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Establishing a one-step marker-free CRISPR/Cas9 system for industrial Aspergillus niger using counter-selectable marker Ang-ace2.
OBJECTIVES: To develop a one-step, marker-free CRISPR/Cas9 system for highly efficient genome editing in industrial Aspergillus niger, with a short genetic operation cycle. RESULTS: Firstly, evaluation of different promoters for sgRNA expression revealed tRNAGly15 as the most efficient, achieving a remarkable 100% gene editing efficiency. Furthermore, a counter-selectable marker, Ang-ace2, was identified for A. niger. Subsequently, a CRISPR/Cas9 plasmid was developed, utilizing a truncated AMA1 element and the Ang-ace2 conditional expression cassette driven by a Tet-on promoter. In the presence of doxycycline, the plasmid demonstrated a 33% loss efficiency in the progeny of A. niger spores after a single generation, resulting in a shortened genetic operation cycle of 16 days for CRISPR/Cas9. CONCLUSIONS: The one-step marker-free CRISPR/Cas9 system was successfully developed in industrial A. niger, allowing for efficient gene editing while simultaneously reducing the editing time.
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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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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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Technique: Genome editing for cell lineage tracing.
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Massively parallel CRISPR-assisted homologous recombination enables saturation editing of full-length endogenous genes in yeast.
Performing saturation editing of chromosomal genes will enable the study of genetic variants in situ and facilitate protein and cell engineering. However, current in vivo editing of endogenous genes either lacks flexibility or is limited to discrete codons and short gene fragments, preventing a comprehensive exploration of genotype-phenotype relationships. To enable facile saturation editing of full-length genes, we used a protospacer adjacent motif-relaxed Cas9 variant and homology-directed repair to achieve above 60% user-defined codon replacement efficiencies in Saccharomyces cerevisiae genome. Coupled with massively parallel DNA design and synthesis, we developed a saturation gene editing method termed CRISPR-Cas9- and homology-directed repair-assisted saturation editing (CHASE) and achieved highly saturated codon swapping of long genomic regions. By applying CHASE to massively edit a well-studied global transcription factor gene, we found known and unreported genetic variants affecting an industrially relevant microbial trait. The user-defined codon editing capability and wide targeting windows of CHASE substantially expand the scope of saturation gene editing.
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Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing.
CRISPR/Cas9-mediated gene editing may involve nonhomologous end-joining to create various insertion/deletions (indels) or may employ homologous recombination to modify precisely the target DNA sequence. Our understanding of these processes has been guided by earlier studies using other site-specific endonucleases, both in model organisms such as budding yeast and in mammalian cells. We briefly review what has been gleaned from such studies using the HO and I-SceI endonucleases and how these findings guide current gene editing strategies.
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EMT signaling: potential contribution of CRISPR/Cas gene editing.
Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.
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Advancements in Nonviral Gene Editing Strategies for Rare Diseases.
Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.
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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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Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing.
CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both Cas9 and sgRNA mediated high gene editing efficiency. ECO and iECO mediated more efficient gene editing than other tested lipids. ECO mediated up to 50% GFP suppression based on observations with confocal microscopy and nearly 80% reduction of GFP mRNA based on RT-PCR measurement in NIH3T3-GFP cells. The multifunctional pH-sensitive amino lipids have the potential for efficient intracellular delivery of CRISPR/Cas9 for effective gene editing.
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