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CRISPR/Cas9 as a Simple Technique for the Generation of Murine Knockout Models for Neuropsychiatric Diseases.
The development of schizophrenia-like rodent models is still a major challenge for the study of this mental disorder. Schizophrenia and other neuropsychiatric disorders are thought to be triggered by multiple factors, and furthermore, the genetic component of schizophrenia is highly complex. The edition of one single gene for mimicking some of the symptoms of the disorder could cause unintended mutations that could influence animal's behavior making it difficult to study. Since 2013, CRISPR-Cas gene-editing technology has been a great improvement in the specificity of transgenic model generation because of its speed, efficiency, cost, and apparent ease. This protocol describes a simple method to generate a knockout mouse model using CRISPR technology, which can be applied to any gene presumably involved in the development of schizophrenia and other neuropsychiatric disorders.
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Editing of DNA Methylation Patterns Using CRISPR-Based Tools.
DNA methylation is an epigenetic modification with an established role in both normal cellular function and mammalian disease. Despite well-characterized associations between aberrant DNA methylation changes and gene expression, evidence for a causal relationship in this context has been difficult to obtain. Early techniques for interrogating the role of DNA methylation in the regulation of gene transcription lack specificity and, where more specific techniques such and ZNFs and TALEs have been developed, they are limited by their extensive cost and labor requirements. However, the recent advent of CRISPR-based technologies has revolutionized our potential for site-specific epigenomic editing. Here, we provide a detailed protocol for the design, construction, and utilization of a transient, CRISPR-based DNA methylation-editing system in mammalian cells.
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Targeted Gene Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases.
Introduction of induced pluripotent stem cell (iPSC) technology and site-directed nucleases brought a major breakthrough in the development of regenerative therapies and biomedical research. With the advancement of ZFNs, TALENs, and the CRISPR/Cas9 technology, straightforward and precise manipulation of the genome of human pluripotent stem cells (PSC) became possible, allowing relatively easy and fast generation of gene knockouts, integration of transgenes, or even introduction of single nucleotide changes for correction or introduction of disease-specific mutations. We review current applications of site-specific nucleases in human PSCs and focus on trends and challenges for efficient gene editing and improvement of targeting strategies. Graphical Abstract.
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Technique: Genome editing for cell lineage tracing.
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Genomic surveillance: protecting sport in the post-CRISPR era.
Personal genomics and gene editing raise the prospect of undetectable gene doping. Athletes' genome sequencing should be integrated into the Athlete Biological Passport to deter future abuse. This provocative vision calls for urgent debate on fairness in sport and for new solutions to share genomic data while preserving privacy.
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SynBioStrainFinder: A microbial strain database of manually curated CRISPR/Cas genetic manipulation system information for biomanufacturing.
BACKGROUND: Microbial strain information databases provide valuable data for microbial basic research and applications. However, they rarely contain information on the genetic operating system of microbial strains. RESULTS: We established a comprehensive microbial strain database, SynBioStrainFinder, by integrating CRISPR/Cas gene-editing system information with cultivation methods, genome sequence data, and compound-related information. It is presented through three modules, Strain2Gms/PredStrain2Gms, Strain2BasicInfo, and Strain2Compd, which combine to form a rapid strain information query system conveniently curated, integrated, and accessible on a single platform. To date, 1426 CRISPR/Cas gene-editing records of 157 microbial strains have been manually extracted from the literature in the Strain2Gms module. For strains without established CRISPR/Cas systems, the PredStrain2Gms module recommends the system of the most closely related strain as a reference to facilitate the construction of a new CRISPR/Cas gene-editing system. The database contains 139,499 records of strain cultivation and genome sequences, and 773,298 records of strain-related compounds. To facilitate simple and intuitive data application, all microbial strains are also labeled with stars based on the order and availability of strain information. SynBioStrainFinder provides a user-friendly interface for querying, browsing, and visualizing detailed information on microbial strains, and it is publicly available at http://design.rxnfinder.org/biosynstrain/ . CONCLUSION: SynBioStrainFinder is the first microbial strain database with manually curated information on the strain CRISPR/Cas system as well as other microbial strain information. It also provides reference information for the construction of new CRISPR/Cas systems. SynBioStrainFinder will serve as a useful resource to extend microbial strain research and application for biomanufacturing.
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Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing.
Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article.
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A Split CRISPR-Cpf1 Platform for Inducible Gene Activation.
The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.
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Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.
Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.
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CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae.
Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021).
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