ability to prime resistance of tobacco against bacterial infection.},
journal = {Physiologia plantarum},
volume = {176},
number = {3},
pages = {e14311},
doi = {10.1111/ppl.14311},
pmid = {38715208},
issn = {1399-3054},
support = {4265668059//Deutsche Forschungsgemeinschaft (DFG)/ ; 18-24397S//Biorefining and Circular Economy for Sustainability/ ; 21-07661S//Grantová agentura České republiky (GAČR)/ ; TN02000044//Technology Agency of the Czech Republic/ ; },
mesh = {*Nicotiana/genetics/microbiology/immunology ; *Cytokinins/metabolism ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/immunology/genetics ; *Disease Resistance/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; Pseudomonas syringae/pathogenicity/physiology ; Mutation ; },
abstract = {Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.},
}

*Nicotiana/genetics/microbiology/immunology
*Cytokinins/metabolism
*CRISPR-Cas Systems
*Plant Diseases/microbiology/immunology/genetics
*Disease Resistance/genetics
*Chlamydomonas reinhardtii/genetics/metabolism
Pseudomonas syringae/pathogenicity/physiology
Mutation

@article {pmid38714951,
year = {2024},
author = {He, Z and Dong, C and Song, T and Zhou, J and Xu, T and He, R and Li, S},
title = {FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {65},
pmid = {38714951},
issn = {1689-1392},
support = {82070723//National Natural Science Foundation of China/ ; 82270797//National Natural Science Foundation of China/ ; 2022CFC020//Natural Science Foundation of Hubei Province/ ; 2022CFB799//Natural Science Foundation of Hubei Province/ ; },
mesh = {Animals ; Humans ; Male ; Mice ; *Calcium Oxalate/metabolism ; *CRISPR-Cas Systems/genetics ; Eukaryotic Initiation Factor-4G/metabolism/genetics ; Ferritins ; Ferroptosis/genetics ; Gene Editing/methods ; HEK293 Cells ; *Kidney/metabolism/pathology ; Kidney Calculi/genetics/metabolism ; Oxidoreductases/metabolism/genetics ; Protein Biosynthesis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.},
}

Animals
Humans
Male
Mice
*Calcium Oxalate/metabolism
*CRISPR-Cas Systems/genetics
Eukaryotic Initiation Factor-4G/metabolism/genetics
Ferritins
Ferroptosis/genetics
Gene Editing/methods
HEK293 Cells
*Kidney/metabolism/pathology
Kidney Calculi/genetics/metabolism
Oxidoreductases/metabolism/genetics
Protein Biosynthesis/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid38714682,
year = {2024},
author = {Prelli Bozzo, C and Laliberté, A and De Luna, A and Pastorio, C and Regensburger, K and Krebs, S and Graf, A and Blum, H and Volcic, M and Sparrer, KMJ and Kirchhoff, F},
title = {Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3813},
pmid = {38714682},
issn = {2041-1723},
support = {CRC 1279, SPP 1923, SP 1600/6-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1279, SPP1923//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Humans ; *HIV-1/genetics/physiology ; *Virus Replication/genetics ; *nef Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; *CD4-Positive T-Lymphocytes/virology/metabolism/immunology ; HEK293 Cells ; CRISPR-Cas Systems ; HIV Infections/virology/genetics/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Phosphoproteins/metabolism/genetics ; rhoA GTP-Binding Protein/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Virus Internalization ; },
abstract = {Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4[+] T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4[+] T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.},
}

Humans
*HIV-1/genetics/physiology
*Virus Replication/genetics
*nef Gene Products, Human Immunodeficiency Virus/genetics/metabolism
*CD4-Positive T-Lymphocytes/virology/metabolism/immunology
HEK293 Cells
CRISPR-Cas Systems
HIV Infections/virology/genetics/immunology
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Phosphoproteins/metabolism/genetics
rhoA GTP-Binding Protein/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Virus Internalization

@article {pmid38714663,
year = {2024},
author = {Scranton, K and John, S and Angelini, M and Steccanella, F and Umar, S and Zhang, R and Goldhaber, JI and Olcese, R and Ottolia, M},
title = {Cardiac function is regulated by the sodium-dependent inhibition of the sodium-calcium exchanger NCX1.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3831},
pmid = {38714663},
issn = {2041-1723},
support = {K08 HL141995/HL/NHLBI NIH HHS/United States ; R01 HL147569/HL/NHLBI NIH HHS/United States ; R01 HL130308/HL/NHLBI NIH HHS/United States ; R01 HL161038/HL/NHLBI NIH HHS/United States ; R01 HL134346/HL/NHLBI NIH HHS/United States ; },
mesh = {*Sodium-Calcium Exchanger/metabolism/genetics ; Animals ; *Myocytes, Cardiac/metabolism ; Male ; *Sodium/metabolism ; Mice ; *Action Potentials ; *Calcium/metabolism ; Myocardial Contraction/physiology/genetics ; Heart/physiology ; Humans ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The Na[+]-Ca[2+] exchanger (NCX1) is the dominant Ca[2+] extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na[+] via a process known as Na[+]-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na[+]-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca[2+] transients, and depressed cell shortening. The results demonstrate that NCX1 Na[+]-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.},
}

*Sodium-Calcium Exchanger/metabolism/genetics
Animals
*Myocytes, Cardiac/metabolism
Male
*Sodium/metabolism
Mice
*Action Potentials
*Calcium/metabolism
Myocardial Contraction/physiology/genetics
Heart/physiology
Humans
Mutation
CRISPR-Cas Systems

@article {pmid38714659,
year = {2024},
author = {Recinos, Y and Ustianenko, D and Yeh, YT and Wang, X and Jacko, M and Yesantharao, LV and Wu, Q and Zhang, C},
title = {CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3839},
pmid = {38714659},
issn = {2041-1723},
support = {S10 RR027050/RR/NCRR NIH HHS/United States ; R01 HG012359/HG/NHGRI NIH HHS/United States ; R56 HG012359/HG/NHGRI NIH HHS/United States ; S10 OD020056/OD/NIH HHS/United States ; R35 GM145279/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA Splicing/genetics ; *CRISPR-Cas Systems ; *Survival of Motor Neuron 2 Protein/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Introns/genetics ; *Exons/genetics ; HEK293 Cells ; Oligonucleotides, Antisense/genetics ; Muscular Atrophy, Spinal/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; RNA Precursors/genetics/metabolism ; },
abstract = {Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.},
}

Humans
*RNA Splicing/genetics
*CRISPR-Cas Systems
*Survival of Motor Neuron 2 Protein/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Introns/genetics
*Exons/genetics
HEK293 Cells
Oligonucleotides, Antisense/genetics
Muscular Atrophy, Spinal/genetics
Regulatory Sequences, Nucleic Acid/genetics
RNA Precursors/genetics/metabolism

@article {pmid38714643,
year = {2024},
author = {Xun, G and Zhu, Z and Singh, N and Lu, J and Jain, PK and Zhao, H},
title = {Harnessing noncanonical crRNA for highly efficient genome editing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3823},
pmid = {38714643},
issn = {2041-1723},
support = {UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
RNA/genetics/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid38712768,
year = {2024},
author = {Zhang, H and Li, X and Bai, J and Zhang, C},
title = {Mice with NOP2/sun RNA methyltransferase 5 deficiency die before reaching puberty due to fatal kidney damage.},
journal = {Renal failure},
volume = {46},
number = {1},
pages = {2349139},
pmid = {38712768},
issn = {1525-6049},
mesh = {Animals ; Mice ; *Apoptosis ; *Mice, Knockout ; *Methyltransferases/genetics/metabolism/deficiency ; *Kidney/pathology ; Disease Models, Animal ; DNA Damage ; Kidney Diseases/genetics/pathology ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Caspase 3/metabolism ; },
abstract = {BACKGROUND: NOP2/Sun RNA methyltransferase 5 (NSUN5) is an RNA methyltransferase that has a broad distribution and plays critical roles in various biological processes. However, our knowledge of the biological functions of NSUN5 in mammals is very limited. Therefore, in this study, we investigate the role of NSUN5 in mice.

METHODS: In the present research, we built a mouse model (Nsun5[-/-]) using the CRISPR/Cas9 system to investigated the specific role of NSUN5.

RESULTS: We observed that Nsun5[-/-] mice had a reduced body weight compared to wild-type mice. Additionally, their survival rate gradually decreased to 20% after postnatal day (PD) 21. Further examination revealed the Nsun5[-/-] mice had multiple organ damage, with the most severe damage occurring in the kidneys. Moreover, we observed glycogen deposition and fibrosis, along with a notable shorting of the primary foot processes of glomeruli in Nsun5[-/-] kidneys. Furthermore, we found that the kidneys of Nsun5[-/-] mice showed increased expression of the apoptotic signal Caspase-3 and accumulated stronger DNA damage at PD 21.

CONCLUSIONS: In our study, we found that mice lacking NSUN5 died before puberty due to kidney fatal damage caused by DNA damage and cell apoptosis. These results suggest that NSUN5 plays a vital role in preventing the accumulation of DNA damage and cell apoptosis in the kidney.},
}

Animals
Mice
*Apoptosis
*Mice, Knockout
*Methyltransferases/genetics/metabolism/deficiency
*Kidney/pathology
Disease Models, Animal
DNA Damage
Kidney Diseases/genetics/pathology
Male
Mice, Inbred C57BL
CRISPR-Cas Systems
Caspase 3/metabolism

@article {pmid38711223,
year = {2024},
author = {Arnold, KA and Moran, MC and Shi, H and van Vlijmen-Willems, IMJJ and Rodijk-Olthuis, D and Smits, JPH and Brewer, MG},
title = {CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.},
journal = {Experimental dermatology},
volume = {33},
number = {5},
pages = {e15084},
doi = {10.1111/exd.15084},
pmid = {38711223},
issn = {1600-0625},
mesh = {*Keratinocytes/metabolism ; *Claudin-1/metabolism/genetics ; Humans ; *Filaggrin Proteins/metabolism ; *Cell Differentiation ; *Epidermis/metabolism/pathology ; Skin Diseases/genetics/metabolism ; Tight Junctions/metabolism ; Keratin-10/metabolism/genetics ; Gene Knockout Techniques ; Cell Proliferation ; CRISPR-Cas Systems ; },
abstract = {The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.},
}

*Keratinocytes/metabolism
*Claudin-1/metabolism/genetics
Humans
*Filaggrin Proteins/metabolism
*Cell Differentiation
*Epidermis/metabolism/pathology
Skin Diseases/genetics/metabolism
Tight Junctions/metabolism
Keratin-10/metabolism/genetics
Gene Knockout Techniques
Cell Proliferation
CRISPR-Cas Systems

@article {pmid38710738,
year = {2024},
author = {Meng, X and Jia, R and Zhao, X and Zhang, F and Chen, S and Yu, S and Liu, X and Dou, H and Feng, X and Zhang, J and Wang, N and Xu, B and Yang, L},
title = {In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3773},
pmid = {38710738},
issn = {2041-1723},
support = {82371074//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; HEK293 Cells ; *Corneal Dystrophies, Hereditary/genetics/therapy/pathology/metabolism ; Mice ; *Induced Pluripotent Stem Cells/metabolism ; *Genetic Therapy/methods ; *Cytochrome P450 Family 4/genetics/metabolism ; Disease Models, Animal ; Mutation ; Retinal Pigment Epithelium/metabolism/pathology ; Genetic Vectors/genetics ; Introns/genetics ; Exons/genetics ; *Retinal Diseases ; },
abstract = {Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3[mut/mut]) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3[mut/mut] mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.},
}

Humans
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
HEK293 Cells
*Corneal Dystrophies, Hereditary/genetics/therapy/pathology/metabolism
Mice
*Induced Pluripotent Stem Cells/metabolism
*Genetic Therapy/methods
*Cytochrome P450 Family 4/genetics/metabolism
Disease Models, Animal
Mutation
Retinal Pigment Epithelium/metabolism/pathology
Genetic Vectors/genetics
Introns/genetics
Exons/genetics
*Retinal Diseases

@article {pmid38709433,
year = {2024},
author = {Yuan, YG and Liu, SZ and Farhab, M and Lv, MY and Zhang, T and Cao, SX},
title = {Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.},
journal = {Functional & integrative genomics},
volume = {24},
number = {3},
pages = {81},
pmid = {38709433},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; Animals ; *Livestock/genetics ; *CRISPR-Cas Systems ; Disease Resistance/genetics ; },
abstract = {One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.},
}

*Gene Editing/methods
Animals
*Livestock/genetics
*CRISPR-Cas Systems
Disease Resistance/genetics

@article {pmid38706796,
year = {2024},
author = {Vondracek, K and Altpeter, F and Liu, T and Lee, S},
title = {Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa).},
journal = {Frontiers in genetics},
volume = {15},
number = {},
pages = {1382445},
pmid = {38706796},
issn = {1664-8021},
abstract = {The cultivated strawberry, Fragaria ×ananassa, is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa. In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species.},
}

@article {pmid38643573,
year = {2024},
author = {Singh, P and Kumar, A and Singh, T and Anto, S and Indoliya, Y and Tiwari, P and Behera, SK and Chakrabarty, D},
title = {Targeting OsNIP3;1 via CRISPR/Cas9: A strategy for minimizing arsenic accumulation and boosting rice resilience.},
journal = {Journal of hazardous materials},
volume = {471},
number = {},
pages = {134325},
doi = {10.1016/j.jhazmat.2024.134325},
pmid = {38643573},
issn = {1873-3336},
mesh = {*Oryza/genetics/metabolism/growth & development ; *CRISPR-Cas Systems ; *Arsenic/metabolism/toxicity ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Aquaporins/genetics/metabolism ; Gene Knockout Techniques ; Silicon/metabolism ; Plants, Genetically Modified/genetics ; Chlorophyll/metabolism ; },
abstract = {Arsenic (As) contamination in rice poses a significant threat to human health due to its toxicity and widespread consumption. Identifying and manipulating key genes governing As accumulation in rice is crucial for reducing this threat. The large NIP gene family of aquaporins in rice presents a promising target due to functional redundancy, potentially allowing for gene manipulation without compromising plant growth. This study aimed to utilize genome editing to generate knock-out (KO) lines of genes of NIP family (OsLsi1, OsNIP3;1) and an anion transporter family (OsLsi2), in order to assess their impact on As accumulation and stress tolerance in rice. KO lines were created using CRISPR/Cas9 technology, and the As accumulation patterns, physiological performance, and grain yield were compared against wild-type (WT) under As-treated conditions. KO lines exhibited significantly reduced As accumulation in grain compared to WT. Notably, Osnip3;1 KO line displayed reduced As in xylem sap (71-74%) and grain (32-46%) upon treatment. Additionally, these lines demonstrated improved silicon (23%) uptake, photosynthetic pigment concentrations (Chl a: 77%; Chl b: 79%, Total Chl: 79% & Carotenoid: 49%) overall physiological and agronomical performance under As stress compared to WT. This study successfully utilized genome editing for the first time to identify OsNIP3;1 as a potential target for manipulating As accumulation in rice without compromising grain yield or plant vigor.},
}

*Oryza/genetics/metabolism/growth & development
*CRISPR-Cas Systems
*Arsenic/metabolism/toxicity
*Gene Editing
*Plant Proteins/genetics/metabolism
Aquaporins/genetics/metabolism
Gene Knockout Techniques
Silicon/metabolism
Plants, Genetically Modified/genetics
Chlorophyll/metabolism

@article {pmid38608704,
year = {2024},
author = {Li, X and Chen, W and Martin, BK and Calderon, D and Lee, C and Choi, J and Chardon, FM and McDiarmid, TA and Daza, RM and Kim, H and Lalanne, JB and Nathans, JF and Lee, DS and Shendure, J},
title = {Chromatin context-dependent regulation and epigenetic manipulation of prime editing.},
journal = {Cell},
volume = {187},
number = {10},
pages = {2411-2427.e25},
doi = {10.1016/j.cell.2024.03.020},
pmid = {38608704},
issn = {1097-4172},
support = {R01 HG010632/HG/NHGRI NIH HHS/United States ; UM1 HG011966/HG/NHGRI NIH HHS/United States ; },
mesh = {*Chromatin/metabolism/genetics ; *Gene Editing/methods ; Humans ; *Epigenesis, Genetic ; *CRISPR-Cas Systems/genetics ; Histones/metabolism ; HEK293 Cells ; Transcription Factors/metabolism ; },
abstract = {We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.},
}

*Chromatin/metabolism/genetics
*Gene Editing/methods
Humans
*Epigenesis, Genetic
*CRISPR-Cas Systems/genetics
Histones/metabolism
HEK293 Cells
Transcription Factors/metabolism

@article {pmid38608448,
year = {2024},
author = {Long, J and Zhao, W and Xiang, Y and Wang, Y and Xiang, W and Liu, X and Jiang, M and Song, Y and Hu, J},
title = {STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1β-induced IL-8 production.},
journal = {International immunopharmacology},
volume = {133},
number = {},
pages = {112065},
doi = {10.1016/j.intimp.2024.112065},
pmid = {38608448},
issn = {1878-1705},
mesh = {Humans ; *STAT3 Transcription Factor/metabolism/genetics ; *Interleukin-1beta/metabolism ; *Interleukin-8/metabolism/genetics ; *ELAV-Like Protein 1/metabolism/genetics ; *Cytoplasm/metabolism ; *Cell Nucleus/metabolism ; Cell Line, Tumor ; Cyclic S-Oxides/pharmacology ; Protein Transport ; Signal Transduction ; Active Transport, Cell Nucleus ; CRISPR-Cas Systems ; },
abstract = {Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1β-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1β-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1β induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1β-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1β-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1β-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1β. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1β-induced IL-8 production through this non-transcriptional mechanism.},
}

Humans
*STAT3 Transcription Factor/metabolism/genetics
*Interleukin-1beta/metabolism
*Interleukin-8/metabolism/genetics
*ELAV-Like Protein 1/metabolism/genetics
*Cytoplasm/metabolism
*Cell Nucleus/metabolism
Cell Line, Tumor
Cyclic S-Oxides/pharmacology
Protein Transport
Signal Transduction
Active Transport, Cell Nucleus
CRISPR-Cas Systems

@article {pmid38509393,
year = {2024},
author = {},
title = {Affinity maturation of CRISPR-engineered B cell receptors in vivo.},
journal = {Nature biomedical engineering},
volume = {8},
number = {4},
pages = {341-342},
pmid = {38509393},
issn = {2157-846X},
support = {U19 AI149646/AI/NIAID NIH HHS/United States ; R01 AI174277/AI/NIAID NIH HHS/United States ; R01 DA056771/DA/NIDA NIH HHS/United States ; U19 AI149646/AI/NIAID NIH HHS/United States ; R01 AI174277/AI/NIAID NIH HHS/United States ; R01 DA056771/DA/NIDA NIH HHS/United States ; R01 AI174277/AI/NIAID NIH HHS/United States ; R01 DA056771/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Receptors, Antigen, B-Cell/genetics/metabolism ; Mice ; Humans ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; B-Lymphocytes/immunology ; },
}

Animals
*CRISPR-Cas Systems/genetics
*Receptors, Antigen, B-Cell/genetics/metabolism
Mice
Humans
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
B-Lymphocytes/immunology

@article {pmid38486104,
year = {2024},
author = {Yin, Y and Guo, Y and Jiang, Y and Quinlan, B and Peng, H and Crynen, G and He, W and Zhang, L and Ou, T and Bailey, CC and Farzan, M},
title = {In vivo affinity maturation of mouse B cells reprogrammed to express human antibodies.},
journal = {Nature biomedical engineering},
volume = {8},
number = {4},
pages = {361-379},
pmid = {38486104},
issn = {2157-846X},
support = {U19 AI149646/AI/NIAID NIH HHS/United States ; R21 AI178031/AI/NIAID NIH HHS/United States ; R01 DA056771/DA/NIDA NIH HHS/United States ; R01 AI174277/AI/NIAID NIH HHS/United States ; U19 AI149646/AI/NIAID NIH HHS/United States ; R21 AI178031/AI/NIAID NIH HHS/United States ; R01 DA056771/DA/NIDA NIH HHS/United States ; R01 AI174277/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; *B-Lymphocytes/immunology ; *HIV-1/immunology ; *SARS-CoV-2/immunology ; *HIV Antibodies/immunology ; *Antibodies, Neutralizing/immunology ; *COVID-19/immunology/virology ; Antibody Affinity/immunology ; CRISPR-Cas Systems/genetics ; COVID-19 Vaccines/immunology ; Antibodies, Viral/immunology ; Mice, Inbred C57BL ; },
abstract = {Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.},
}

Animals
Humans
Mice
*B-Lymphocytes/immunology
*HIV-1/immunology
*SARS-CoV-2/immunology
*HIV Antibodies/immunology
*Antibodies, Neutralizing/immunology
*COVID-19/immunology/virology
Antibody Affinity/immunology
CRISPR-Cas Systems/genetics
COVID-19 Vaccines/immunology
Antibodies, Viral/immunology
Mice, Inbred C57BL

@article {pmid38326496,
year = {2024},
author = {Barriga, FM and Lowe, SW},
title = {Engineering megabase-sized genomic deletions with MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements).},
journal = {Nature protocols},
volume = {19},
number = {5},
pages = {1381-1399},
pmid = {38326496},
issn = {1750-2799},
mesh = {*CRISPR-Cas Systems ; Humans ; Gene Editing/methods ; Genetic Engineering/methods ; Chromosomes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Deletion ; },
abstract = {The elimination of large genomic regions has been enabled by the advent of site-specific nucleases. However, as the intended deletions get larger, the efficiency of successful engineering decreases to a point where it is not feasible to retrieve edited cells due to the rarity of on-target events. To address this issue, we developed a system called molecular alteration of chromosomes with engineered tandem elements (MACHETE). MACHETE is a CRISPR-Cas9-based system involving two stages: the initial insertion of a bicistronic positive/negative selection cassette to the locus of interest. This is followed by the introduction of single-guide RNAs flanking the knockin cassette to engineer the intended deletion, where only cells that have lost the locus survive the negative selection. In contrast to other approaches optimizing the activity of sequence-specific nucleases, MACHETE selects for the deletion event itself, thus greatly enriching for cells with the engineered alteration. The procedure routinely takes 4-6 weeks from design to selection of polyclonal populations bearing the deletion of interest. We have successfully deployed MACHETE to engineer deletions of up to 45 Mb, as well as the rapid creation of allelic series to map the relevant activities within a locus. This protocol details the design and step-by-step procedure to engineer megabase-sized deletions in cells of interest, with potential application for cancer genetics, transcriptional regulation, genome architecture and beyond.},
}

*CRISPR-Cas Systems
Humans
Gene Editing/methods
Genetic Engineering/methods
Chromosomes/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Sequence Deletion

@article {pmid38062131,
year = {2024},
author = {Chang, PC and Yuan, X and Zampieri, A and Towns, C and Yoo, SP and Engstrom, C and Tsai, S and Robles, CR and Zhu, Y and Lopez, S and Montel-Hagen, A and Seet, CS and Crooks, GM},
title = {Generation of antigen-specific mature T cells from RAG1[-/-]RAG2[-/-]B2M[-/-] stem cells by engineering their microenvironment.},
journal = {Nature biomedical engineering},
volume = {8},
number = {4},
pages = {461-478},
pmid = {38062131},
issn = {2157-846X},
support = {T32 GM008042/GM/NIGMS NIH HHS/United States ; U01 CA233074/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Homeodomain Proteins/genetics/metabolism ; *T-Lymphocytes/immunology ; Mice ; DNA-Binding Proteins/genetics/deficiency ; Receptors, Antigen, T-Cell/metabolism/immunology ; Cell Differentiation ; CRISPR-Cas Systems/genetics ; Nuclear Proteins ; },
abstract = {Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1[-/-]RAG2[-/-]B2M[-/-] human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.},
}

Animals
Humans
*Homeodomain Proteins/genetics/metabolism
*T-Lymphocytes/immunology
Mice
DNA-Binding Proteins/genetics/deficiency
Receptors, Antigen, T-Cell/metabolism/immunology
Cell Differentiation
CRISPR-Cas Systems/genetics
Nuclear Proteins

@article {pmid38012307,
year = {2024},
author = {Stranford, DM and Simons, LM and Berman, KE and Cheng, L and DiBiase, BN and Hung, ME and Lucks, JB and Hultquist, JF and Leonard, JN},
title = {Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells.},
journal = {Nature biomedical engineering},
volume = {8},
number = {4},
pages = {397-414},
pmid = {38012307},
issn = {2157-846X},
support = {P30 AI117943/AI/NIAID NIH HHS/United States ; R01 AI150998/AI/NIAID NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; },
mesh = {*Extracellular Vesicles/metabolism ; Humans ; *Biological Products ; CD4-Positive T-Lymphocytes/metabolism ; T-Lymphocytes/immunology/metabolism ; HEK293 Cells ; Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; Drug Delivery Systems/methods ; },
abstract = {The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)-which naturally encapsulate and transfer proteins and nucleic acids between cells-for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9-single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4[+] T cells. The strategy is amenable to the targeted delivery of biologics to other cell types.},
}

*Extracellular Vesicles/metabolism
Humans
*Biological Products
CD4-Positive T-Lymphocytes/metabolism
T-Lymphocytes/immunology/metabolism
HEK293 Cells
Genetic Engineering/methods
CRISPR-Cas Systems/genetics
Drug Delivery Systems/methods

@article {pmid38704787,
year = {2024},
author = {Wang, Y and Wang, D and Du, J and Wang, Y and Shao, C and Cui, C and Xiao, J and Wang, X},
title = {Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.},
journal = {Plant cell reports},
volume = {43},
number = {5},
pages = {135},
pmid = {38704787},
issn = {1432-203X},
support = {BJK2022028//Science Research Project of Hebei Education Department/ ; },
mesh = {*Arabidopsis/genetics/growth & development/metabolism ; *Chloroplasts/metabolism/ultrastructure ; *Arabidopsis Proteins/metabolism/genetics ; *Gene Expression Regulation, Plant ; Plant Leaves/genetics/metabolism/growth & development/ultrastructure ; Cotyledon/genetics/metabolism/growth & development ; Proteomics ; Chloroplast Proteins/metabolism/genetics ; Organelle Biogenesis ; Chlorophyll/metabolism ; CRISPR-Cas Systems ; },
abstract = {The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.},
}

*Arabidopsis/genetics/growth & development/metabolism
*Chloroplasts/metabolism/ultrastructure
*Arabidopsis Proteins/metabolism/genetics
*Gene Expression Regulation, Plant
Plant Leaves/genetics/metabolism/growth & development/ultrastructure
Cotyledon/genetics/metabolism/growth & development
Proteomics
Chloroplast Proteins/metabolism/genetics
Organelle Biogenesis
Chlorophyll/metabolism
CRISPR-Cas Systems

@article {pmid38702571,
year = {2024},
author = {Yang, H and Patel, DJ},
title = {Structures, mechanisms and applications of RNA-centric CRISPR-Cas13.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {38702571},
issn = {1552-4469},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
abstract = {Prokaryotes are equipped with a variety of resistance strategies to survive frequent viral attacks or invading mobile genetic elements. Among these, CRISPR-Cas surveillance systems are abundant and have been studied extensively. This Review focuses on CRISPR-Cas type VI Cas13 systems that use single-subunit RNA-guided Cas endonucleases for targeting and subsequent degradation of foreign RNA, thereby providing adaptive immunity. Notably, distinct from single-subunit DNA-cleaving Cas9 and Cas12 systems, Cas13 exhibits target RNA-activated substrate RNase activity. This Review outlines structural, biochemical and cell biological studies toward elucidation of the unique structural and mechanistic principles underlying surveillance effector complex formation, precursor CRISPR RNA (pre-crRNA) processing, self-discrimination and RNA degradation in Cas13 systems as well as insights into suppression by bacteriophage-encoded anti-CRISPR proteins and regulation by endogenous accessory proteins. Owing to its programmable ability for RNA recognition and cleavage, Cas13 provides powerful RNA targeting, editing, detection and imaging platforms with emerging biotechnological and therapeutic applications.},
}

@article {pmid38701251,
year = {2024},
author = {Hu, J and Zhong, Y and Xu, P and Xin, L and Zhu, X and Jiang, X and Gao, W and Yang, B and Chen, Y},
title = {β-Thalassemia gene editing therapy: Advancements and difficulties.},
journal = {Medicine},
volume = {103},
number = {18},
pages = {e38036},
pmid = {38701251},
issn = {1536-5964},
mesh = {*beta-Thalassemia/therapy/genetics ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {β-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of β-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent β-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of β-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of β-thalassemia diseases.},
}

*beta-Thalassemia/therapy/genetics
Humans
*Gene Editing/methods
*Genetic Therapy/methods
CRISPR-Cas Systems
Transcription Activator-Like Effector Nucleases/genetics
Zinc Finger Nucleases/genetics

@article {pmid38698311,
year = {2024},
author = {Jia, S and Liang, R and Chen, J and Liao, S and Lin, J and Li, W},
title = {Emerging technology has a brilliant future: the CRISPR-Cas system for senescence, inflammation, and cartilage repair in osteoarthritis.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {64},
pmid = {38698311},
issn = {1689-1392},
support = {SZSM202211019//Shenzhen"San-Ming" Project of Medicine/ ; 2023A1515220257//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2023A1515220019//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2022A1515220056//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
mesh = {Humans ; *Osteoarthritis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Inflammation/genetics ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; Cartilage/metabolism/pathology ; Cellular Senescence/genetics ; Cartilage, Articular/pathology/metabolism ; },
abstract = {Osteoarthritis (OA), known as one of the most common types of aseptic inflammation of the musculoskeletal system, is characterized by chronic pain and whole-joint lesions. With cellular and molecular changes including senescence, inflammatory alterations, and subsequent cartilage defects, OA eventually leads to a series of adverse outcomes such as pain and disability. CRISPR-Cas-related technology has been proposed and explored as a gene therapy, offering potential gene-editing tools that are in the spotlight. Considering the genetic and multigene regulatory mechanisms of OA, we systematically review current studies on CRISPR-Cas technology for improving OA in terms of senescence, inflammation, and cartilage damage and summarize various strategies for delivering CRISPR products, hoping to provide a new perspective for the treatment of OA by taking advantage of CRISPR technology.},
}

Humans
*Osteoarthritis/genetics/therapy
*CRISPR-Cas Systems/genetics
*Inflammation/genetics
*Gene Editing/methods
Animals
Genetic Therapy/methods
Cartilage/metabolism/pathology
Cellular Senescence/genetics
Cartilage, Articular/pathology/metabolism

@article {pmid38698035,
year = {2024},
author = {Bhoobalan-Chitty, Y and Xu, S and Martinez-Alvarez, L and Karamycheva, S and Makarova, KS and Koonin, EV and Peng, X},
title = {Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3699},
pmid = {38698035},
issn = {2041-1723},
support = {NNF21OC0067491//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF17OC0031154//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; DFF-0135-00402//Det Frie Forskningsråd (Danish Council for Independent Research)/ ; },
mesh = {*Archaeal Viruses/genetics ; *Archaea/genetics/virology/immunology ; Promoter Regions, Genetic/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Viral Proteins/genetics ; Archaeal Proteins/genetics/metabolism ; Metagenome/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.},
}

*Archaeal Viruses/genetics
*Archaea/genetics/virology/immunology
Promoter Regions, Genetic/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Regulatory Sequences, Nucleic Acid/genetics
Viral Proteins/genetics
Archaeal Proteins/genetics/metabolism
Metagenome/genetics
CRISPR-Associated Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid38696670,
year = {2024},
author = {Tanveer, M and Abidin, ZU and Alawadi, HFN and Shahzad, AN and Mahmood, A and Khan, BA and Qari, S and Oraby, HF},
title = {Recent advances in genome editing strategies for balancing growth and defence in sugarcane (Saccharum officinarum).},
journal = {Functional plant biology : FPB},
volume = {51},
number = {},
pages = {},
doi = {10.1071/FP24036},
pmid = {38696670},
issn = {1445-4416},
mesh = {*Saccharum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genome, Plant ; Crops, Agricultural/genetics/growth & development ; },
abstract = {Sugarcane (Saccharum officinarum) has gained more attention worldwide in recent decades because of its importance as a bioenergy resource and in producing table sugar. However, the production capabilities of conventional varieties are being challenged by the changing climates, which struggle to meet the escalating demands of the growing global population. Genome editing has emerged as a pivotal field that offers groundbreaking solutions in agriculture and beyond. It includes inserting, removing or replacing DNA in an organism's genome. Various approaches are employed to enhance crop yields and resilience in harsh climates. These techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas). Among these, CRISPR/Cas is one of the most promising and rapidly advancing fields. With the help of these techniques, several crops like rice (Oryza sativa), tomato (Solanum lycopersicum), maize (Zea mays), barley (Hordeum vulgare) and sugarcane have been improved to be resistant to viral diseases. This review describes recent advances in genome editing with a particular focus on sugarcane and focuses on the advantages and limitations of these approaches while also considering the regulatory and ethical implications across different countries. It also offers insights into future prospects and the application of these approaches in agriculture.},
}

*Saccharum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Genome, Plant
Crops, Agricultural/genetics/growth & development

@article {pmid38695626,
year = {2024},
author = {Wirtz, L and Casanova, F and Schaffrath, U and Wegner, A},
title = {Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.},
journal = {Molecular plant pathology},
volume = {25},
number = {5},
pages = {e13460},
pmid = {38695626},
issn = {1364-3703},
support = {//RWTH Aachen University scholarships for Doctoral Students/ ; SCHA 631/11-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Telomere/genetics ; *Phenotype ; *Genes, Essential ; *Genetic Vectors/genetics ; CRISPR-Cas Systems/genetics ; Genes, Fungal/genetics ; Gene Deletion ; Magnaporthe/genetics/pathogenicity ; *Ascomycota ; },
abstract = {Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.},
}

*Telomere/genetics
*Phenotype
*Genes, Essential
*Genetic Vectors/genetics
CRISPR-Cas Systems/genetics
Genes, Fungal/genetics
Gene Deletion
Magnaporthe/genetics/pathogenicity
*Ascomycota

@article {pmid38498115,
year = {2024},
author = {Hoekstra, M and Van Eck, M},
title = {Gene Editing for the Treatment of Hypercholesterolemia.},
journal = {Current atherosclerosis reports},
volume = {26},
number = {5},
pages = {139-146},
pmid = {38498115},
issn = {1534-6242},
mesh = {*Gene Editing/methods ; Humans ; Animals ; *Hypercholesterolemia/therapy/genetics ; *Genetic Therapy/methods ; *Proprotein Convertase 9/genetics ; Cholesterol, LDL/blood ; CRISPR-Cas Systems ; },
abstract = {PURPOSE OF REVIEW: Here, we summarize the key findings from preclinical studies that tested the concept that editing of hepatic genes can lower plasma low-density lipoprotein (LDL)-cholesterol levels to subsequently reduce atherosclerotic cardiovascular disease risk.

RECENT FINDINGS: Selective delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing tools targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) to hepatocytes, i.e., through encapsulation into N-acetylgalactosamine-coupled lipid nanoparticles, is able to induce a stable ~ 90% decrease in plasma PCSK9 levels and a concomitant 60% reduction in LDL-cholesterol levels in mice and non-humane primates. Studies in mice have shown that this state-of-the-art technology can be extended to include additional targets related to dyslipidemia such as angiopoietin-like 3 and several apolipoproteins. The use of gene editors holds great promise to lower plasma LDL-cholesterol levels also in the human setting. However, gene editing safety has to be guaranteed before this approach can become a clinical success.},
}

*Gene Editing/methods
Humans
Animals
*Hypercholesterolemia/therapy/genetics
*Genetic Therapy/methods
*Proprotein Convertase 9/genetics
Cholesterol, LDL/blood
CRISPR-Cas Systems

@article {pmid38695159,
year = {2024},
author = {Sharrar, A and Meacham, Z and Staples-Ager, J and Arake de Tacca, L and Rabuka, D and Collingwood, T and Schelle, M},
title = {Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0010},
pmid = {38695159},
issn = {2573-1602},
abstract = {Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying CRISPR editing to diverse genetic sequences and organs in the body.},
}

@article {pmid38695127,
year = {2024},
author = {Zheng, M and Li, Y and Zhang, L and Li, C and Liu, M and Tang, H},
title = {Detection of free DNA based on metal-enhanced fluorescence triggered by CRISPR-Cas12a and colorimetric analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4ay00149d},
pmid = {38695127},
issn = {1759-9679},
abstract = {The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.},
}

@article {pmid38694604,
year = {2024},
author = {Davis, DJ and Yeddula, SGR},
title = {CRISPR Advancements for Human Health.},
journal = {Missouri medicine},
volume = {121},
number = {2},
pages = {170-176},
pmid = {38694604},
issn = {0026-6620},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Neoplasms/genetics/therapy ; Genetic Therapy/methods/trends ; Clustered Regularly Interspaced Short Palindromic Repeats ; Regenerative Medicine/methods/trends ; Precision Medicine/methods/trends ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Neoplasms/genetics/therapy
Genetic Therapy/methods/trends
Clustered Regularly Interspaced Short Palindromic Repeats
Regenerative Medicine/methods/trends
Precision Medicine/methods/trends

@article {pmid38692475,
year = {2024},
author = {Brehm, JN and Sorg, JA},
title = {Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange.},
journal = {Anaerobe},
volume = {},
number = {},
pages = {102858},
doi = {10.1016/j.anaerobe.2024.102858},
pmid = {38692475},
issn = {1095-8274},
abstract = {Historically, mutagenesis in the non-model enteropathogenic bacterium Clostridioides difficile has been challenging. Developing a versatile and reliable method of generating targeted mutations in C. difficile is important to further our understanding of its pathogenesis. Some of the most common targeted mutagenesis systems rely on allelic exchange mediated by either uracil auxotrophy combined with a toxic uracil precursor, a toxin / anti-toxin system, group II introns, or CRISPR/Cas mutagenesis. However, each of these methods suffers from its own issues. Here, we develop and test an allelic exchange strategy which better facilitates screening for integration and selecting for excision than previous systems. This is achieved by controlling plasmid replication with a theophylline-dependent riboswitch cloned upstream of repA, the gene whose product controls plasmid replication. This allows efficient mutant generation, can be performed in a wild-type strain of C. difficile, does not have the off-target effects inherent to group II introns, and alleviates the problem of testing multiple gRNA targets in CRISPR mutagenesis.},
}

@article {pmid38691604,
year = {2024},
author = {Ma, Y and Schwager Karpukhina, A and Dib, C and Gautier, C and Hermine, O and Allemand, E and Vassetzky, YS},
title = {Exchange of subtelomeric regions between chromosomes 4q and 10q reverts the FSHD genotype and phenotype.},
journal = {Science advances},
volume = {10},
number = {18},
pages = {eadl1922},
pmid = {38691604},
issn = {2375-2548},
mesh = {Humans ; *Chromosomes, Human, Pair 10/genetics ; *Chromosomes, Human, Pair 4/genetics ; CRISPR-Cas Systems ; *Genotype ; *Homeodomain Proteins/genetics/metabolism ; *Muscular Dystrophy, Facioscapulohumeral/genetics ; Myoblasts/metabolism ; PAX7 Transcription Factor/genetics/metabolism ; *Phenotype ; *Telomere/genetics/metabolism ; Translocation, Genetic ; },
abstract = {The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.},
}

Humans
*Chromosomes, Human, Pair 10/genetics
*Chromosomes, Human, Pair 4/genetics
CRISPR-Cas Systems
*Genotype
*Homeodomain Proteins/genetics/metabolism
*Muscular Dystrophy, Facioscapulohumeral/genetics
Myoblasts/metabolism
PAX7 Transcription Factor/genetics/metabolism
*Phenotype
*Telomere/genetics/metabolism
Translocation, Genetic

@article {pmid38651297,
year = {2024},
author = {Lin, CL and Chen, WD and Liu, L and Cheng, L},
title = {Chemical control of CRISPR/Cpf1 editing via orthogonal activation and deactivation of crosslinked crRNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {39},
pages = {5197-5200},
doi = {10.1039/d4cc01106f},
pmid = {38651297},
issn = {1364-548X},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Light ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/metabolism/chemistry ; RNA/chemistry/genetics ; },
abstract = {Through the integration of CRISPR/Cpf1 with optogenetics and a reduction-responsive motif, we have developed a photoactivatable cross-linked crRNA that enables precise genome editing upon light exposure. This system also allows for termination of editing activity through external application of reducing agent. The dual-stimuli-responsive CRISPR/Cpf1 editing process operates in a unique OFF → ON → OFF sequence, making it a valuable tool for investigating time-sensitive biological events.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Light
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins/metabolism/chemistry
RNA/chemistry/genetics

@article {pmid38621334,
year = {2024},
author = {Miao, P and Sun, Y and Zheng, G and Wang, B and Wang, W and Zhang, J and Yan, M and Lv, Y},
title = {Near-infrared light-induced homogeneous photoelectrochemical biosensor based on 3D walking nanomotor-assisted CRISPR/Cas12a for ultrasensitive microRNA-155 detection.},
journal = {Journal of colloid and interface science},
volume = {667},
number = {},
pages = {82-90},
doi = {10.1016/j.jcis.2024.04.012},
pmid = {38621334},
issn = {1095-7103},
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques ; *Electrochemical Techniques ; *Infrared Rays ; Humans ; *CRISPR-Cas Systems ; *Photochemical Processes ; Limit of Detection ; },
abstract = {The dysregulation of microRNA (miRNA) expression levels is intricately linked to a myriad of human diseases, and the precise and delicate detection thereof holds paramount significance in the realm of clinical diagnosis and therapy. Herein, a near-infrared (NIR) light-mediated homogeneous photoelectrochemical (PEC) biosensor was constructed for miRNA-155 detection based on NaYF4: Yb, Tm@ZnIn2S4 (NYF@ ZIS) coupled with a three-dimensional (3D) walking nanomotor-assisted CRISPR/Cas12a strategy. The upconverted light emitted by the NYF in the visible and UV region upon NIR light excitation could be utilized to excite ZIS to produce a photocurrent response. The presence of target miRNA-155 initiated an amplification reaction within the 3D walking nanomotor, resulting in the production of multiple nucleic acid fragments. These fragments could activate the collateral cleavage capability of CRISPR/Cas12a, leading to the indiscriminate cleavage of single-stranded DNA (ssDNA) on ALP-ssDNA-modified magnetic beads and the subsequent liberation of alkaline phosphatase (ALP). The released ALP facilitated the catalysis of ascorbic acid 2-phosphate to generate ascorbic acid as the electron donor to capture the photogenerated holes on the NYF@ZIS surface, resulting in a positively correlated alteration in the photocurrent response. Under optimal conditions, the NIR light-initiated homogeneous PEC biosensor had the merits of good linear range (0.1 fM to 100 pM), an acceptable limit of detection (65.77 aM) for miRNA-155 detection. Considering the pronounced sensitivity, light stability, and low photodamage, this strategy presents a promising platform for detecting various other miRNA biomarkers in molecular diagnostic practice.},
}

*MicroRNAs/analysis
*Biosensing Techniques
*Electrochemical Techniques
*Infrared Rays
Humans
*CRISPR-Cas Systems
*Photochemical Processes
Limit of Detection

@article {pmid38604459,
year = {2024},
author = {Maliszewska-Olejniczak, K and Pytlak, K and Dabrowska, A and Zochowska, M and Hoser, J and Lukasiak, A and Zajac, M and Kulawiak, B and Bednarczyk, P},
title = {Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.},
journal = {Mitochondrion},
volume = {76},
number = {},
pages = {101880},
doi = {10.1016/j.mito.2024.101880},
pmid = {38604459},
issn = {1872-8278},
mesh = {Humans ; *Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism/genetics ; *Bronchi/metabolism/cytology ; *Epithelial Cells/metabolism ; Cell Line ; Mitochondria/metabolism ; CRISPR-Cas Systems ; Respiratory Mucosa/metabolism/cytology ; Cell Membrane/metabolism ; Mitochondrial Membranes/metabolism/physiology ; },
abstract = {Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.},
}

Humans
*Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism/genetics
*Bronchi/metabolism/cytology
*Epithelial Cells/metabolism
Cell Line
Mitochondria/metabolism
CRISPR-Cas Systems
Respiratory Mucosa/metabolism/cytology
Cell Membrane/metabolism
Mitochondrial Membranes/metabolism/physiology

@article {pmid38302097,
year = {2024},
author = {Wang, S and Xiao, Y and Tian, J and Dai, B and Tao, Z and Liu, J and Sun, Z and Liu, X and Li, Y and Zhao, G and Cui, Y and Wang, F and Liu, S},
title = {Targeted Macrophage CRISPR-Cas13 mRNA Editing in Immunotherapy for Tendon Injury.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {36},
number = {19},
pages = {e2311964},
doi = {10.1002/adma.202311964},
pmid = {38302097},
issn = {1521-4095},
support = {82172408//National Natural Science Foundation of China/ ; 81922045//National Natural Science Foundation of China/ ; 81772314//National Natural Science Foundation of China/ ; 22ZR1480300//Original Exploration project/ ; 21XD1422900//Outstanding Academic Leaders (Youth) project of Shanghai Science and Technology Innovation Action Plan/ ; 2022YQ073//Shanghai Municipal Health Commission/ ; 20191829//Shanghai Jiao Tong University Medical College/ ; },
mesh = {*Macrophages/metabolism ; *CRISPR-Cas Systems ; Animals ; Mice ; *Tendon Injuries/therapy/genetics ; Immunotherapy ; RNA Editing ; RNA, Messenger/genetics/metabolism ; RAW 264.7 Cells ; Osteopontin/genetics/metabolism ; Reactive Oxygen Species/metabolism ; },
abstract = {CRISPR-Cas13 holds substantial promise for tissue repair through its RNA editing capabilities and swift catabolism. However, conventional delivery methods fall short in addressing the heightened inflammatory response orchestrated by macrophages during the acute stages of tendon injury. In this investigation, macrophage-targeting cationic polymers are systematically screened to facilitate the entry of Cas13 ribonucleic-protein complex (Cas13 RNP) into macrophages. Notably, SPP1 (OPN encoding)-producing macrophages are recognized as a profibrotic subtype that emerges during the inflammatory stage. By employing ROS-responsive release mechanisms tailored for macrophage-targeted Cas13 RNP editing systems, the overactivation of SPP1 is curbed in the face of an acute immune microenvironment. Upon encapsulating this composite membrane around the tendon injury site, the macrophage-targeted Cas13 RNP effectively curtails the emergence of injury-induced SPP1-producing macrophages in the acute phase, leading to diminished fibroblast activation and mitigated peritendinous adhesion. Consequently, this study furnishes a swift RNA editing strategy for macrophages in the inflammatory phase triggered by ROS in tendon injury, along with a pioneering macrophage-targeted carrier proficient in delivering Cas13 into macrophages efficiently.},
}

*Macrophages/metabolism
*CRISPR-Cas Systems
Animals
Mice
*Tendon Injuries/therapy/genetics
Immunotherapy
RNA Editing
RNA, Messenger/genetics/metabolism
RAW 264.7 Cells
Osteopontin/genetics/metabolism
Reactive Oxygen Species/metabolism

@article {pmid36794352,
year = {2024},
author = {Qian, W and Wang, X and Wang, T and Huang, J and Zhang, Q and Li, Y and Chen, S},
title = {Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1.},
journal = {Animal models and experimental medicine},
volume = {7},
number = {2},
pages = {179-188},
doi = {10.1002/ame2.12298},
pmid = {36794352},
issn = {2576-2095},
support = {81973531//Natural Science Foundation of China/ ; 22GXFW0007//Science and Technology Plan Project of Xi'an/ ; 20200812211704001//Shenzhen Science and Technology Innovation Commission/ ; A2019502//Medical Scientific Research Foundation of Guangdong Province/ ; NS2022022//Nanshan District Science and Technology Plan Project/ ; 22JC010//Scientific Research Program Funded by Shaanxi Provincial Education Department/ ; },
mesh = {Humans ; *Human bocavirus/genetics/isolation & purification ; Parvoviridae Infections/diagnosis ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Fluorescence ; CRISPR-Cas Systems ; Recombinases/metabolism ; DNA, Viral/genetics/analysis ; },
abstract = {Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.},
}

Humans
*Human bocavirus/genetics/isolation & purification
Parvoviridae Infections/diagnosis
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Fluorescence
CRISPR-Cas Systems
Recombinases/metabolism
DNA, Viral/genetics/analysis

@article {pmid38691600,
year = {2024},
author = {Kim, J and Kratz, AF and Chen, S and Sheng, J and Kim, HK and Zhang, L and Singh, BK and Chavez, A},
title = {High-throughput tagging of endogenous loci for rapid characterization of protein function.},
journal = {Science advances},
volume = {10},
number = {18},
pages = {eadg8771},
pmid = {38691600},
issn = {2375-2548},
mesh = {Humans ; *Genetic Loci ; CRISPR-Cas Systems ; Proteins/genetics/metabolism ; High-Throughput Screening Assays/methods ; Cytoplasmic Granules/metabolism/genetics ; },
abstract = {To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.},
}

Humans
*Genetic Loci
CRISPR-Cas Systems
Proteins/genetics/metabolism
High-Throughput Screening Assays/methods
Cytoplasmic Granules/metabolism/genetics

@article {pmid38691425,
year = {2024},
author = {Richter, I and Hasan, M and Kramer, JW and Wein, P and Krabbe, J and Woitas, KP and Stinear, TP and Pidot, SJ and Kloss, F and Hertweck, C and Lackner, G},
title = {Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrae074},
pmid = {38691425},
issn = {1751-7370},
abstract = {The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.},
}

@article {pmid38689239,
year = {2024},
author = {Zhang, W and Qu, H and Wu, X and Shi, J and Wang, X},
title = {Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system.},
journal = {BMC infectious diseases},
volume = {24},
number = {1},
pages = {458},
pmid = {38689239},
issn = {1471-2334},
support = {2021GGJS085//The youth backbone teacher training program in colleges and universities of Henan province/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/isolation & purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Pseudomonas Infections/microbiology/diagnosis ; Humans ; Limit of Detection ; Recombinases/metabolism ; *Bacterial Proteins ; *Endodeoxyribonucleases ; *CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention.

METHODS: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples).

RESULTS: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 10[0] copies/µL for fluorescence assay and 10[1] copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements.

CONCLUSIONS: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.},
}

*Pseudomonas aeruginosa/genetics/isolation & purification
*CRISPR-Cas Systems
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Pseudomonas Infections/microbiology/diagnosis
Humans
Limit of Detection
Recombinases/metabolism
*Bacterial Proteins
*Endodeoxyribonucleases
*CRISPR-Associated Proteins

@article {pmid38688988,
year = {2024},
author = {Singh, A and Smedley, GD and Rose, JG and Fredriksen, K and Zhang, Y and Li, L and Yuan, SH},
title = {A high efficiency precision genome editing method with CRISPR in iPSCs.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {9933},
pmid = {38688988},
issn = {2045-2322},
support = {P50 AG016573/AG/NIA NIH HHS/United States ; R03 AG070415/AG/NIA NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; 1R03AG070415/GF/NIH HHS/United States ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Homologous Recombination ; },
abstract = {The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Tumor Suppressor Protein p53/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Homologous Recombination