@article {pmid39042694,
year = {2024},
author = {Zhou, B and Purmann, C and Guo, H and Shin, G and Huang, Y and Pattni, R and Meng, Q and Greer, SU and Roychowdhury, T and Wood, RN and Ho, M and Dohna, HZ and Abyzov, A and Hallmayer, JF and Wong, WH and Ji, HP and Urban, AE},
title = {Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {31},
pages = {e2322834121},
doi = {10.1073/pnas.2322834121},
pmid = {39042694},
issn = {1091-6490},
support = {K01MH129758//HHS | NIH | National Institute of Mental Health (NIMH)/ ; Instructor K Support Award//SU | SOM | Stanford Maternal and Child Health Research Institute (MCHRI)/ ; Uytengsu-Hamilton 22q11 Neuropsychiatry Research Award//SU | SOM | Stanford Maternal and Child Health Research Institute (MCHRI)/ ; R01HG006137//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; P01HG00205ESH//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG010963//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; P50HG00773506//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; DP2 MH100010-01//HHS | NIH | National Institute of Mental Health (NIMH)/ ; RSG-13-297-01-TBG//American Cancer Society (ACS)/ ; 1807371//National Science Foundation (NSF)/ ; R01MH100900//HHS | NIH | National Institute of Mental Health (NIMH)/ ; HG010359//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *DiGeorge Syndrome/genetics ; CRISPR-Cas Systems ; Chromosome Breakpoints ; Chromosomes, Human, Pair 22/genetics ; Genome, Human ; Gene Rearrangement ; Sequence Analysis, DNA/methods ; Chromosome Deletion ; },
abstract = {We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.},
}

Humans
*DiGeorge Syndrome/genetics
CRISPR-Cas Systems
Chromosome Breakpoints
Chromosomes, Human, Pair 22/genetics
Genome, Human
Gene Rearrangement
Sequence Analysis, DNA/methods
Chromosome Deletion

@article {pmid39039550,
year = {2024},
author = {Song, P and Zhang, Q and Xu, Z and Shi, Y and Jing, R and Luo, D},
title = {Correction: CRISPR/Cas-based CAR-T cells: production and application.},
journal = {Biomarker research},
volume = {12},
number = {1},
pages = {69},
doi = {10.1186/s40364-024-00616-7},
pmid = {39039550},
issn = {2050-7771},
}

@article {pmid39039415,
year = {2024},
author = {Meertens, L and Couture, L and Amara, A},
title = {Genome-Wide CRISPR-Cas9 Screening for the Identification of Host Dependency Factors of Emerging Viruses.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2824},
number = {},
pages = {203-219},
pmid = {39039415},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Rift Valley fever virus/genetics ; Virus Replication/genetics ; Host-Pathogen Interactions/genetics ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Like all the RNA viruses, Rift Valley fever virus (RVFV) encodes only few viral proteins and relies heavily on the host cellular machinery for productive infection. This dependence creates a potential "Achille's heel" that may be exploited to develop new approaches to treat RVFV infection. The recent development of lentiviral sgRNAs pool has enabled the creation of genome-scale CRISPR-Cas9 knockout libraries that has been used to identify host factors required for virus replication. In this chapter, we describe the preparation and execution of a pooled CRISPR-Cas9 loss-of-function screen using virus-induced cell death phenotypic readout. Using this technique, we outline a strategy for the identification of host factors essential for important human emerging viruses such as RVFV.},
}

*CRISPR-Cas Systems
Humans
*Rift Valley fever virus/genetics
Virus Replication/genetics
Host-Pathogen Interactions/genetics
Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39037655,
year = {2024},
author = {Cortina, C and Cañellas-Socias, A},
title = {CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2811},
number = {},
pages = {137-154},
pmid = {39037655},
issn = {1940-6029},
mesh = {*Organoids/metabolism/pathology ; *CRISPR-Cas Systems ; Humans ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; Animals ; Neoplasms/genetics/pathology ; Genes, Reporter ; },
abstract = {The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.},
}

*Organoids/metabolism/pathology
*CRISPR-Cas Systems
Humans
*Gene Knock-In Techniques/methods
*Gene Editing/methods
Animals
Neoplasms/genetics/pathology
Genes, Reporter

@article {pmid39037483,
year = {2024},
author = {de Carvalho, A and Giambiagi-deMarval, M and Rossi, CC},
title = {Mammaliicoccus sciuri's Pan-Immune System and the Dynamics of Horizontal Gene Transfer Among Staphylococcaceae: a One-Health CRISPR Tale.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {},
number = {},
pages = {},
pmid = {39037483},
issn = {1976-3794},
support = {408564/2023-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 304839/2022-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; E-26/010.000172/2016; 010.00128/2016; E-26.210.875/2016//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; 101056/2018; 001463/2019; 211.554/2019; 201.071/2020//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; 200.895/2021//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E-26/204.925/2022//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; APQ-03498-22//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; 23038.002486/2018-26//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
abstract = {Recently emancipated from the Staphylococcus genus due to genomic differences, Mammaliicoccus sciuri, previously classified as an occasional pathogen, emerges as a significant player in the landscape of resistance gene dissemination among Staphylococcaceae. Despite its classification, its role remained enigmatic. In this study, we delved into the genomic repertoire of M. sciuri to unravel its contribution to resistance and virulence gene transfer in the context of One Health. Through comprehensive analysis of publicly available genomes, we unveiled a diverse pan-immune system adept at defending against exogenous genetic elements, yet concurrently fostering horizontal gene transfer (HGT). Specifically, exploration of CRISPR-Cas systems, with spacer sequences as molecular signatures, elucidated a global dissemination pattern spanning environmental, animal, and human hosts. Notably, we identified the integration of CRISPR-Cas systems within SCCmecs (Staphylococcal Cassette Chromosome mec), harboring key genes associated with pathogenicity and resistance, especially the methicillin resistance gene mecA, suggesting a strategic adaptation to outcompete other mobile genetic elements. Our findings underscored M. sciuri's active engagement in HGT dynamics and evolutionary trajectories within Staphylococcaceae, emphasizing its central role in shaping microbial communities and highlighting the significance of understanding its implications in the One Health framework, an interdisciplinary approach that recognizes the interconnectedness of human, animal, and environmental health to address global health challenges.},
}

@article {pmid39035353,
year = {2024},
author = {Zuberi, A and Ahmad, N and Ahmad, H and Saeed, M and Ahmad, I},
title = {Beyond antibiotics: CRISPR/Cas9 triumph over biofilm-associated antibiotic resistance infections.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1408569},
pmid = {39035353},
issn = {2235-2988},
mesh = {*Biofilms/drug effects/growth & development ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Bacteria/drug effects/genetics ; Drug Resistance, Bacterial/genetics ; Bacterial Infections/microbiology/drug therapy ; Gene Editing ; },
abstract = {A complex structure known as a biofilm is formed when a variety of bacterial colonies or a single type of cell in a group sticks to a surface. The extracellular polymeric compounds that encase these cells, often consisting of proteins, eDNA, and polysaccharides, exhibit strong antibiotic resistance. Concerns about biofilm in the pharmaceutical industry, public health, and medical fields have sparked a lot of interest, as antibiotic resistance is a unique capacity exhibited by these biofilm-producing bacteria, which increases morbidity and death. Biofilm formation is a complicated process that is controlled by several variables. Insights into the processes to target for the therapy have been gained from multiple attempts to dissect the biofilm formation process. Targeting pathogens within a biofilm is profitable because the bacterial pathogens become considerably more resistant to drugs in the biofilm state. Although biofilm-mediated infections can be lessened using the currently available medications, there has been a lot of focus on the development of new approaches, such as bioinformatics tools, for both treating and preventing the production of biofilms. Technologies such as transcriptomics, metabolomics, nanotherapeutics and proteomics are also used to develop novel anti-biofilm agents. These techniques help to identify small compounds that can be used to inhibit important biofilm regulators. The field of appropriate control strategies to avoid biofilm formation is expanding quickly because of this spurred study. As a result, the current article addresses our current knowledge of how biofilms form, the mechanisms by which bacteria in biofilms resist antibiotics, and cutting-edge treatment approaches for infections caused by biofilms. Furthermore, we have showcased current ongoing research utilizing the CRISPR/Cas9 gene editing system to combat bacterial biofilm infections, particularly those brought on by lethal drug-resistant pathogens, concluded the article with a novel hypothesis and aspirations, and acknowledged certain limitations.},
}

*Biofilms/drug effects/growth & development
*CRISPR-Cas Systems
*Anti-Bacterial Agents/pharmacology
Humans
Bacteria/drug effects/genetics
Drug Resistance, Bacterial/genetics
Bacterial Infections/microbiology/drug therapy
Gene Editing

@article {pmid39035184,
year = {2024},
author = {Rooholamini, Z and Dianat-Moghadam, H and Esmaeilifallah, M and Khanahmad, H},
title = {From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization.},
journal = {Frontiers in public health},
volume = {12},
number = {},
pages = {1382996},
pmid = {39035184},
issn = {2296-2565},
mesh = {*Vaccines, Attenuated/immunology ; *Leishmaniasis, Cutaneous/prevention & control ; Humans ; *Leishmaniasis Vaccines/immunology ; *Genetic Engineering ; *Leishmania major/immunology/genetics ; Animals ; Immunization ; Gene Editing ; },
abstract = {Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis.},
}

*Vaccines, Attenuated/immunology
*Leishmaniasis, Cutaneous/prevention & control
Humans
*Leishmaniasis Vaccines/immunology
*Genetic Engineering
*Leishmania major/immunology/genetics
Animals
Immunization
Gene Editing

@article {pmid39034763,
year = {2024},
author = {Ou, X and Li, K and Liu, M and Song, J and Zuo, Z and Guo, Y},
title = {EXPAR for biosensing: recent developments and applications.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4an00609g},
pmid = {39034763},
issn = {1364-5528},
abstract = {Emerging as a promising novel amplification technique, the exponential amplification reaction (EXPAR) offers significant advantages due to its potent exponential amplification capability, straightforward reaction design, rapid reaction kinetics, and isothermal operation. The past few years have witnessed swift advancements and refinements in EXPAR-based technologies, with numerous high-performance biosensing systems documented. A deeper understanding of the EXPAR mechanism has facilitated the proposal of novel strategies to overcome limitations inherent to traditional EXPAR. Furthermore, the synergistic integration of EXPAR with diverse amplification methodologies, including the use of a CRISPR/Cas system, metal nanoparticles, aptamers, alternative isothermal amplification techniques, and enzymes, has significantly bolstered analytical efficacy, aiming to enhance specificity, sensitivity, and amplification efficiency. This comprehensive review presents a detailed exposition of the EXPAR mechanism and analyzes its primary challenges. Additionally, we summarize the latest research advancements in the biomedical field concerning the integration of EXPAR with diverse amplification technologies for sensing strategies. Finally, we discuss the challenges and future prospects of EXPAR technology in the realms of biosensing and clinical applications.},
}

@article {pmid39034336,
year = {2024},
author = {Tarafder, E and Nizamani, MM and Karunarathna, SC and Das, D and Zeng, X and Rind, RA and Wang, Y and Tian, F},
title = {Advancements in genetic studies of mushrooms: a comprehensive review.},
journal = {World journal of microbiology & biotechnology},
volume = {40},
number = {9},
pages = {275},
pmid = {39034336},
issn = {1573-0972},
mesh = {*Agaricales/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA Interference ; Genome, Fungal ; Agriculture/methods ; Gene Knockout Techniques ; },
abstract = {Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world.},
}

*Agaricales/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
RNA Interference
Genome, Fungal
Agriculture/methods
Gene Knockout Techniques

@article {pmid39034325,
year = {2024},
author = {Maurizio, M and Masid, M and Woods, K and Caldelari, R and Doench, JG and Naguleswaran, A and Joly, D and González-Fernández, M and Zemp, J and Borteele, M and Hatzimanikatis, V and Heussler, V and Rottenberg, S and Olias, P},
title = {Host cell CRISPR genomics and modelling reveal shared metabolic vulnerabilities in the intracellular development of Plasmodium falciparum and related hemoparasites.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {6145},
pmid = {39034325},
issn = {2041-1723},
support = {173972//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 189127//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Plasmodium falciparum/genetics ; Humans ; *Hepatocytes/parasitology/metabolism ; *Malaria, Falciparum/parasitology ; *CRISPR-Cas Systems ; *Theileria/genetics ; Genomics/methods ; Heme/metabolism ; Host-Parasite Interactions/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Gene Knockout Techniques ; },
abstract = {Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum's adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.},
}

*Plasmodium falciparum/genetics
Humans
*Hepatocytes/parasitology/metabolism
*Malaria, Falciparum/parasitology
*CRISPR-Cas Systems
*Theileria/genetics
Genomics/methods
Heme/metabolism
Host-Parasite Interactions/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
Gene Knockout Techniques

@article {pmid39034177,
year = {2024},
author = {Ye, X and Wu, H and Liu, J and Xiang, J and Feng, Y and Liu, Q},
title = {One-pot diagnostic methods based on CRISPR/Cas and Argonaute nucleases: strategies and perspectives.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2024.06.009},
pmid = {39034177},
issn = {1879-3096},
abstract = {CRISPR/Cas and Argonaute (Ago) proteins, which target specific nucleic acid sequences, can be applied as diagnostic tools. Despite high specificity and efficiency, achieving sensitive detection often necessitates a preamplification step that involves opening the lid and multistep operation, which may elevate the risk of contamination and prove inadequate for point-of-care testing. Hence, various one-pot detection strategies have been developed that enable preamplification and sensing in a single operation. We outline the challenges of one-pot detection with Cas and Ago proteins, present several main implementation strategies, and discuss future prospects. This review offers comprehensive insights into this vital field and explores potential improvements to detection methods that will be beneficial for human health.},
}

@article {pmid39033258,
year = {2024},
author = {Reyhani-Ardabili, M and Fathi, M and Ghafouri-Fard, S},
title = {CRISPR/Cas9 technology in the modeling of and evaluation of possible treatments for Niemann-Pick C.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {828},
pmid = {39033258},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Niemann-Pick Disease, Type C/therapy/genetics/metabolism ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Cholesterol/metabolism ; Animals ; Niemann-Pick C1 Protein ; Disease Models, Animal ; },
abstract = {Niemann-Pick disease type C (NPC) is a rare neurodegenerative condition resulted from mutations in NPC1 and NPC2 genes. This cellular lipid transferring disorder mainly involves endocytosed cholesterol trafficking. The accumulation of cholesterol and glycolipids in late endosomes and lysosomes results in progressive neurodegeneration and death. Recently, genome editing technologies, particularly CRISPR/Cas9 have offered the opportunity to create disease models to screen novel therapeutic options for this disorder. Moreover, these methods have been used for the purpose of gene therapy. This review summarizes the studies that focused on the application of CRISPR/Cas9 technology for exploring the mechanism of intracellular cholesterol transferring, and screening of novel agents for treatment of NPC.},
}

*CRISPR-Cas Systems/genetics
Humans
*Niemann-Pick Disease, Type C/therapy/genetics/metabolism
*Gene Editing/methods
*Genetic Therapy/methods
*Cholesterol/metabolism
Animals
Niemann-Pick C1 Protein
Disease Models, Animal

@article {pmid39033086,
year = {2024},
author = {Zimmerman, E and Sturrock, A and Reilly, CA and Burrell-Gerbers, KL and Warren, K and Mir-Kasimov, M and Zhang, MA and Pierce, MS and Helms, MN and Paine, R},
title = {Aryl Hydrocarbon Receptor Activation in Pulmonary Alveolar Epithelial Cells Limits Inflammation and Preserves Lung Epithelial Cell Integrity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {},
number = {},
pages = {},
doi = {10.4049/jimmunol.2300325},
pmid = {39033086},
issn = {1550-6606},
support = {5I01BX001777//VA Merit Grant/ ; },
abstract = {The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-β-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes.},
}

@article {pmid39032106,
year = {2024},
author = {Bircheneder, M and Schreiber, T and Tissier, A and Parniske, M},
title = {A quantitative assay for the efficiency of RNA-guided genome editing in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.16931},
pmid = {39032106},
issn = {1365-313X},
abstract = {RNA-guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double-strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch-on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20-bp spacer either side to preferentially detect full-deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas-mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4-HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.},
}

@article {pmid39030569,
year = {2024},
author = {Vinceti, A and Iannuzzi, RM and Boyle, I and Trastulla, L and Campbell, CD and Vazquez, F and Dempster, JM and Iorio, F},
title = {A benchmark of computational methods for correcting biases of established and unknown origin in CRISPR-Cas9 screening data.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {192},
pmid = {39030569},
issn = {1474-760X},
support = {28772//Fondazione AIRC per la ricerca sul cancro ETS/ ; 101125051//HORIZON EUROPE European Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Benchmarking ; Computational Biology/methods ; Bias ; },
abstract = {BACKGROUND: CRISPR-Cas9 dropout screens are formidable tools for investigating biology with unprecedented precision and scale. However, biases in data lead to potential confounding effects on interpretation and compromise overall quality. The activity of Cas9 is influenced by structural features of the target site, including copy number amplifications (CN bias). More worryingly, proximal targeted loci tend to generate similar gene-independent responses to CRISPR-Cas9 targeting (proximity bias), possibly due to Cas9-induced whole chromosome-arm truncations or other genomic structural features and different chromatin accessibility levels.

RESULTS: We benchmarked eight computational methods, rigorously evaluating their ability to reduce both CN and proximity bias in the two largest publicly available cell-line-based CRISPR-Cas9 screens to date. We also evaluated the capability of each method to preserve data quality and heterogeneity by assessing the extent to which the processed data allows accurate detection of true positive essential genes, established oncogenetic addictions, and known/novel biomarkers of cancer dependency. Our analysis sheds light on the ability of each method to correct biases under different scenarios. AC-Chronos outperforms other methods in correcting both CN and proximity biases when jointly processing multiple screens of models with available CN information, whereas CRISPRcleanR is the top performing method for individual screens or when CN information is not available. In addition, Chronos and AC-Chronos yield a final dataset better able to recapitulate known sets of essential and non-essential genes.

CONCLUSIONS: Overall, our investigation provides guidance for the selection of the most appropriate bias-correction method, based on its strengths, weaknesses and experimental settings.},
}

*CRISPR-Cas Systems
Humans
*Benchmarking
Computational Biology/methods
Bias

@article {pmid39030018,
year = {2024},
author = {Sun, H and Zhang, X and Ma, H and Zhang, L and Zhang, Y and Sun, R and Zheng, H and Wang, H and Guo, J and Liu, Y and Wang, Y and Qi, Y},
title = {A programmable sensitive platform for pathogen detection based on CRISPR/Cas12a -hybridization chain reaction-poly T-Cu.},
journal = {Analytica chimica acta},
volume = {1317},
number = {},
pages = {342888},
doi = {10.1016/j.aca.2024.342888},
pmid = {39030018},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Copper/chemistry ; *Nucleic Acid Hybridization ; Poly T/chemistry ; Staphylococcus aureus/isolation & purification/genetics ; DNA, Bacterial ; Limit of Detection ; Fluorescent Dyes/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe. The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus, respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.},
}

*CRISPR-Cas Systems
*Copper/chemistry
*Nucleic Acid Hybridization
Poly T/chemistry
Staphylococcus aureus/isolation & purification/genetics
DNA, Bacterial
Limit of Detection
Fluorescent Dyes/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39028827,
year = {2024},
author = {Patel, RP and Ghilardi, G and Zhang, Y and Chiang, YH and Xie, W and Guruprasad, P and Kim, KH and Chun, I and Angelos, MG and Pajarillo, R and Hong, SJ and Lee, YG and Shestova, O and Shaw, C and Cohen, I and Gupta, A and Vu, T and Qian, D and Yang, S and Nimmagadda, A and Snook, AE and Siciliano, N and Rotolo, A and Inamdar, A and Harris, J and Ugwuanyi, O and Wang, M and Carturan, A and Paruzzo, L and Chen, L and Ballard, HJ and Blanchard, T and Xu, C and Abdel-Mohsen, M and Gabunia, K and Wysocka, M and Linette, GP and Carreno, B and Barrett, DM and Teachey, DT and Posey, AD and Powell, DJ and Sauter, CT and Pileri, S and Pillai, V and Scholler, J and Rook, AH and Schuster, SJ and Barta, SK and Porazzi, P and Ruella, M},
title = {CD5 deletion enhances the antitumor activity of adoptive T cell therapies.},
journal = {Science immunology},
volume = {9},
number = {97},
pages = {eadn6509},
doi = {10.1126/sciimmunol.adn6509},
pmid = {39028827},
issn = {2470-9468},
mesh = {Animals ; *Immunotherapy, Adoptive/methods ; *CD5 Antigens/immunology ; Mice ; Humans ; *T-Lymphocytes/immunology/transplantation ; Receptors, Chimeric Antigen/immunology/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Female ; },
abstract = {Most patients treated with US Food and Drug Administration (FDA)-approved chimeric antigen receptor (CAR) T cells eventually experience disease progression. Furthermore, CAR T cells have not been curative against solid cancers and several hematological malignancies such as T cell lymphomas, which have very poor prognoses. One of the main barriers to the clinical success of adoptive T cell immunotherapies is CAR T cell dysfunction and lack of expansion and/or persistence after infusion. In this study, we found that CD5 inhibits CAR T cell activation and that knockout (KO) of CD5 using CRISPR-Cas9 enhances the antitumor effect of CAR T cells in multiple hematological and solid cancer models. Mechanistically, CD5 KO drives increased T cell effector function with enhanced cytotoxicity, in vivo expansion, and persistence, without apparent toxicity in preclinical models. These findings indicate that CD5 is a critical inhibitor of T cell function and a potential clinical target for enhancing T cell therapies.},
}

Animals
*Immunotherapy, Adoptive/methods
*CD5 Antigens/immunology
Mice
Humans
*T-Lymphocytes/immunology/transplantation
Receptors, Chimeric Antigen/immunology/genetics
Cell Line, Tumor
CRISPR-Cas Systems
Female

@article {pmid39026343,
year = {2024},
author = {Zhou, H and Ye, P and Xiong, W and Duan, X and Jing, S and He, Y and Zeng, Z and Wei, Y and Ye, Q},
title = {Genome-scale CRISPR-Cas9 screening in stem cells: theories, applications and challenges.},
journal = {Stem cell research & therapy},
volume = {15},
number = {1},
pages = {218},
pmid = {39026343},
issn = {1757-6512},
support = {2022YFC2504200//Chinese Academy of Sciences Key Project/ ; 2022BCA029//Science and Technology Program of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Stem Cells/metabolism/cytology ; Animals ; },
abstract = {Due to the rapid development of stem cell technology, there have been tremendous advances in molecular biological and pathological research, cell therapy as well as organoid technologies over the past decades. Advances in genome editing technology, particularly the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9), have further facilitated the rapid development of stem cell researches. The CRISPR-Cas9 technology now goes beyond creating single gene editing to enable the inhibition or activation of endogenous gene loci by fusing inhibitory (CRISPRi) or activating (CRISPRa) domains with deactivated Cas9 proteins (dCas9). These tools have been utilized in genome-scale CRISPRi/a screen to recognize hereditary modifiers that are synergistic or opposing to malady mutations in an orderly and fair manner, thereby identifying illness mechanisms and discovering novel restorative targets to accelerate medicinal discovery investigation. However, the application of this technique is still relatively rare in stem cell research. There are numerous specialized challenges in applying large-scale useful genomics approaches to differentiated stem cell populations. Here, we present the first comprehensive review on CRISPR-based functional genomics screening in the field of stem cells, as well as practical considerations implemented in a range of scenarios, and exploration of the insights of CRISPR-based screen into cell fates, disease mechanisms and cell treatments in stem cell models. This review will broadly benefit scientists, engineers and medical practitioners in the areas of stem cell research.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
*Stem Cells/metabolism/cytology
Animals

@article {pmid39025833,
year = {2024},
author = {Ge, W and Gou, S and Zhao, X and Jin, Q and Zhuang, Z and Zhao, Y and Liang, Y and Ouyang, Z and Liu, X and Chen, F and Shi, H and Yan, H and Wu, H and Lai, L and Wang, K},
title = {In vivo evaluation of guide-free Cas9-induced safety risks in a pig model.},
journal = {Signal transduction and targeted therapy},
volume = {9},
number = {1},
pages = {184},
pmid = {39025833},
issn = {2059-3635},
support = {32300426//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Genetic Therapy ; },
abstract = {The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Gene Editing
*CRISPR-Associated Protein 9/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Genetic Therapy

@article {pmid39024377,
year = {2024},
author = {Yoon, PH and Zhang, Z and Loi, KJ and Adler, BA and Lahiri, A and Vohra, K and Shi, H and Rabelo, DB and Trinidad, M and Boger, RS and Al-Shimary, MJ and Doudna, JA},
title = {Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {eadq0553},
doi = {10.1126/science.adq0553},
pmid = {39024377},
issn = {1095-9203},
abstract = {The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an), and further trace Cas13 origins to defense-associated ribonucleases. Despite being one third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.},
}

@article {pmid39023769,
year = {2024},
author = {He, X and Deng, L and Zhou, S and Dong, J and Zhu, S and Li, J and Li, X and Huo, D and Hou, C},
title = {CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.},
journal = {Mikrochimica acta},
volume = {191},
number = {8},
pages = {470},
pmid = {39023769},
issn = {1436-5073},
support = {2023-K08//Hubei Key Laboratory of Intelligent Geo-Information Processing/ ; CSTB2022NSCO-BHX0727//Chongqing Natural Science Foundation/ ; 2022CDJYGRH-013//Fundamental Research Founds for the Central Universities/ ; },
mesh = {*MicroRNAs/analysis/blood/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.},
}

*MicroRNAs/analysis/blood/genetics
Humans
*CRISPR-Cas Systems/genetics
DNA, Single-Stranded/chemistry/genetics
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39023219,
year = {2024},
author = {López-Beltrán, A and Botelho, J and Iranzo, J},
title = {Dynamics of CRISPR-mediated virus-host interactions in the human gut microbiome.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrae134},
pmid = {39023219},
issn = {1751-7370},
abstract = {Arms races between mobile genetic elements and prokaryotic hosts are major drivers of ecological and evolutionary change in microbial communities. Prokaryotic defense systems such as CRISPR-Cas have the potential to regulate microbiome composition by modifying the interactions among bacteria, plasmids, and phages. Here, we used longitudinal metagenomic data from 130 healthy and diseased individuals to study how the interplay of genetic parasites and CRISPR-Cas immunity reflects on the dynamics and composition of the human gut microbiome. Based on the coordinated study of 80 000 CRISPR-Cas loci and their targets, we show that CRISPR-Cas immunity effectively modulates bacteriophage abundances in the gut. Acquisition of CRISPR-Cas immunity typically leads to a decrease in the abundance of lytic phages but does not necessarily cause their complete disappearance. Much smaller effects are observed for lysogenic phages and plasmids. Conversely, phage-CRISPR interactions shape bacterial microdiversity by producing weak selective sweeps that benefit immune host lineages. We also show that distal (and chronologically older) regions of CRISPR arrays are enriched in spacers that are potentially functional and target crass-like phages and local prophages. This suggests that exposure to reactivated prophages and other endemic viruses is a major selective pressure in the gut microbiome that drives the maintenance of long-lasting immune memory.},
}

@article {pmid39023080,
year = {2024},
author = {Sha, L and Yao, J and Yang, S and Hu, M and Zhou, Q and Zhao, J and Bei, Y and Cao, Y},
title = {Collaborative CRISPR-Cas System-Enabled Detection of Circulating Circular RNA for Reliable Monitoring of Acute Myocardial Infarction.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2402895},
doi = {10.1002/smll.202402895},
pmid = {39023080},
issn = {1613-6829},
support = {81972799//National Natural Science Foundation of China/ ; 81970335//National Natural Science Foundation of China/ ; 82170285//National Natural Science Foundation of China/ ; 20ZR1443300//Natural Science Foundation of Shanghai/ ; 23ZR1421400//Natural Science Foundation of Shanghai/ ; XZ2020ZR-YZ35//Natural Science Foundation of Xizang/ ; },
abstract = {Acute myocardial infarction (AMI) is one of the major causes of death worldwide, posing significant global health challenges. Circular RNA (circRNA) has recently emerged as a potential diagnostic biomarker for AMI, providing valuable information for timely medical care. In this work, a new electrochemical method for circRNA detection by engineering a collaborative CRISPR-Cas system is developed. This system integrates the unique circRNA-targeting ability with cascade trans-cleavage activities of Cas effectors, using an isothermal primer exchange reaction as the bridge. Using cZNF292, a circulating circRNA biomarker for AMI is identified by this group; as a model, the collaborative CRISPR-Cas system-based method exhibits excellent accuracy and sensitivity with a low detection limit of 2.13 × 10[-15] m. Moreover, the method demonstrates a good diagnostic performance for AMI when analyzing whole blood samples. Therefore, the method may provide new insight into the detection of circRNA biomarkers and is expected to have great potential in AMI diagnosis in the future.},
}

@article {pmid39021763,
year = {2024},
author = {Asadbeigi, A and Bakhtiarizadeh, MR and Saffari, M and Modarressi, MH and Sadri, N and Kafi, ZZ and Fazilaty, H and Ghalyanchilangeroudi, A and Esmaeili, H},
title = {Protection of animals against devastating RNA viruses using CRISPR-Cas13s.},
journal = {Molecular therapy. Nucleic acids},
volume = {35},
number = {3},
pages = {102235},
pmid = {39021763},
issn = {2162-2531},
abstract = {The intrinsic nature of CRISPR-Cas in conferring immunity to bacteria and archaea has been repurposed to combat pathogenic agents in mammalian and plant cells. In this regard, CRISPR-Cas13 systems have proved their remarkable potential for single-strand RNA viruses targeting. Here, different types of Cas13 orthologs were applied to knockdown foot-and-mouth disease virus (FMDV), a highly contagious disease of a wide variety of species with genetically diverse strains and is widely geographically distributed. Using programmable CRISPR RNAs capable of targeting conserved regions of the viral genome, all Cas13s from CRISPR system type VI (subtype A/B/D) could comprehensively target and repress different serotypes of FMDV virus. This approach has the potential to destroy all strains of a virus as targets the ultra-conserved regions of genome. We experimentally compared the silencing efficiency of CRISPR and RNAi by designing the most effective short hairpin RNAs according to our developed scoring system and observed comparable results. This study showed successful usage of various Cas13 enzymes for suppression of FMDV, which provides a flexible strategy to battle with other animal infectious RNA viruses, an underdeveloped field in the biotechnology scope.},
}

@article {pmid39020310,
year = {2024},
author = {Collier, TC and Lee, Y and Mathias, DK and Del Amo, VL},
title = {CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {700},
pmid = {39020310},
issn = {1471-2164},
mesh = {Animals ; *Anopheles/genetics ; *CRISPR-Cas Systems ; *Aedes/genetics ; Genetic Variation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Genome, Insect ; Mosquito Vectors/genetics ; Gene Editing ; Bacterial Proteins ; },
abstract = {Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.},
}

Animals
*Anopheles/genetics
*CRISPR-Cas Systems
*Aedes/genetics
Genetic Variation
RNA, Guide, CRISPR-Cas Systems/genetics
Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Genome, Insect
Mosquito Vectors/genetics
Gene Editing
Bacterial Proteins

@article {pmid39019776,
year = {2024},
author = {Nguyen, GT and Schelling, MA and Raju, A and Buscher, KA and Sritharan, A and Sashital, DG},
title = {CRISPR-Cas12a exhibits metal-dependent specificity switching.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae613},
pmid = {39019776},
issn = {1362-4962},
support = {1652661//National Science Foundation/ ; GM140876/NH/NIH HHS/United States ; //Iowa State University/ ; },
abstract = {Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.},
}

@article {pmid39018342,
year = {2024},
author = {Bubeck, F and Grimm, D},
title = {When size matters: A novel compact Cas12a variant for in vivo genome editing.},
journal = {PLoS biology},
volume = {22},
number = {7},
pages = {e3002637},
pmid = {39018342},
issn = {1545-7885},
mesh = {*Gene Editing/methods ; *Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Animals ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; },
abstract = {A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.},
}

*Gene Editing/methods
*Dependovirus/genetics
*CRISPR-Cas Systems/genetics
*Genetic Vectors/genetics
Animals
CRISPR-Associated Proteins/genetics/metabolism
Humans
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism

@article {pmid39018338,
year = {2024},
author = {Vatanparast, M and Esmaeily, M and Stanley, D and Kim, Y},
title = {A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.},
journal = {PloS one},
volume = {19},
number = {7},
pages = {e0304958},
pmid = {39018338},
issn = {1932-6203},
mesh = {Animals ; *Eicosanoids/metabolism ; *CRISPR-Cas Systems ; *Spodoptera ; *Phospholipases A2/genetics/metabolism ; Signal Transduction ; Larva/genetics/immunology ; Insect Proteins/genetics/metabolism ; Sequence Deletion ; Genes, Insect ; Gene Editing ; Gene Deletion ; },
abstract = {Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.},
}

Animals
*Eicosanoids/metabolism
*CRISPR-Cas Systems
*Spodoptera
*Phospholipases A2/genetics/metabolism
Signal Transduction
Larva/genetics/immunology
Insect Proteins/genetics/metabolism
Sequence Deletion
Genes, Insect
Gene Editing
Gene Deletion

@article {pmid39018310,
year = {2024},
author = {Lee, HY and Min, YH and Lee, DG and Lee, KH and Kim, J and Lee, MK and Byun, JY and Shin, YB},
title = {CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c01246},
pmid = {39018310},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.},
}

@article {pmid39017887,
year = {2024},
author = {Anupam, K and Laidlaw, BJ},
title = {In Vivo CRISPR/Cas9-Mediated Gene Ablation in Murine B Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2826},
number = {},
pages = {79-91},
pmid = {39017887},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *B-Lymphocytes/metabolism/immunology ; *Cell Differentiation/genetics ; *Gene Editing/methods ; Germinal Center/immunology/metabolism/cytology ; Gene Deletion ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.},
}

Animals
*CRISPR-Cas Systems
Mice
*B-Lymphocytes/metabolism/immunology
*Cell Differentiation/genetics
*Gene Editing/methods
Germinal Center/immunology/metabolism/cytology
Gene Deletion
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39017845,
year = {2024},
author = {Zheng, G and Orkin, SH},
title = {Transcriptional Repressor BCL11A in Erythroid Cells.},
journal = {Advances in experimental medicine and biology},
volume = {1459},
number = {},
pages = {199-215},
pmid = {39017845},
issn = {0065-2598},
mesh = {*Repressor Proteins/genetics/metabolism ; Humans ; Animals ; *Erythroid Cells/metabolism ; Anemia, Sickle Cell/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Mice ; beta-Thalassemia/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Nuclear Proteins/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; gamma-Globins/genetics/metabolism ; Gene Expression Regulation ; Genome-Wide Association Study ; },
abstract = {BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and β-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and β-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.},
}

*Repressor Proteins/genetics/metabolism
Humans
Animals
*Erythroid Cells/metabolism
Anemia, Sickle Cell/genetics/metabolism
Fetal Hemoglobin/genetics/metabolism
Mice
beta-Thalassemia/genetics/metabolism
CRISPR-Cas Systems
Gene Editing/methods
Nuclear Proteins/genetics/metabolism
Carrier Proteins/genetics/metabolism
gamma-Globins/genetics/metabolism
Gene Expression Regulation
Genome-Wide Association Study

@article {pmid39017814,
year = {2024},
author = {He, Q and Chen, Q and Lian, L and Qu, J and Yuan, X and Wang, C and Xu, L and Wei, J and Zeng, S and Yu, D and Dong, Y and Zhang, Y and Deng, L and Du, K and Zhang, C and Pandey, V and Gul, I and Qin, P},
title = {Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.},
journal = {Mikrochimica acta},
volume = {191},
number = {8},
pages = {466},
pmid = {39017814},
issn = {1436-5073},
support = {31970752//National Natural Science Foundation of China/ ; 32350410397//National Natural Science Foundation of China/ ; JCYJ20190809180003689//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JSGG20191129110812708//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JSGG20200225150707332; JCYJ20220530143014032//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20230807113017035//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; WDZC20200820173710001//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; SZBL2020090501004//Shenzhen Bay Laboratory Open Funding/ ; 2020M680023//China Postdoctoral Science Foundation/ ; D2301002//Shenzhen Medical Research Funds/ ; DCE-iBHE-2022-3//Department of Chemical Engineering-iBHE special cooperation joint fund project/ ; JC2022009//Tsinghua Shenzhen International Graduate School/ ; (2022) 207//Ministry of Land and Resources of the People's Republic of China/ ; },
mesh = {*SARS-CoV-2/genetics ; *CRISPR-Cas Systems ; *RNA, Viral/analysis/genetics ; Humans ; COVID-19/diagnosis/virology ; Biosensing Techniques/methods ; CRISPR-Associated Proteins ; Microscopy, Fluorescence ; Lab-On-A-Chip Devices ; Limit of Detection ; Magnesium/chemistry ; COVID-19 Nucleic Acid Testing/methods ; },
abstract = {The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg[2+] concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.},
}

*SARS-CoV-2/genetics
*CRISPR-Cas Systems
*RNA, Viral/analysis/genetics
Humans
COVID-19/diagnosis/virology
Biosensing Techniques/methods
CRISPR-Associated Proteins
Microscopy, Fluorescence
Lab-On-A-Chip Devices
Limit of Detection
Magnesium/chemistry
COVID-19 Nucleic Acid Testing/methods

@article {pmid39017499,
year = {2024},
author = {Makin, S},
title = {Rapid Answers: Gene-editing technology gives diagnostic tests a boost.},
journal = {Scientific American},
volume = {331},
number = {1},
pages = {12},
doi = {10.1038/scientificamerican072024-2MXPoUcSWyso9xhGc3BU5U},
pmid = {39017499},
issn = {0036-8733},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; },
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems

@article {pmid39014928,
year = {2024},
author = {Huang, L and Li, N and Song, Y and Gao, J and Nian, L and Zhou, J and Zhang, B and Liu, Z and Zheng, Y},
title = {Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.},
journal = {Biotechnology journal},
volume = {19},
number = {7},
pages = {e2400164},
doi = {10.1002/biot.202400164},
pmid = {39014928},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Fusarium/genetics ; Plasmids/genetics ; Metabolic Engineering/methods ; Genetic Markers/genetics ; },
abstract = {Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50 bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Fusarium/genetics
Plasmids/genetics
Metabolic Engineering/methods
Genetic Markers/genetics

@article {pmid39012602,
year = {2024},
author = {Sarno, F and Koncz, M and Eilers, RE and Verschure, PJ and Rots, MG},
title = {Generation of Cell Lines Stably Expressing a dCas9-Fusion or sgRNA to Address Dynamics of Long-Term Effects of Epigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {289-307},
pmid = {39012602},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Epigenesis, Genetic
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells

@article {pmid39012601,
year = {2024},
author = {Woodward, EA and Wang, E and Wallis, C and Sharma, R and Tie, AWJ and Murthy, N and Blancafort, P},
title = {Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {267-287},
pmid = {39012601},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/chemistry ; Animals ; *Neoplasms/genetics/therapy ; Epigenesis, Genetic ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Liposomes/chemistry ; Cell Line, Tumor ; Lipids/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Therapy/methods ; Gene Transfer Techniques ; },
abstract = {Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
*Nanoparticles/chemistry
Animals
*Neoplasms/genetics/therapy
Epigenesis, Genetic
Mice
RNA, Guide, CRISPR-Cas Systems/genetics
Liposomes/chemistry
Cell Line, Tumor
Lipids/chemistry
CRISPR-Associated Protein 9/genetics/metabolism
Genetic Therapy/methods
Gene Transfer Techniques

@article {pmid39012600,
year = {2024},
author = {van den Berg van Saparoea, ACH and van Loosen, QC and Sarno, F and Ntini, E and Rots, MG and Gjaltema, RAF and Verschure, PJ},
title = {Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {255-265},
pmid = {39012600},
issn = {1940-6029},
mesh = {Humans ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; *Transfection/methods ; *Epigenesis, Genetic ; *Single-Cell Analysis/methods ; Epigenomics/methods ; Flow Cytometry ; },
abstract = {To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.},
}

Humans
*Plasmids/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
HEK293 Cells
*Transfection/methods
*Epigenesis, Genetic
*Single-Cell Analysis/methods
Epigenomics/methods
Flow Cytometry

@article {pmid39012598,
year = {2024},
author = {Azcona, MSR and Mussolino, C},
title = {Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {209-223},
pmid = {39012598},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Gene Silencing ; T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; Programmed Cell Death 1 Receptor/genetics ; Epigenome ; },
abstract = {Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.},
}

*Gene Editing/methods
Humans
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Gene Silencing
T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods
Programmed Cell Death 1 Receptor/genetics
Epigenome

@article {pmid39012597,
year = {2024},
author = {Oberkofler, V and Bäurle, I},
title = {Plant Epigenetic Editing to Analyze the Function of Histone Modifications in Gene-Specific Regulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {193-207},
pmid = {39012597},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Histones/metabolism/genetics ; *Epigenesis, Genetic ; *Gene Expression Regulation, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Histone Code ; Chromatin/genetics/metabolism ; Promoter Regions, Genetic ; Plants, Genetically Modified/genetics ; Plants/genetics/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Epigenetic editing enables the locus-specific manipulation of chromatin modifications. It allows the functional analysis of interactions between chromatin modifications and epigenetically stable gene expression states, thus establishing causal relationships, where previously correlations were suspected. Here, we describe the procedures for gene-specific epigenetic editing in plants that are based on targeting a histone modifier using an inactive dCas9 fusion protein that is recruited by a set of three distinct single guide RNAs (sgRNAs) that all target a region within the promoter of the target gene. We outline design principles and emphasize the need for suitable control constructs. In summary, the protocol will be widely useful for plant scientists looking to manipulate chromatin modifications in a locus-specific manner.},
}

*Gene Editing/methods
*Histones/metabolism/genetics
*Epigenesis, Genetic
*Gene Expression Regulation, Plant
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Histone Code
Chromatin/genetics/metabolism
Promoter Regions, Genetic
Plants, Genetically Modified/genetics
Plants/genetics/metabolism
Arabidopsis/genetics/metabolism

@article {pmid39012596,
year = {2024},
author = {Rajaram, N and Bashtrykov, P and Jeltsch, A},
title = {Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {179-192},
pmid = {39012596},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Alleles ; *DNA Methylation ; HEK293 Cells ; *Epigenome ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenomics/methods ; Epigenesis, Genetic ; },
abstract = {The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Alleles
*DNA Methylation
HEK293 Cells
*Epigenome
RNA, Guide, CRISPR-Cas Systems/genetics
Epigenomics/methods
Epigenesis, Genetic

@article {pmid39012595,
year = {2024},
author = {Sekita, Y and Kimura, T},
title = {Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {167-178},
pmid = {39012595},
issn = {1940-6029},
mesh = {*Genomic Imprinting ; *DNA Methylation ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Mice ; Genetic Loci ; Calcium-Binding Proteins/genetics/metabolism ; Iodide Peroxidase/genetics ; Alleles ; Humans ; },
abstract = {In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.},
}

*Genomic Imprinting
*DNA Methylation
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
Mice
Genetic Loci
Calcium-Binding Proteins/genetics/metabolism
Iodide Peroxidase/genetics
Alleles
Humans

@article {pmid39012594,
year = {2024},
author = {Morita, S and Horii, T and Hatada, I},
title = {Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {155-165},
pmid = {39012594},
issn = {1940-6029},
mesh = {*DNA Methylation ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression Regulation ; Epigenesis, Genetic ; Mixed Function Oxygenases/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.},
}

*DNA Methylation
Humans
*CRISPR-Cas Systems
Gene Editing/methods
Gene Expression Regulation
Epigenesis, Genetic
Mixed Function Oxygenases/genetics/metabolism
Proto-Oncogene Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39012593,
year = {2024},
author = {Hamilton, PJ and Lim, CJ and Nestler, EJ and Heller, EA},
title = {Neuroepigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {129-152},
pmid = {39012593},
issn = {1940-6029},
mesh = {*Epigenesis, Genetic ; Animals ; Humans ; *Gene Editing/methods ; Neurons/metabolism ; Brain/metabolism ; Histones/metabolism ; Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Epigenetic regulation is intrinsic to basic neurobiological function as well as neurological disease. Regulation of chromatin-modifying enzymes in the brain is critical during both development and adulthood and in response to external stimuli. Biochemical studies are complemented by numerous next-generation sequencing (NGS) studies that quantify global changes in gene expression, chromatin accessibility, histone and DNA modifications in neurons and glial cells. Neuroepigenetic editing tools are essential to distinguish between the mere presence and functional relevance of histone and DNA modifications to gene transcription in the brain and animal behavior. This review discusses current advances in neuroepigenetic editing, highlighting methodological considerations pertinent to neuroscience, such as delivery methods and the spatiotemporal specificity of editing and it demonstrates the enormous potential of epigenetic editing for basic neurobiological research and therapeutic application.},
}

*Epigenesis, Genetic
Animals
Humans
*Gene Editing/methods
Neurons/metabolism
Brain/metabolism
Histones/metabolism
Chromatin/genetics/metabolism
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing/methods

@article {pmid39012592,
year = {2024},
author = {Kim, SH and Haynes, KA},
title = {Reader-Effectors as Actuators of Epigenome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {103-127},
pmid = {39012592},
issn = {1940-6029},
mesh = {Animals ; Humans ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; Epigenomics/methods ; *Gene Editing/methods ; Gene Expression Regulation ; },
abstract = {Epigenome editing applications are gaining broader use for targeted transcriptional control as more enzymes with diverse chromatin-modifying functions are being incorporated into fusion proteins. Development of these fusion proteins, called epigenome editors, has outpaced the study of proteins that interact with edited chromatin. One type of protein that acts downstream of chromatin editing is the reader-effector, which bridges epigenetic marks with biological effects like gene regulation. As the name suggests, a reader-effector protein is generally composed of a reader domain and an effector domain. Reader domains directly bind epigenetic marks, while effector domains often recruit protein complexes that mediate transcription, chromatin remodeling, and DNA repair. In this chapter, we discuss the role of reader-effectors in driving the outputs of epigenome editing and highlight instances where abnormal and context-specific reader-effectors might impair the effects of epigenome editing. Lastly, we discuss how engineered reader-effectors may complement the epigenome editing toolbox to achieve robust and reliable gene regulation.},
}

Animals
Humans
Chromatin/genetics/metabolism
Chromatin Assembly and Disassembly
CRISPR-Cas Systems
*Epigenesis, Genetic
*Epigenome
Epigenomics/methods
*Gene Editing/methods
Gene Expression Regulation

@article {pmid39012590,
year = {2024},
author = {Noviello, G and Gjaltema, RAF},
title = {Fine-Tuning the Epigenetic Landscape: Chemical Modulation of Epigenome Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {57-77},
pmid = {39012590},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; *Epigenesis, Genetic/drug effects ; Epigenome ; CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; Epigenomics/methods ; Animals ; },
abstract = {Epigenome editing has emerged as a powerful technique for targeted manipulation of the chromatin and transcriptional landscape, employing designer DNA binding domains fused with effector domains, known as epi-editors. However, the constitutive expression of dCas9-based epi-editors presents challenges, including off-target activity and lack of temporal resolution. Recent advancements of dCas9-based epi-editors have addressed these limitations by introducing innovative switch systems that enable temporal control of their activity. These systems allow precise modulation of gene expression over time and offer a means to deactivate epi-editors, thereby reducing off-target effects associated with prolonged expression. The development of novel dCas9 effectors regulated by exogenous chemical signals has revolutionized temporal control in epigenome editing, significantly expanding the researcher's toolbox. Here, we provide a comprehensive review of the current state of these cutting-edge systems and specifically discuss their advantages and limitations, offering context to better understand their capabilities.},
}

*Gene Editing/methods
Humans
*Epigenesis, Genetic/drug effects
Epigenome
CRISPR-Cas Systems
Chromatin/genetics/metabolism
Epigenomics/methods
Animals

@article {pmid39012589,
year = {2024},
author = {Yagci, ZB and Kelkar, GR and Johnson, TJ and Sen, D and Keung, AJ},
title = {Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {23-55},
pmid = {39012589},
issn = {1940-6029},
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Gene Editing/methods ; Epigenome ; Epigenomics/methods ; Epigenesis, Genetic ; Genetic Loci ; Animals ; CRISPR-Cas Systems ; },
abstract = {The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors (EEs) enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus-specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here, we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to: account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus-specificity by considering concentration, affinity, avidity, and sequestration effects.},
}

Humans
*Chromatin/genetics/metabolism
*Gene Editing/methods
Epigenome
Epigenomics/methods
Epigenesis, Genetic
Genetic Loci
Animals
CRISPR-Cas Systems

@article {pmid39012588,
year = {2024},
author = {Rots, MG and Jeltsch, A},
title = {Development of Locus-Directed Editing of the Epigenome from Basic Mechanistic Engineering to First Clinical Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {3-20},
pmid = {39012588},
issn = {1940-6029},
mesh = {Animals ; Humans ; *Chromatin/genetics/metabolism ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; Epigenomics/methods ; *Gene Editing/methods ; },
abstract = {The introduction of CRISPR/Cas systems has resulted in a strong impulse for the field of gene-targeted epigenome/epigenetic reprogramming (EpiEditing), where EpiEditors consisting of a DNA binding part for targeting and an enzymatic part for rewriting of chromatin modifications are applied in cells to alter chromatin modifications at targeted genome loci in a directed manner. Pioneering studies preceding this era indicated causal relationships of chromatin marks instructing gene expression. The accumulating evidence of chromatin reprogramming of a given genomic locus resulting in gene expression changes opened the field for mainstream applications of this technology in basic and clinical research. The growing knowledge on chromatin biology and application of EpiEditing tools, however, also revealed a lack of predictability of the efficiency of EpiEditing in some cases. In this perspective, the dependence of critical parameters such as specificity, effectivity, and sustainability of EpiEditing on experimental settings and conditions including the expression levels and expression times of the EpiEditors, their chromatin binding affinity and specificity, and the crosstalk between EpiEditors and cellular epigenome modifiers are discussed. These considerations highlight the intimate connection between the outcome of epigenome reprogramming and the details of the technical approaches toward EpiEditing, which are the main topic of this volume of Methods in Molecular Biology. Once established in a fully functional "plug-and-play" mode, EpiEditing will allow to better understand gene expression control and to translate such knowledge into therapeutic tools. These expectations are beginning to be met as shown by various in vivo EpiEditing applications published in recent years, several companies aiming to exploit the therapeutic power of EpiEditing and the first clinical trial initiated.},
}

Animals
Humans
*Chromatin/genetics/metabolism
*CRISPR-Cas Systems
*Epigenesis, Genetic
*Epigenome
Epigenomics/methods
*Gene Editing/methods

@article {pmid39012436,
year = {2024},
author = {Wang, F and Ma, S and Zhang, S and Ji, Q and Hu, C},
title = {CRISPR beyond: harnessing compact RNA-guided endonucleases for enhanced genome editing.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {39012436},
issn = {1869-1889},
abstract = {The CRISPR-Cas system, an adaptive immunity system in prokaryotes designed to combat phages and foreign nucleic acids, has evolved into a groundbreaking technology enabling gene knockout, large-scale gene insertion, base editing, and nucleic acid detection. Despite its transformative impact, the conventional CRISPR-Cas effectors face a significant hurdle-their size poses challenges in effective delivery into organisms and cells. Recognizing this limitation, the imperative arises for the development of compact and miniature gene editors to propel advancements in gene-editing-related therapies. Two strategies were accepted to develop compact genome editors: harnessing OMEGA (Obligate Mobile Element-guided Activity) systems, or engineering the existing CRISPR-Cas system. In this review, we focus on the advances in miniature genome editors based on both of these strategies. The objective is to unveil unprecedented opportunities in genome editing by embracing smaller, yet highly efficient genome editors, promising a future characterized by enhanced precision and adaptability in the genetic interventions.},
}

@article {pmid39012160,
year = {2024},
author = {Zhang, J and Zhao, G and Bai, W and Chen, Y and Zhang, Y and Li, F and Wang, M and Shen, Y and Wang, Y and Wang, X and Li, C},
title = {A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00017},
pmid = {39012160},
issn = {2161-5063},
abstract = {Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae, a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.},
}

@article {pmid39012150,
year = {2024},
author = {Peng, S and Xu, Y and Qu, H and Nong, F and Shu, F and Yuan, G and Ruan, L and Zheng, D},
title = {Trojan Horse virus delivering CRISPR-AsCas12f1 controls plant bacterial wilt caused by Ralstonia solanacearum.},
journal = {mBio},
volume = {},
number = {},
pages = {e0061924},
doi = {10.1128/mbio.00619-24},
pmid = {39012150},
issn = {2150-7511},
abstract = {Plant bacterial wilt caused by Ralstonia solanacearum results in huge losses. Accordingly, developing an effective control method for this disease is urgently required. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential biocontrol solution. A filamentous phage RSCq that infects R. solanacearum was isolated in this study through genome mining. We constructed engineered filamentous phages based on RSCq by employing our proposed approach with wide applicability to non-model phages, enabling the exogenous genes delivery into bacterial cells. CRISPR-AsCas12f1 is a miniature class 2 type V-F CRISPR-Cas system. A CRISPR-AsCas12f1-based gene editing system that targets the key virulence regulator gene hrpB was developed, generating the engineered phage RSCqCRISPR-Cas. Similar to the Greek soldiers in the Trojan Horse, our findings demonstrated that the engineered phage-delivered CRISPR-Cas system could disarm the key "weapon," hrpB, of R. solanacearum, in medium and plants. Remarkably, pretreatment with RSCqCRISPR-Cas significantly controlled tobacco bacterial wilt, highlighting the potential of engineered filamentous phages as promising biocontrol agents against plant bacterial diseases.IMPORTANCEBacterial disease, one of the major plant diseases, causes huge food and economic losses. Phage therapy, an environmentally friendly control strategy, has been frequently reported in plant bacterial disease control. However, host specificity, sensitivity to ultraviolet light and certain conditions, and bacterial resistance to phage impede the widespread application of phage therapy in crop production. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential solution to overcome the limitations of lytic phage biocontrol. This study developed a genetic engineering approach with wide applicability to non-model filamentous phages and proved the application possibility of engineered phage-based gene delivery in plant bacterial disease biocontrol for the first.},
}

@article {pmid39011680,
year = {2024},
author = {Jia, X and Lu, K and Liang, X},
title = {Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout.},
journal = {Journal of Zhejiang University. Science. B},
volume = {25},
number = {7},
pages = {605-616},
pmid = {39011680},
issn = {1862-1783},
support = {31972809//the National Natural Science Foundation of China/ ; 2022BBA0051//the Key Research & Development Program of Hubei Province/ ; },
mesh = {Animals ; *Oryzias/genetics ; *Receptors, Neuropeptide Y/genetics ; *Larva/genetics ; *Gene Knockout Techniques ; *Digestion ; CRISPR-Cas Systems ; Feeding Behavior ; Fish Proteins/genetics/metabolism ; },
abstract = {Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br[-/-] Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br[-/-] medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br[-/-] medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.},
}

Animals
*Oryzias/genetics
*Receptors, Neuropeptide Y/genetics
*Larva/genetics
*Gene Knockout Techniques
*Digestion
CRISPR-Cas Systems
Feeding Behavior
Fish Proteins/genetics/metabolism

@article {pmid39011146,
year = {2024},
author = {Yu, T and Huang, J and Huang, X and Hao, J and Zhang, P and Guo, T and Bao, G and Li, G},
title = {Sub-MIC antibiotics increased the fitness cost of CRISPR-Cas in Acinetobacter baumannii.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1381749},
pmid = {39011146},
issn = {1664-302X},
abstract = {INTRODUCTION: The escalating prevalence of bacterial resistance, particularly multidrug-resistant bacteria like Acinetobacter baumannii, has become a significant global public health concern. The CRISPR-Cas system, a crucial defense mechanism in bacteria against foreign genetic elements, provides a competitive advantage. Type I-Fb and Type I-Fa are two subtypes of CRISPR-Cas systems that were found in A. baumannii, and the I-Fb CRISPR-Cas system regulates antibiotic resistance in A. baumannii. However, it is noteworthy that a majority of clinical isolates of A. baumannii lack or have incomplete CRISPR-Cas systems and most of them are multidrug-resistant. In light of this, our study aimed to examine the impact of antibiotic pressure on the fitness cost of the I-Fb CRISPR-Cas system in A. baumannii.

METHODS AND RESULTS: In the study, we conducted in vitro competition experiments to investigate the influence of sub-minimum inhibitory concentration (sub-MIC) on the CRISPR-Cas systems' fitness cost in A. baumannii. We found that the fitness cost of the CRISPR-Cas system was increased under sub-MIC conditions. The expression of CRISPR-Cas-related genes was decreased, while the conjugation frequency was increased in AB43 under sub-MIC conditions. Through metabolomic analysis, we identified that sub-MIC conditions primarily affected energy metabolism pathways. In particular, we observed increased carbon metabolism, nitrogen metabolism, and intracellular ATP. Notably, the CRISPR-Cas system demonstrated resistance to the efflux pump-mediated resistance. Furthermore, the expression of efflux pump-related genes was increased under sub-MIC conditions.

CONCLUSION: Our findings suggest that the I-Fb CRISPR-Cas system confers a significant competitive advantage in A. baumanni. However, under sub-MIC conditions, its function and the ability to inhibit the energy required for efflux pumps are reduced, resulting in an increased fitness cost and loss of competitive advantage.},
}

@article {pmid39011108,
year = {2024},
author = {Hosur, V and Erhardt, V and Hartig, E and Lorenzo, K and Megathlin, H and Tarchini, B},
title = {Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.},
journal = {Research square},
volume = {},
number = {},
pages = {},
pmid = {39011108},
issn = {2693-5015},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 CA265978/CA/NCI NIH HHS/United States ; R01 DC015242/DC/NIDCD NIH HHS/United States ; R01 DC018304/DC/NIDCD NIH HHS/United States ; },
abstract = {The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.},
}

@article {pmid39009678,
year = {2024},
author = {Lin, J and Jin, M and Yang, D and Li, Z and Zhang, Y and Xiao, Q and Wang, Y and Yu, Y and Zhang, X and Shao, Z and Shi, L and Zhang, S and Chen, WJ and Wang, N and Wu, S and Yang, H and Xu, C and Li, G},
title = {Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5927},
pmid = {39009678},
issn = {2041-1723},
mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics/therapy ; *Dystrophin/genetics/metabolism ; *Exons/genetics ; Humans ; Male ; *Gene Editing/methods ; *Disease Models, Animal ; Mice ; *Adenine/metabolism ; *CRISPR-Cas Systems ; Muscle, Skeletal/metabolism ; Dependovirus/genetics ; Genetic Therapy/methods ; },
abstract = {Duchenne muscular dystrophy (DMD) affecting 1 in 3500-5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient's DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.},
}

Animals
*Muscular Dystrophy, Duchenne/genetics/therapy
*Dystrophin/genetics/metabolism
*Exons/genetics
Humans
Male
*Gene Editing/methods
*Disease Models, Animal
Mice
*Adenine/metabolism
*CRISPR-Cas Systems
Muscle, Skeletal/metabolism
Dependovirus/genetics
Genetic Therapy/methods

@article {pmid39009379,
year = {2024},
author = {Sinan, S and Appleby, NM and Chou, CW and Finkelstein, IJ and Russell, R},
title = {Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.},
journal = {RNA (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1261/rna.080088.124},
pmid = {39009379},
issn = {1469-9001},
abstract = {CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may pre-order the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA binding specificity is robust to changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.},
}

@article {pmid39007834,
year = {2024},
author = {Li, D and Cheng, K and Zhu, X},
title = {Construction and Identification of a Novel Mice Model of Microphthalmia.},
journal = {Translational vision science & technology},
volume = {13},
number = {7},
pages = {11},
pmid = {39007834},
issn = {2164-2591},
mesh = {Animals ; *Microphthalmos/genetics/pathology/metabolism ; *Disease Models, Animal ; Mice ; *Zinc Finger Protein Gli3/genetics/metabolism ; Hedgehog Proteins/genetics/metabolism ; Lens, Crystalline/metabolism/pathology ; Signal Transduction ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; },
abstract = {PURPOSE: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia.

METHODS: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice.

RESULTS: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation.

CONCLUSIONS: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease.

TRANSLATIONAL RELEVANCE: This study developed a novel animal model to facilitate clinical research on microphthalmia.},
}

Animals
*Microphthalmos/genetics/pathology/metabolism
*Disease Models, Animal
Mice
*Zinc Finger Protein Gli3/genetics/metabolism
Hedgehog Proteins/genetics/metabolism
Lens, Crystalline/metabolism/pathology
Signal Transduction
CRISPR-Cas Systems
Mice, Inbred C57BL
Nerve Tissue Proteins

@article {pmid39007743,
year = {2024},
author = {Li, S and Jin, B and Ma, Y and Yang, X and Fan, J and Xie, Y and Xu, C and Dai, X and Wang, M and Liu, Q and Fu, T and Liu, Y and Tan, W},
title = {Proteome Fishing for CRISPR/Cas12a-Based Orthogonal Multiplex Aptamer Sensing.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {29},
pages = {19874-19885},
doi = {10.1021/jacs.4c03061},
pmid = {39007743},
issn = {1520-5126},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Carcinoma, Non-Small-Cell Lung/diagnosis/blood ; *CRISPR-Cas Systems ; Proteome/analysis ; Lung Neoplasms/blood/diagnosis ; Biomarkers, Tumor/blood ; Magnetite Nanoparticles/chemistry ; Protein Corona/chemistry ; },
abstract = {Detection of serum protein biomarkers is extremely challenging owing to the superior complexity of serum. Here, we report a method of proteome fishing from the serum. It uses a magnetic nanoparticle-protein corona and a multiplexed aptamer panel, which we incubated with the nanoparticle-protein corona for biomarker recognition. To transfer protein biomarker detection to aptamer detection, we established a CRISPR/Cas12a-based orthogonal multiplex aptamer sensing (COMPASS) platform by profiling the aptamers of protein corona with clinical nonsmall cell lung cancer (NSCLC) serum samples. Furthermore, we determined the four out of nine (FOON) panel (including HE4, NSE, AFP, and VEGF165) to be the most cost-effective and accurate panel for COMPASS in NSCLC diagnosis. The diagnostic accuracy of NSCLC by the FOON panel with internal and external cohorts was 95.56% (ROC-AUC = 99.40%) and 89.58% (ROC-AUC = 95.41%), respectively. Our developed COMPASS technology circumvents the otherwise challenging multiplexed serum protein amplification problem and avoids aptamer degradation in serum. Therefore, this novel COMPASS could lead to the development of a facile, cost-effective, intelligent, and high-throughput diagnostic platform for large-cohort cancer screening.},
}

*Aptamers, Nucleotide/chemistry
Humans
*Carcinoma, Non-Small-Cell Lung/diagnosis/blood
*CRISPR-Cas Systems
Proteome/analysis
Lung Neoplasms/blood/diagnosis
Biomarkers, Tumor/blood
Magnetite Nanoparticles/chemistry
Protein Corona/chemistry

@article {pmid39005118,
year = {2024},
author = {Ng, MG and Tan, HY and Ng, PY and Koh, RY and Voon, KGL and Chye, SM},
title = {Cancer Antibody Engineering: Comparison of Mammalian, Yeast, Bacterial, Plants, Cell-free and Hybridoma Expression Systems.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010307146240626080746},
pmid = {39005118},
issn = {1873-4316},
abstract = {BACKGROUND: Cancer is a significant issue worldwide. Generally, commercially available treatments, such as surgery, radiotherapy, and chemotherapy, are associated with undesirable complications. Hence, immunotherapy serves as a crucial alternative to those treatment options.

OBJECTIVE: This modality is aimed to boost the immune system through the application of engineered antibodies, which can be produced using recombinant DNA technology.

RESULTS: The discussion of the technologies leads to an introduction of the single-chain variable fragment (scFv). Thereafter, the advantages, disadvantages, and challenges associated with different expression systems, such as mammalian cells, yeast cells, bacterial cells, plant cells, and phage display were discussed comprehensively.

CONCLUSION: Furthermore, conventional approaches such as hybridoma and modern approaches such as cell-free protein synthesis (CFPS) and simple colony assays are included. In short, this article has compiled evidence relating to each display system and may serve as a reference for those who aim to explore antibody engineering using one of the methods listed in this article.},
}

@article {pmid39005116,
year = {2024},
author = {Tee, PYE and Chu, SYC and Kok, CCY and Foo, M and Tan, CZJ and Foo, JB and Mohd Hatta, FH and Tan, LKS and Hamzah, S},
title = {Applications of CRISPR in Parasitology.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010316710240626042205},
pmid = {39005116},
issn = {1873-4316},
abstract = {Clustered Regions of Interspersed Palindromic Repeat (CRISPR)-based techniques have been utilized in various research areas, including agriculture, biotechnology, and medicine. With the use of a short sequence guide RNA and CRISPR-associated (Cas) protein, this technique allows for robust, site-specific manipulation of the genome, aiding researchers in making important biomedical discoveries and scientific advancements. In this review, we explored the applications of CRISPR/Cas systems in the field of parasitology for the identification and validation of novel functional genes, diagnosis of parasitic infections, reduction of parasite virulence, and the disruption of disease transmission. We also discussed how CRISPR can be used for the development of therapeutics, vaccines, and drug discovery. Furthermore, the challenges and future perspectives of this technology are also highlighted.},
}

@article {pmid39003402,
year = {2024},
author = {Hu, T and Ji, Q and Ke, X and Zhou, H and Zhang, S and Ma, S and Yu, C and Ju, W and Lu, M and Lin, Y and Ou, Y and Zhou, Y and Xiao, Y and Xu, C and Hu, C},
title = {Repurposing Type I-A CRISPR-Cas3 for a robust diagnosis of human papillomavirus (HPV).},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {858},
pmid = {39003402},
issn = {2399-3642},
support = {23-0178-A0003//National University of Singapore (NUS)/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Papillomavirus Infections/diagnosis/virology/genetics ; Papillomaviridae/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Human Papillomavirus Viruses ; },
abstract = {R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes-responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE,). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.},
}

*CRISPR-Cas Systems
Humans
*Papillomavirus Infections/diagnosis/virology/genetics
Papillomaviridae/genetics
CRISPR-Associated Proteins/metabolism/genetics
Human Papillomavirus Viruses

@article {pmid39003336,
year = {2024},
author = {Shimamori, Y and Tan, XE and Li, FY and Nishikawa, Y and Watanabe, S and Sasahara, T and Miyanaga, K and Aiba, Y and Veeranarayanan, S and Thitiananpakorn, K and Nguyen, HM and Batbold, A and Nayanjin, T and Lian, AYS and Hossain, S and Kawaguchi, T and Alessa, O and Kumwenda, G and Sarangi, J and Revilleza, JEC and Baranwal, P and Arbaah, M and Maniruzzaman, and Yi, L and Duyen, HTM and Sugano, T and Sultana, S and Faruk, MO and Hidaka, Y and Thu, M and Shimojyo, T and Kiga, K and Cui, L},
title = {Efficient synthesis of CRISPR-Cas13a-antimicrobial capsids against MRSA facilitated by silent mutation incorporation.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {16225},
pmid = {39003336},
issn = {2045-2322},
support = {22K15673//JSPS KAKENHI/ ; 22K19386//JSPS KAKENHI/ ; 21K19666//JSPS KAKENHI/ ; 21K19488//JSPS KAKENHI/ ; JP21fk0108497//Japan Agency for Medical Research and Development/ ; JP23wm0325065//Japan Agency for Medical Research and Development/ ; JP21ae0121045//Japan Agency for Medical Research and Development/ ; JPJ009237//Moonshot Research and Development Program/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/drug effects ; *CRISPR-Cas Systems ; *Capsid/metabolism ; *Mutation ; Anti-Bacterial Agents/pharmacology ; Bacteriophages/genetics ; },
abstract = {In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.},
}

*Methicillin-Resistant Staphylococcus aureus/genetics/drug effects
*CRISPR-Cas Systems
*Capsid/metabolism
*Mutation
Anti-Bacterial Agents/pharmacology
Bacteriophages/genetics

@article {pmid39003305,
year = {2024},
author = {Guo, X and Hong, P and Xiong, S and Yan, Y and Xie, H and Guan, JS},
title = {Kdm4a is an activity downregulated barrier to generate engrams for memory separation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5887},
pmid = {39003305},
issn = {2041-1723},
support = {32225023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31970903//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Hippocampus/metabolism ; Mice ; *Memory/physiology ; *TRPM Cation Channels/metabolism/genetics ; Jumonji Domain-Containing Histone Demethylases/metabolism/genetics ; Humans ; Down-Regulation/genetics ; Neurons/metabolism ; Male ; Mice, Inbred C57BL ; Rats ; CRISPR-Cas Systems ; RNA-Binding Proteins/metabolism/genetics ; Neuronal Plasticity/genetics ; HEK293 Cells ; Histone Demethylases ; },
abstract = {Memory engrams are a subset of learning activated neurons critical for memory recall, consolidation, extinction and separation. While the transcriptional profile of engrams after learning suggests profound neural changes underlying plasticity and memory formation, little is known about how memory engrams are selected and allocated. As epigenetic factors suppress memory formation, we developed a CRISPR screening in the hippocampus to search for factors controlling engram formation. We identified histone lysine-specific demethylase 4a (Kdm4a) as a negative regulator for engram formation. Kdm4a is downregulated after neural activation and controls the volume of mossy fiber boutons. Mechanistically, Kdm4a anchors to the exonic region of Trpm7 gene loci, causing the stalling of nascent RNAs and allowing burst transcription of Trpm7 upon the dismissal of Kdm4a. Furthermore, the YTH domain containing protein 2 (Ythdc2) recruits Kdm4a to the Trpm7 gene and stabilizes nascent RNAs. Reducing the expression of Kdm4a in the hippocampus via genetic manipulation or artificial neural activation facilitated the ability of pattern separation in rodents. Our work indicates that Kdm4a is a negative regulator of engram formation and suggests a priming state to generate a separate memory.},
}

Animals
*Hippocampus/metabolism
Mice
*Memory/physiology
*TRPM Cation Channels/metabolism/genetics
Jumonji Domain-Containing Histone Demethylases/metabolism/genetics
Humans
Down-Regulation/genetics
Neurons/metabolism
Male
Mice, Inbred C57BL
Rats
CRISPR-Cas Systems
RNA-Binding Proteins/metabolism/genetics
Neuronal Plasticity/genetics
HEK293 Cells
Histone Demethylases

@article {pmid39003282,
year = {2024},
author = {Jiao, C and Peeck, NL and Yu, J and Ghaem Maghami, M and Kono, S and Collias, D and Martinez Diaz, SL and Larose, R and Beisel, CL},
title = {TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5909},
pmid = {39003282},
issn = {2041-1723},
support = {865973//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 031B0989//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; 16LW0132//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; 031B0989//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; 16LW0132//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; RNA, Ribosomal, 16S/genetics/metabolism ; DNA/metabolism/genetics ; RNA/metabolism/genetics ; DNA Cleavage ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/metabolism/genetics ; Gene Editing/methods ; Endodeoxyribonucleases/metabolism/genetics ; Francisella/genetics ; },
abstract = {Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
CRISPR-Associated Proteins/metabolism/genetics
RNA, Ribosomal, 16S/genetics/metabolism
DNA/metabolism/genetics
RNA/metabolism/genetics
DNA Cleavage
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacterial Proteins/metabolism/genetics
Gene Editing/methods
Endodeoxyribonucleases/metabolism/genetics
Francisella/genetics

@article {pmid39002873,
year = {2024},
author = {Sheykholeslami, N and Mirzaei, H and Nami, Y and Khandaghi, J and Javadi, A},
title = {Ecological and evolutionary dynamics of CRISPR-Cas systems in Clostridium botulinum: Insights from genome mining and comparative analysis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {123},
number = {},
pages = {105638},
doi = {10.1016/j.meegid.2024.105638},
pmid = {39002873},
issn = {1567-7257},
abstract = {Understanding the prevalence and distribution of CRISPR-Cas systems across different strains can illuminate the ecological and evolutionary dynamics of Clostridium botulinum populations. In this study, we conducted genome mining to characterize the CRISPR-Cas systems of C. botulinum strains. Our analysis involved retrieving complete genome sequences of these strains and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Subsequently, we performed an analysis of homology in spacer sequences from identified CRISPR arrays to investigate and characterize the range of targeted phages and plasmids. Additionally, we investigated the evolutionary trajectory of C. botulinum strains under selective pressures from foreign invasive DNA. Our findings revealed that 306 strains possessed complete CRISPR-Cas structures, comprising 58% of the studied C. botulinum strains. Secondary structure prediction of consensus repeats indicated that subtype II-C, with longer stems compared to subtypes ID and IB, tended to form more stable RNA secondary structures. Moreover, protospacer motif analysis demonstrated that strains with subtype IB CRISPR-Cas systems exhibited 5'-CGG-3', 5'-CC-3', and 5'-CAT-3' motifs in the 3' flanking regions of protospacers. The diversity observed in CRISPR-Cas systems indicated their classification into subtypes IB, ID, II-C, III-B, and III-D. Furthermore, our results showed that systems with subtype ID and III-D frequently harbored similar spacer patterns. Moreover, analysis of spacer sequences homology with phage and prophage genomes highlighted the specific activities exhibited by subtype IB and III-B against phages and plasmids, providing valuable insights into the functional specialization within these systems.},
}

@article {pmid39001969,
year = {2024},
author = {Khudhair, YI and Alsultan, A and Hussain, MH and Ayez, FJ},
title = {Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.},
journal = {Tropical animal health and production},
volume = {56},
number = {6},
pages = {211},
pmid = {39001969},
issn = {1573-7438},
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/isolation & purification/genetics ; *Diarrhea/veterinary/virology/diagnosis ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; *Cattle Diseases/virology/diagnosis ; Coronavirus Infections/veterinary/virology/diagnosis ; },
abstract = {Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.},
}

Animals
Cattle
*Coronavirus, Bovine/isolation & purification/genetics
*Diarrhea/veterinary/virology/diagnosis
*CRISPR-Cas Systems
*Sensitivity and Specificity
*Cattle Diseases/virology/diagnosis
Coronavirus Infections/veterinary/virology/diagnosis

@article {pmid39001947,
year = {2024},
author = {Yoo, M and Bunkowski, K and Lie, A and Junn, E},
title = {Regulation of MicroRNA-4697-3p by Parkinson's disease-associated SNP rs329648 and its impact on SNCA112 mRNA.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {797},
pmid = {39001947},
issn = {1573-4978},
mesh = {Humans ; Alleles ; *alpha-Synuclein/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression Regulation/genetics ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; *MicroRNAs/genetics/metabolism ; *Parkinson Disease/genetics/metabolism ; *Polymorphism, Single Nucleotide/genetics ; *RNA, Messenger/genetics/metabolism ; },
abstract = {BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a multifaceted genetic foundation. Genome-Wide Association Studies (GWAS) have played a crucial role in pinpointing genetic variants linked to PD susceptibility. Current study aims to delve into the mechanistic aspects through which the PD-associated Single Nucleotide Polymorphism (SNP) rs329648, identified in prior GWAS, influences the pathogenesis of PD.

METHODS AND RESULTS: Employing the CRISPR/Cas9-mediated genome editing mechanism, we demonstrated the association of the disease-associated allele of rs329648 with increased expression of miR-4697-3p in differentiated SH-SY5Y cells. We revealed that miR-4697-3p contributes to the formation of high molecular weight complexes of α-Synuclein (α-Syn), indicative of α-Syn aggregate formation, as evidenced by Western blot analysis. Furthermore, our study unveiled that miR-4697-3p elevates SNCA112 mRNA levels. The resultant protein product, α-Syn 112, a variant of α-Syn with 112 amino acids, is recognized for augmenting α-Syn aggregation. Notably, this regulatory effect minimally impacts the levels of full-length SNCA140 mRNA, as evidenced by qRT-PCR. Additionally, we observed a correlation between the disease-associated allele and miR-4697-3p with increased cell death, substantiated by assessments including cell viability assays, alterations in cell morphology, and TUNEL assays.

CONCLUSION: Our research reveals that the disease-associated allele of rs329648 is linked to higher levels of miR-4697-3p. This increase in miR-4697-3p leads to elevated SNCA112 mRNA levels, consequently promoting the formation of α-Syn aggregates. Furthermore, miR-4697-3p appears to play a role in increased cell death, potentially contributing to the pathogenesis of PD.},
}

Humans
Alleles
*alpha-Synuclein/genetics/metabolism
Cell Line, Tumor
CRISPR-Cas Systems/genetics
Gene Editing/methods
Gene Expression Regulation/genetics
Genetic Predisposition to Disease
Genome-Wide Association Study
*MicroRNAs/genetics/metabolism
*Parkinson Disease/genetics/metabolism
*Polymorphism, Single Nucleotide/genetics
*RNA, Messenger/genetics/metabolism

@article {pmid39001804,
year = {2024},
author = {Wang, H and Wang, H and Pian, H and Su, F and Tang, F and Chen, D and Chen, J and Wen, Y and Le, XC and Li, Z},
title = {CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor for Point-of-Care Detection of RNA.},
journal = {Analytical chemistry},
volume = {96},
number = {29},
pages = {12022-12029},
doi = {10.1021/acs.analchem.4c02087},
pmid = {39001804},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems ; *Hydrogels/chemistry ; *DNA/chemistry/analysis ; *SARS-CoV-2/genetics/isolation & purification ; Point-of-Care Systems ; RNA, Viral/analysis ; Biosensing Techniques ; COVID-19/diagnosis/virology ; RNA/analysis ; },
abstract = {Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the trans-cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.},
}

Humans
*CRISPR-Cas Systems
*Hydrogels/chemistry
*DNA/chemistry/analysis
*SARS-CoV-2/genetics/isolation & purification
Point-of-Care Systems
RNA, Viral/analysis
Biosensing Techniques
COVID-19/diagnosis/virology
RNA/analysis

@article {pmid39000440,
year = {2024},
author = {Taghdiri, M and Mussolino, C},
title = {Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000440},
issn = {1422-0067},
support = {101072427//European Union's Horizon 2020/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *Gene Transfer Techniques ; Animals ; Genetic Vectors/genetics ; Viruses/genetics ; },
abstract = {Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.},
}

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

@article {pmid39000386,
year = {2024},
author = {Chen, L and Elizalde, M and Dubois, LJ and Roeth, AA and Neumann, UP and Olde Damink, SWM and Schaap, FG and Alvarez-Sola, G},
title = {GAL3ST1 Deficiency Reduces Epithelial-Mesenchymal Transition and Tumorigenic Capacity in a Cholangiocarcinoma Cell Line.},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000386},
issn = {1422-0067},
mesh = {*Cholangiocarcinoma/metabolism/pathology/genetics ; Humans ; *Epithelial-Mesenchymal Transition/genetics ; *Bile Duct Neoplasms/metabolism/pathology/genetics ; Cell Line, Tumor ; *Cell Proliferation ; Sulfotransferases/metabolism/genetics/deficiency ; Sulfoglycosphingolipids/metabolism ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism/pathology ; },
abstract = {Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1-deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.},
}

*Cholangiocarcinoma/metabolism/pathology/genetics
Humans
*Epithelial-Mesenchymal Transition/genetics
*Bile Duct Neoplasms/metabolism/pathology/genetics
Cell Line, Tumor
*Cell Proliferation
Sulfotransferases/metabolism/genetics/deficiency
Sulfoglycosphingolipids/metabolism
CRISPR-Cas Systems
Carcinogenesis/genetics/metabolism/pathology

@article {pmid39000346,
year = {2024},
author = {García-López, M and Jiménez-Vicente, L and González-Jabardo, R and Dorado, H and Gómez-Manjón, I and Martín, MÁ and Ayuso, C and Arenas, J and Gallardo, ME},
title = {Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene.},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000346},
issn = {1422-0067},
support = {PI15/00484, CP16/00046, PI18/00151 PI21/00162, CPII21/00011, FI19/00043//Instituto de Salud Carlos III/ ; PCI2022-132920//Agencia Estatal de Investigación/ ; PI15/00484, CP16/00046, and PI18/00151//European Regional Development Fund/ ; PI21/00162 and CPII21/00011//European Union/ ; 2021/0146; 2022/0152; 2023/0121//Organización Nacional de Ciegos Españoles/ ; },
mesh = {Humans ; *Optic Atrophy, Autosomal Dominant/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; *GTP Phosphohydrolases/genetics/metabolism ; *Retinal Ganglion Cells/metabolism/pathology ; CRISPR-Cas Systems ; Gene Editing/methods ; Mutation ; Apoptosis/genetics ; Reactive Oxygen Species/metabolism ; Mitochondria/metabolism/genetics ; },
abstract = {Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.},
}

Humans
*Optic Atrophy, Autosomal Dominant/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism
*GTP Phosphohydrolases/genetics/metabolism
*Retinal Ganglion Cells/metabolism/pathology
CRISPR-Cas Systems
Gene Editing/methods
Mutation
Apoptosis/genetics
Reactive Oxygen Species/metabolism
Mitochondria/metabolism/genetics

@article {pmid39000251,
year = {2024},
author = {Kim, MS and Le, VT and Jung, YJ and Kang, KK and Cho, YG},
title = {OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000251},
issn = {1422-0067},
support = {NRF, 2022R1I1A3071999//This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF, 2022R1I1A3071999)./ ; },
mesh = {*Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ubiquitin-Protein Ligases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.},
}

*Oryza/genetics/microbiology
*CRISPR-Cas Systems
*Gene Editing/methods
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
*Ubiquitin-Protein Ligases/genetics/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified/genetics

@article {pmid39000067,
year = {2024},
author = {Ferrando, J and Miñana-Galbis, D and Picart, P},
title = {The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000067},
issn = {1422-0067},
support = {2021 DI 77//Pla de Doctorats Industrials del Departament de Recerca/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; *CRISPR-Cas Systems ; *alpha-Amylases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Secretory Pathway/genetics ; Promoter Regions, Genetic ; Recombinant Proteins/metabolism/genetics ; Gene Expression Regulation, Bacterial ; Lipoproteins ; Membrane Proteins ; },
abstract = {Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.},
}

*Bacillus subtilis/genetics/metabolism
*CRISPR-Cas Systems
*alpha-Amylases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Secretory Pathway/genetics
Promoter Regions, Genetic
Recombinant Proteins/metabolism/genetics
Gene Expression Regulation, Bacterial
Lipoproteins
Membrane Proteins

@article {pmid39000052,
year = {2024},
author = {Lee, KR and Yeo, Y and Lee, J and Kim, S and Im, C and Kim, I and Lee, J and Lee, SK and Suh, MC and Kim, HU},
title = {Functional Characterization of the Effects of CsDGAT1 and CsDGAT2 on Fatty Acid Composition in Camelina sativa.},
journal = {International journal of molecular sciences},
volume = {25},
number = {13},
pages = {},
pmid = {39000052},
issn = {1422-0067},
support = {PJ01318501//Next-Generation BioGreen21 Program, SSAC/ ; NRF-2021R1A2C1006049//the Mid-Career Researcher Program of the National Research Foundation of Korea/ ; NRF-2020R1A2C2008175//the Mid-Career Researcher Program of the National Research Foundation of Korea/ ; },
mesh = {*Fatty Acids/metabolism ; *Plant Proteins/metabolism/genetics ; *Diacylglycerol O-Acyltransferase/metabolism/genetics ; *Seeds/metabolism/genetics ; *Brassicaceae/genetics/metabolism ; CRISPR-Cas Systems ; Triglycerides/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Mutation ; Gene Editing ; },
abstract = {Triacylglycerols (TAGs) are the storage oils of plant seeds, and these lipids provide energy for seed germination and valuable oils for human consumption. Three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) and phospholipid:diacylglycerol acyltransferases participate in the biosynthesis of TAGs. DGAT1 and DGAT2 participate in the biosynthesis of TAGs through the endoplasmic reticulum (ER) pathway. In this study, we functionally characterized CsDGAT1 and CsDGAT2 from camelina (Camelina sativa). Green fluorescent protein-fused CsDGAT1 and CsDGAT2 localized to the ER when transiently expressed in Nicotiana benthamiana leaves. To generate Csdgat1 and Csdgat2 mutants using the CRISPR/Cas9 system, camelina was transformed with a binary vector carrying Cas9 and the respective guide RNAs targeting CsDGAT1s and CsDGAT2s via the Agrobacterium-mediated floral dip method. The EDD1 lines had missense and nonsense mutations in the CsDGAT1 homoeologs, suggesting that they retained some CsDGAT1 function, and their seeds showed decreased eicosaenoic acid (C20:1) contents and increased C18:3 contents compared to the wild type (WT). The EDD2 lines had a complete knockout of all CsDGAT2 homoeologs and a slightly decreased C18:3 content compared to the WT. In conclusion, CsDGAT1 and CsDGAT2 have a small influence on the seed oil content and have an acyl preference for C20:1 and C18:3, respectively. This finding can be applied to develop oilseed plants containing high omega-3 fatty acids or high oleic acid.},
}

*Fatty Acids/metabolism
*Plant Proteins/metabolism/genetics
*Diacylglycerol O-Acyltransferase/metabolism/genetics
*Seeds/metabolism/genetics
*Brassicaceae/genetics/metabolism
CRISPR-Cas Systems
Triglycerides/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Mutation
Gene Editing

@article {pmid38997669,
year = {2024},
author = {Li, J and Wei, Q and Cheng, Y and Kong, D and Kong, Z and Ke, Y and Dang, X and Zhu, JK and Shimada, H and Miki, D},
title = {Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana.},
journal = {BMC plant biology},
volume = {24},
number = {1},
pages = {665},
pmid = {38997669},
issn = {1471-2229},
mesh = {*Arabidopsis/genetics ; *Gene Targeting/methods ; Transformation, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.},
}

*Arabidopsis/genetics
*Gene Targeting/methods
Transformation, Genetic
CRISPR-Associated Proteins/genetics/metabolism
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
Plants, Genetically Modified/genetics

@article {pmid38997592,
year = {2024},
author = {Mukhopadhyay, M},
title = {Multiplexed base editing.},
journal = {Nature methods},
volume = {21},
number = {7},
pages = {1144},
doi = {10.1038/s41592-024-02357-9},
pmid = {38997592},
issn = {1548-7105},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; },
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans

@article {pmid38997519,
year = {2024},
author = {Stoltzfus, MJ and Workman, RE and Keith, NC and Modell, JW},
title = {A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {38997519},
issn = {2058-5276},
support = {90094894//Rita Allen Foundation (RAF)/ ; R35GM142731//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {Many CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems, which provide bacteria with adaptive immunity against phages, are transcriptionally repressed in their native hosts. How CRISPR-Cas expression is induced as needed, for example, during a bacteriophage infection, remains poorly understood. In Streptococcus pyogenes, a non-canonical guide RNA tracr-L directs Cas9 to autorepress its own promoter. Here we describe a dynamic subpopulation of cells harbouring single mutations that disrupt Cas9 binding and cause CRISPR-Cas overexpression. Cas9 actively expands this population by elevating mutation rates at the tracr-L target site. Overexpressers show higher rates of memory formation, stronger potency of old memories and a larger memory storage capacity relative to wild-type cells, which are surprisingly vulnerable to phage infection. However, in the absence of phage, CRISPR-Cas overexpression reduces fitness. We propose that CRISPR-Cas overexpressers are critical players in phage defence, enabling bacterial populations to mount rapid transcriptional responses to phage without requiring transient changes in any one cell.},
}

@article {pmid38997291,
year = {2024},
author = {Zahraeifard, S and Xiao, Z and So, JY and Ahad, A and Montoya, S and Park, WY and Sornapudi, T and Andohkow, T and Read, A and Kedei, N and Koparde, V and Yang, H and Lee, M and Wong, N and Cam, M and Wang, K and Ruppin, E and Luo, J and Hollander, C and Yang, L},
title = {Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5873},
pmid = {38997291},
issn = {2041-1723},
mesh = {*Tumor Microenvironment/immunology ; Animals ; *Lymphocyte Activation Gene 3 Protein ; Mice ; Female ; Humans ; *Triple Negative Breast Neoplasms/immunology/pathology/genetics ; *Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; *Tuberous Sclerosis Complex 1 Protein/genetics/metabolism ; *B7-H1 Antigen/metabolism/genetics ; Neurofibromin 1/genetics/metabolism ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/immunology ; Inflammation/immunology ; CD4-Positive T-Lymphocytes/immunology ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.},
}

*Tumor Microenvironment/immunology
Animals
*Lymphocyte Activation Gene 3 Protein
Mice
Female
Humans
*Triple Negative Breast Neoplasms/immunology/pathology/genetics
*Immune Checkpoint Inhibitors/pharmacology/therapeutic use
*Tuberous Sclerosis Complex 1 Protein/genetics/metabolism
*B7-H1 Antigen/metabolism/genetics
Neurofibromin 1/genetics/metabolism
Cell Line, Tumor
CD8-Positive T-Lymphocytes/immunology
Inflammation/immunology
CD4-Positive T-Lymphocytes/immunology
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid38996645,
year = {2024},
author = {Hoepers, AM and Heinemann, JA and Zanatta, CB and Chu, P and Hiscox, TC and Agapito-Tenfen, SZ},
title = {Predicted multispecies unintended effects from outdoor genome editing.},
journal = {Ecotoxicology and environmental safety},
volume = {282},
number = {},
pages = {116707},
doi = {10.1016/j.ecoenv.2024.116707},
pmid = {38996645},
issn = {1090-2414},
abstract = {CRISPR/Cas9, a potent genetic engineering tool widely adopted in agriculture, is capable of introducing new characteristics into plants on a large scale and without conventional breeding methods. Despite its remarkable efficiency, concerns have arisen regarding unintended consequences in uncontrolled environments. Our aim was to assess potential activity in organisms that could be exposed to genome editing in uncontrolled environments. We developed three scenarios, using irrigation, fumigation and fertilization as delivery methods, based on outdoor uses in agriculture, namely pest and disease control. Using publicly available software (Cas-OFFinder, NCBI Genome Data Viewer and STRING), off-target effects were predicted in multiple species commonly found in the agroecosystem, including humans (16 of 38 (42 %) sampled). Metabolic enrichment analysis (gene IDs), by connecting off-target genes into a physiological network, predicted effects on the development of nervous and respiratory systems. Our findings emphasize the importance of exercising caution when considering the use of this genome editing in uncontrolled environments. Unintended genomic alterations may occur in unintended organisms, underscoring the significance of understanding potential hazards and implementing safety measures to protect human health and the environment.},
}

@article {pmid38996066,
year = {2024},
author = {Bilger, R and Migur, A and Wulf, A and Steglich, C and Urlaub, H and Hess, WR},
title = {A type III-Dv CRISPR-Cas system is controlled by the transcription factor RpaB and interacts with the DEAD-box RNA helicase CrhR.},
journal = {Cell reports},
volume = {43},
number = {7},
pages = {114485},
doi = {10.1016/j.celrep.2024.114485},
pmid = {38996066},
issn = {2211-1247},
abstract = {How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions. The cas operon promoter of this system is controlled by the light- and redox-responsive transcription factor RpaB binding to an HLR1 motif, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, the leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled with mass spectrometry analysis and site-directed mutagenesis revealed six residues involved in the CrhR-RNA interaction, with C371 being critically important. Thus, the expression of a type III-Dv CRISPR-Cas system is linked to the redox status of the photosynthetic cell at the transcriptional and post-transcriptional levels.},
}

@article {pmid38995475,
year = {2024},
author = {Chen, Y and Jiang, Q and Xue, Y and Chen, W and Hua, M},
title = {CRISPR-Cas9-mediated deletion enhancer of MECOM play a tumor suppressor role in ovarian cancer.},
journal = {Functional & integrative genomics},
volume = {24},
number = {4},
pages = {125},
pmid = {38995475},
issn = {1438-7948},
mesh = {Female ; Humans ; Animals ; *Ovarian Neoplasms/genetics/drug therapy/metabolism/pathology ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; *Azepines/pharmacology ; Enhancer Elements, Genetic ; Triazoles/pharmacology ; MDS1 and EVI1 Complex Locus Protein/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; },
abstract = {MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.},
}

Female
Humans
Animals
*Ovarian Neoplasms/genetics/drug therapy/metabolism/pathology
Mice
Cell Line, Tumor
*CRISPR-Cas Systems
*Azepines/pharmacology
Enhancer Elements, Genetic
Triazoles/pharmacology
MDS1 and EVI1 Complex Locus Protein/genetics/metabolism
Gene Expression Regulation, Neoplastic
Genes, Tumor Suppressor

@article {pmid38995331,
year = {2024},
author = {Kiyokawa, K and Sakuma, T and Moriguchi, K and Sugiyama, M and Akao, T and Yamamoto, T and Suzuki, K},
title = {Conversion of polyploid and alloploid Saccharomyces sensu stricto strains to leu2 mutants by genome DNA editing.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {416},
pmid = {38995331},
issn = {1432-0614},
support = {21H02167//Ministry of Education, Sports and Culture, Japan/ ; 24K09069//Japan Society for the Promotion of Science/ ; JPMJPF2010//Program on Open Innovation Platform with Enterprises, Research Institute and Academia/ ; },
mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Polyploidy ; *Mutation ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces/genetics ; Saccharomyces cerevisiae/genetics ; 3-Isopropylmalate Dehydrogenase/genetics/metabolism ; Genome, Fungal/genetics ; },
abstract = {A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2[-] mutants produced in this study will be deposited in several repository organizations. KEY POINTS: • All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains • Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains • The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.},
}

*Plasmids/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Polyploidy
*Mutation
Saccharomyces cerevisiae Proteins/genetics
Saccharomyces/genetics
Saccharomyces cerevisiae/genetics
3-Isopropylmalate Dehydrogenase/genetics/metabolism
Genome, Fungal/genetics

@article {pmid38994567,
year = {2024},
author = {Kim, H and Marraffini, LA},
title = {Cas9 interaction with the tracrRNA nexus modulates the repression of type II-A CRISPR-cas genes.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae597},
pmid = {38994567},
issn = {1362-4962},
support = {T32GM007739/GM/NIGMS NIH HHS/United States ; /NH/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
abstract = {Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity.},
}

@article {pmid38994558,
year = {2024},
author = {Xing, W and Li, D and Wang, W and Liu, JG and Chen, C},
title = {Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae604},
pmid = {38994558},
issn = {1362-4962},
support = {21877069//National Natural Science Foundation of China/ ; //Ministry of Agriculture/ ; 2022YFF1002801//National Key Research and Development Program/ ; //Beijing Frontier Research Center for Biological Structure/ ; //Tsinghua-Peking Joint Center for Life Sciences/ ; },
abstract = {CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.},
}

@article {pmid38994027,
year = {2024},
author = {Li, K and Li, M and Luo, Y and Zou, D and Li, Y and Mang, X and Zhang, Z and Li, P and Lu, Y and Miao, S and Song, W},
title = {Adeno-associated-virus-mediated delivery of CRISPR-CasRx induces efficient RNA knockdown in the mouse testis.},
journal = {Theranostics},
volume = {14},
number = {10},
pages = {3827-3842},
pmid = {38994027},
issn = {1838-7640},
mesh = {Male ; Animals ; *Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; *Testis/metabolism ; *Gene Knockdown Techniques/methods ; *Spermatogenesis/genetics ; RNA/genetics ; Genetic Vectors/genetics/administration & dosage ; },
abstract = {Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.},
}

Male
Animals
*Dependovirus/genetics
*CRISPR-Cas Systems/genetics
Mice
*Testis/metabolism
*Gene Knockdown Techniques/methods
*Spermatogenesis/genetics
RNA/genetics
Genetic Vectors/genetics/administration & dosage

@article {pmid38994004,
year = {2024},
author = {Tan, M and Liang, L and Liao, C and Zhou, Z and Long, S and Yi, X and Wang, C and Wei, C and Cai, J and Li, X and Wei, G},
title = {A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1362513},
pmid = {38994004},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Limit of Detection ; Klebsiella Infections/diagnosis/microbiology ; Recombinases/metabolism/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Associated Proteins/genetics ; DNA, Bacterial/genetics ; Endodeoxyribonucleases ; },
abstract = {The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.},
}

*Klebsiella pneumoniae/genetics/isolation & purification
*CRISPR-Cas Systems
*Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Humans
*Limit of Detection
Klebsiella Infections/diagnosis/microbiology
Recombinases/metabolism/genetics
Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Associated Proteins/genetics
DNA, Bacterial/genetics
Endodeoxyribonucleases

@article {pmid38994001,
year = {2024},
author = {Al-Fadhli, AH and Jamal, WY},
title = {Recent advances in gene-editing approaches for tackling antibiotic resistance threats: a review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1410115},
pmid = {38994001},
issn = {2235-2988},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Animals ; *Bacteria/drug effects/genetics ; *Bacterial Infections/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial/genetics ; Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic resistance, a known global health challenge, involves the flow of bacteria and their genes among animals, humans, and their surrounding environment. It occurs when bacteria evolve and become less responsive to the drugs designated to kill them, making infections harder to treat. Despite several obstacles preventing the spread of genes and bacteria, pathogens regularly acquire novel resistance factors from other species, which reduces their ability to prevent and treat such bacterial infections. This issue requires coordinated efforts in healthcare, research, and public awareness to address its impact on human health worldwide. This review outlines how recent advances in gene editing technology, especially CRISPR/Cas9, unveil a breakthrough in combating antibiotic resistance. Our focus will remain on the relationship between CRISPR/cas9 and its impact on antibiotic resistance and its related infections. Moreover, the prospects of this new advanced research and the challenges of adopting these technologies against infections will be outlined by exploring its different derivatives and discussing their advantages and limitations over others, thereby providing a corresponding reference for the control and prevention of the spread of antibiotic resistance.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
Animals
*Bacteria/drug effects/genetics
*Bacterial Infections/microbiology/drug therapy
*Anti-Bacterial Agents/pharmacology
Drug Resistance, Bacterial/genetics
Drug Resistance, Microbial/genetics

@article {pmid38992762,
year = {2024},
author = {Rao, J and Wang, X and Chen, X and Liu, Y and Jiang, J and Wang, Z},
title = {Multi-omics analysis reveals that Cas13d contributes to PI3K-AKT signaling and facilitates cell proliferation via PFKFB4 upregulation.},
journal = {Gene},
volume = {927},
number = {},
pages = {148760},
doi = {10.1016/j.gene.2024.148760},
pmid = {38992762},
issn = {1879-0038},
mesh = {Humans ; HeLa Cells ; *Proto-Oncogene Proteins c-akt/metabolism/genetics ; *Cell Proliferation ; *Phosphatidylinositol 3-Kinases/metabolism/genetics ; *Signal Transduction ; *Phosphofructokinase-2/genetics/metabolism ; *Up-Regulation ; *CRISPR-Cas Systems ; Proteomics/methods ; Gene Editing/methods ; Transcriptome ; Multiomics ; },
abstract = {The CRISPR-Cas system is a powerful gene editing technology, the clinical application of which is currently constrained due to safety concerns. A substantial body of safety research concerning Cas9 exists; however, scant attention has been directed toward investigating the safety profile of the emergent Cas13 system, which confers RNA editing capabilities. In particular, uncertainties persist regarding the potential cellular impacts of Cas13d in the absence of reliance on a cleavage effect. In this study, we conducted an initial exploration of the effects of Cas13d on HeLa cells. Total RNA and protein samples were extracted after transfection with a Cas13d-expressing plasmid construct, followed by transcriptomic and proteomic sequencing. Differential gene expression analysis identified 94 upregulated and 847 downregulated genes, while differential protein expression analysis identified 185 upregulated and 231 downregulated proteins. Subsequently, enrichment analysis was conducted on the transcriptome and proteome sequencing data, revealing that the PI3K-Akt signaling pathway is a common term. After intersecting the differentially expressed genes enriched in the PI3K-Akt signaling pathway with all the differentially expressed proteins, it was found that the expression of the related regulatory gene PFKFB4 was upregulated. Moreover, western blot analysis demonstrated that Cas13d can mediate PI3K-Akt signaling upregulation through overexpression of PFKFB4. CCK-8 assay, colony formation, and EdU experiments showed that Cas13d can promote cell proliferation. Our data demonstrate, for the first time, that Cas13d significantly impacts the transcriptomic and proteomic profiles, and proliferation phenotype, of HeLa cells, thus offering novel insights into safety considerations regarding gene editing systems.},
}

Humans
HeLa Cells
*Proto-Oncogene Proteins c-akt/metabolism/genetics
*Cell Proliferation
*Phosphatidylinositol 3-Kinases/metabolism/genetics
*Signal Transduction
*Phosphofructokinase-2/genetics/metabolism
*Up-Regulation
*CRISPR-Cas Systems
Proteomics/methods
Gene Editing/methods
Transcriptome
Multiomics

@article {pmid38992003,
year = {2024},
author = {Neumayer, G and Torkelson, JL and Li, S and McCarthy, K and Zhen, HH and Vangipuram, M and Mader, MM and Gebeyehu, G and Jaouni, TM and Jacków-Malinowska, J and Rami, A and Hansen, C and Guo, Z and Gaddam, S and Tate, KM and Pappalardo, A and Li, L and Chow, GM and Roy, KR and Nguyen, TM and Tanabe, K and McGrath, PS and Cramer, A and Bruckner, A and Bilousova, G and Roop, D and Tang, JY and Christiano, A and Steinmetz, LM and Wernig, M and Oro, AE},
title = {A scalable and cGMP-compatible autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5834},
pmid = {38992003},
issn = {2041-1723},
support = {R01 GM121932/GM/NIGMS NIH HHS/United States ; ARO73170//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; DISC2-12590//California Institute for Regenerative Medicine (CIRM)/ ; TRAN1-10416//California Institute for Regenerative Medicine (CIRM)/ ; },
mesh = {Humans ; *Epidermolysis Bullosa Dystrophica/therapy/genetics ; Animals ; *Induced Pluripotent Stem Cells/transplantation/metabolism/cytology ; Mice ; *Collagen Type VII/genetics/metabolism ; *Cell- and Tissue-Based Therapy/methods ; Fibroblasts/metabolism ; Cell Differentiation ; Keratinocytes/metabolism/transplantation ; Skin/metabolism ; Transplantation, Autologous ; Male ; Mutation ; Female ; Skin Transplantation/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.},
}

Humans
*Epidermolysis Bullosa Dystrophica/therapy/genetics
Animals
*Induced Pluripotent Stem Cells/transplantation/metabolism/cytology
Mice
*Collagen Type VII/genetics/metabolism
*Cell- and Tissue-Based Therapy/methods
Fibroblasts/metabolism
Cell Differentiation
Keratinocytes/metabolism/transplantation
Skin/metabolism
Transplantation, Autologous
Male
Mutation
Female
Skin Transplantation/methods
Gene Editing/methods
CRISPR-Cas Systems

@article {pmid38991068,
year = {2024},
author = {Žedaveinytė, R and Meers, C and Le, HC and Mortman, EE and Tang, S and Lampe, GD and Pesari, SR and Gelsinger, DR and Wiegand, T and Sternberg, SH},
title = {Antagonistic conflict between transposon-encoded introns and guide RNAs.},
journal = {Science (New York, N.Y.)},
volume = {385},
number = {6705},
pages = {eadm8189},
doi = {10.1126/science.adm8189},
pmid = {38991068},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; *DNA Transposable Elements ; Homologous Recombination ; *Introns ; RNA Splicing ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Transposases/metabolism/genetics ; *Clostridium botulinum/genetics ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.},
}

CRISPR-Cas Systems
*DNA Transposable Elements
Homologous Recombination
*Introns
RNA Splicing
*RNA, Guide, CRISPR-Cas Systems/genetics
Transposases/metabolism/genetics
*Clostridium botulinum/genetics
*Bacterial Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid38990000,
year = {2024},
author = {Roberts, A and Spang, D and Sanozky-Dawes, R and Nethery, MA and Barrangou, R},
title = {Characterization of Ligilactobacillus salivarius CRISPR-Cas systems.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0017124},
doi = {10.1128/msphere.00171-24},
pmid = {38990000},
issn = {2379-5042},
abstract = {UNLABELLED: Ligilactobacillus is a diverse genus among lactobacilli with phenotypes that reflect adaptation to various hosts. CRISPR-Cas systems are highly prevalent within lactobacilli, and Ligilactobacillus salivarius, the most abundant species of Ligilactobacillus, possesses both DNA- and RNA-targeting CRISPR-Cas systems. In this study, we explore the presence and functional properties of I-B, I-C, I-E, II-A, and III-A CRISPR-Cas systems in over 500 Ligilactobacillus genomes, emphasizing systems found in L. salivarius. We examined the I-E, II-A, and III-A CRISPR-Cas systems of two L. salivarius strains and observed occurrences of split cas genes and differences in CRISPR RNA maturation in native hosts. This prompted testing of the single Cas9 and multiprotein Cascade and Csm CRISPR-Cas effector complexes in a cell-free context to demonstrate the functionality of these systems. We also predicted self-targeting spacers within L. salivarius CRISPR-Cas systems and found that nearly a third of L. salivarius genomes possess unique self-targeting spacers that generally target elements other than prophages. With these two L. salivarius strains, we performed prophage induction coupled with RNA sequencing and discovered that the prophages residing within these strains are inducible and likely active elements, despite targeting by CRISPR-Cas systems. These findings deepen our comprehension of CRISPR-Cas systems in L. salivarius, further elucidating their relationship with associated prophages and providing a functional basis for the repurposing of these Cas effectors for bacterial manipulation.

IMPORTANCE: Ligilactobacillus salivarius is a diverse bacterial species widely used in the food and dietary supplement industries. In this study, we investigate the occurrence and diversity of their adaptive immune systems, CRISPR-Cas, in over 500 genomes. We establish their function and provide insights into their role in the interplay between the bacterial host and the predatory phages that infect them. Such findings expand our knowledge about these important CRISPR-Cas immune systems widespread across the bacterial tree of life and also provide a technical basis for the repurposing of these molecular machines for the development of molecular biology tools and the manipulation and engineering of bacteria and other life forms.},
}

@article {pmid38989619,
year = {2024},
author = {Goswami, HN and Ahmadizadeh, F and Wang, B and Addo-Yobo, D and Zhao, Y and Whittington, AC and He, H and Terns, MP and Li, H},
title = {Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae603},
pmid = {38989619},
issn = {1362-4962},
support = {R35 GM152081/NH/NIH HHS/United States ; },
abstract = {The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length.},
}

@article {pmid38989031,
year = {2024},
author = {Rathore, P and Basnet, A and Kilonzo-Nthenge, A and Dumenyo, K and Yadegari, Z and Taheri, A},
title = {Rapid detection of pathogenic E. coli based on CRISPR Cas system.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1423478},
pmid = {38989031},
issn = {1664-302X},
abstract = {Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.},
}

@article {pmid38989015,
year = {2024},
author = {Barrio-Pujante, A and Bleriot, I and Blasco, L and Fernández-Garcia, L and Pacios, O and Ortiz-Cartagena, C and Cuenca, FF and Oteo-Iglesias, J and Tomás, M},
title = {Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1416628},
pmid = {38989015},
issn = {1664-302X},
abstract = {BACKGROUND: Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms.

RESULTS: Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected.

CONCLUSION: Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.},
}

@article {pmid38987591,
year = {2024},
author = {Birkholz, N and Kamata, K and Feussner, M and Wilkinson, ME and Cuba Samaniego, C and Migur, A and Kimanius, D and Ceelen, M and Went, SC and Usher, B and Blower, TR and Brown, CM and Beisel, CL and Weinberg, Z and Fagerlund, RD and Jackson, SA and Fineran, PC},
title = {Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.},
journal = {Nature},
volume = {631},
number = {8021},
pages = {670-677},
pmid = {38987591},
issn = {1476-4687},
mesh = {*Cryoelectron Microscopy ; *Bacteriophages/metabolism/genetics/chemistry ; *Models, Molecular ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism/chemistry ; Protein Biosynthesis ; Helix-Turn-Helix Motifs ; Ribosomes/metabolism/chemistry ; Binding Sites ; Protein Domains ; Viral Proteins/metabolism/chemistry ; DNA-Binding Proteins/metabolism/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Associated Proteins/metabolism/chemistry ; Nucleic Acid Conformation ; RNA-Binding Proteins/metabolism/chemistry ; RNA, Viral/metabolism/genetics/chemistry ; Transcription, Genetic ; },
abstract = {In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins[1]. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression[2-5]. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.},
}

*Cryoelectron Microscopy
*Bacteriophages/metabolism/genetics/chemistry
*Models, Molecular
*CRISPR-Cas Systems/genetics
RNA, Messenger/genetics/metabolism/chemistry
Protein Biosynthesis
Helix-Turn-Helix Motifs
Ribosomes/metabolism/chemistry
Binding Sites
Protein Domains
Viral Proteins/metabolism/chemistry
DNA-Binding Proteins/metabolism/chemistry
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Associated Proteins/metabolism/chemistry
Nucleic Acid Conformation
RNA-Binding Proteins/metabolism/chemistry
RNA, Viral/metabolism/genetics/chemistry
Transcription, Genetic

@article {pmid38987539,
year = {2024},
author = {Reginato, G and Dello Stritto, MR and Wang, Y and Hao, J and Pavani, R and Schmitz, M and Halder, S and Morin, V and Cannavo, E and Ceppi, I and Braunshier, S and Acharya, A and Ropars, V and Charbonnier, JB and Jinek, M and Nussenzweig, A and Ha, T and Cejka, P},
title = {HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {5789},
pmid = {38987539},
issn = {2041-1723},
support = {ANR 23-CE11-0033//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-10-INBS-0005//Agence Nationale de la Recherche (French National Research Agency)/ ; 310030_207588//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; R35 GM122569/GM/NIGMS NIH HHS/United States ; R35 GM 122569//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; ALTF 710-2021//European Molecular Biology Organization (EMBO)/ ; 310030_205199//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; *DNA/metabolism/genetics ; MRE11 Homologue Protein/metabolism/genetics ; DNA-Binding Proteins/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing ; Endonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; DNA End-Joining Repair ; DNA Cleavage ; Transcription Factors/metabolism/genetics ; },
abstract = {The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.},
}

*DNA Breaks, Double-Stranded
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics
*DNA/metabolism/genetics
MRE11 Homologue Protein/metabolism/genetics
DNA-Binding Proteins/metabolism/genetics
CRISPR-Associated Proteins/metabolism/genetics
Gene Editing
Endonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
DNA End-Joining Repair
DNA Cleavage
Transcription Factors/metabolism/genetics

@article {pmid38987356,
year = {2024},
author = {Mondal, P and Alyateem, G and Mitchell, AV and Gottesman, MM},
title = {A whole-genome CRISPR screen identifies the spindle accessory checkpoint as a locus of nab-paclitaxel resistance in a pancreatic cancer cell line.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {15912},
pmid = {38987356},
issn = {2045-2322},
mesh = {*Paclitaxel/pharmacology ; Humans ; *Pancreatic Neoplasms/genetics/drug therapy/pathology ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; *Albumins/pharmacology ; M Phase Cell Cycle Checkpoints/drug effects/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Pancreatic adenocarcinoma is one of the most aggressive and lethal forms of cancer. Chemotherapy is the primary treatment for pancreatic cancer, but resistance to the drugs used remains a major challenge. A genome-wide CRISPR interference and knockout screen in the PANC-1 cell line with the drug nab-paclitaxel has identified a group of spindle assembly checkpoint (SAC) genes that enhance survival in nab-paclitaxel. Knockdown of these SAC genes (BUB1B, BUB3, and TTK) attenuates paclitaxel-induced cell death. Cells treated with the small molecule inhibitors BAY 1217389 or MPI 0479605, targeting the threonine tyrosine kinase (TTK), also enhance survival in paclitaxel. Overexpression of these SAC genes does not affect sensitivity to paclitaxel. These discoveries have helped to elucidate the mechanisms behind paclitaxel cytotoxicity. The outcomes of this investigation may pave the way for a deeper comprehension of the diverse responses of pancreatic cancer to therapies including paclitaxel. Additionally, they could facilitate the formulation of novel treatment approaches for pancreatic cancer.},
}

*Paclitaxel/pharmacology
Humans
*Pancreatic Neoplasms/genetics/drug therapy/pathology
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
*Albumins/pharmacology
M Phase Cell Cycle Checkpoints/drug effects/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38987223,
year = {2024},
author = {Wu, LY and Xu, Y and Yu, XW},
title = {Efficient CRISPR-mediated C-to-T base editing in Komagataella phaffii.},
journal = {Biotechnology journal},
volume = {19},
number = {7},
pages = {e2400115},
doi = {10.1002/biot.202400115},
pmid = {38987223},
issn = {1860-7314},
support = {2021YFC2100203//National Key Research and Development Program of China/ ; 32072162//National Natural Science Foundation of China/ ; KYCX21_2030//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Gene Editing/methods ; *Saccharomycetales/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Cytidine Deaminase/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Proteins ; },
abstract = {The nonconventional methylotrophic yeast Komagataella phaffii is widely applied in the production of industrial enzymes, pharmaceutical proteins, and various high-value chemicals. The development of robust and versatile genome editing tools for K. phaffii is crucial for the design of increasingly advanced cell factories. Here, we first developed a base editing method for K. phaffii based on the CRISPR-nCas9 system. We engineered 24 different base editor constructs, using a variety of promoters and cytidine deaminases (CDAs). The optimal base editor (PAOX2*-KpA3A-nCas9-KpUGI-DAS1TT) comprised a truncated AOX2 promoter (PAOX2*), a K. phaffii codon-optimized human APOBEC3A CDA (KpA3A), human codon-optimized nCas9 (D10A), and a K. phaffii codon-optimized uracil glycosylase inhibitor (KpUGI). This optimal base editor efficiently performed C-to-T editing in K. phaffii, with single-, double-, and triple-locus editing efficiencies of up to 96.0%, 65.0%, and 5.0%, respectively, within a 7-nucleotide window from C-18 to C-12. To expand the targetable genomic region, we also replaced nCas9 in the optimal base editor with nSpG and nSpRy, and achieved 50.0%-60.0% C-to-T editing efficiency for NGN-protospacer adjacent motif (PAM) sites and 20.0%-93.2% C-to-T editing efficiency for NRN-PAM sites, respectively. Therefore, these constructed base editors have emerged as powerful tools for gene function research, metabolic engineering, genetic improvement, and functional genomics research in K. phaffii.},
}

*Gene Editing/methods
*Saccharomycetales/genetics
*CRISPR-Cas Systems/genetics
Humans
Cytidine Deaminase/genetics/metabolism
Promoter Regions, Genetic/genetics
Proteins

@article {pmid38987221,
year = {2024},
author = {Cui, C and Guo, G and Chen, TH},
title = {Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.},
journal = {Biotechnology journal},
volume = {19},
number = {7},
pages = {e2400097},
doi = {10.1002/biot.202400097},
pmid = {38987221},
issn = {1860-7314},
support = {//COCHE/ ; 11217820//Hong Kong Research Grants Council/ ; N_CityU119/19//Hong Kong Research Grants Council/ ; JCYJ20210324134006017//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 9678242//City University of Hong Kong/ ; 9609336//City University of Hong Kong/ ; 7020072//City University of Hong Kong/ ; },
mesh = {*Uracil-DNA Glycosidase/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins/genetics/metabolism ; DNA, Single-Stranded/metabolism/genetics ; },
abstract = {DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL[-1]. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.},
}

*Uracil-DNA Glycosidase/metabolism/genetics
*CRISPR-Cas Systems/genetics
Humans
CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins/genetics/metabolism
DNA, Single-Stranded/metabolism/genetics

@article {pmid38986742,
year = {2024},
author = {Pedersen, JS and Carstens, AB and Rothgard, MM and Roy, C and Viry, A and Papudeshi, B and Kot, W and Hille, F and Franz, CMAP and Edwards, R and Hansen, LH},
title = {A novel genus of Pectobacterium bacteriophages display broad host range by targeting several species of Danish soft rot isolates.},
journal = {Virus research},
volume = {347},
number = {},
pages = {199435},
doi = {10.1016/j.virusres.2024.199435},
pmid = {38986742},
issn = {1872-7492},
abstract = {The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39 kb to 43 kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes.},
}

@article {pmid38986250,
year = {2024},
author = {Ma, H and Tian, Y and Kong, D and Guo, M and Dai, C and Wang, Q and Li, S and Tian, Z and Liu, Y and Wei, D},
title = {One-base-mismatch CRISPR-based transistors for single nucleotide resolution assay.},
journal = {Biosensors & bioelectronics},
volume = {262},
number = {},
pages = {116548},
doi = {10.1016/j.bios.2024.116548},
pmid = {38986250},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation/methods ; *Transistors, Electronic ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Viral/genetics/isolation & purification/analysis ; *Polymorphism, Single Nucleotide/genetics ; SARS-CoV-2/genetics/isolation & purification ; Base Pair Mismatch ; Limit of Detection ; COVID-19/virology/diagnosis ; Graphite/chemistry ; },
abstract = {An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 μL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 10[5] copy mL[-1] in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 μL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics.},
}

*Biosensing Techniques/instrumentation/methods
*Transistors, Electronic
Humans
*CRISPR-Cas Systems/genetics
*RNA, Viral/genetics/isolation & purification/analysis
*Polymorphism, Single Nucleotide/genetics
SARS-CoV-2/genetics/isolation & purification
Base Pair Mismatch
Limit of Detection
COVID-19/virology/diagnosis
Graphite/chemistry