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CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material. Methods: In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs. Results: amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both in vitro and in vivo and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs. Conclusion: Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.
Pubmed ID: 32550896
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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
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View all literature mentionsCommercial organization which provides reagents and services for molecular biology research. Its services include clone collections, microRNA solutions, genome editing, qPCR products, and fluorescent labeling and detection.
View all literature mentionsCentral online repository for microRNA nomenclature, sequence data, annotation and target prediction.Collection of published miRNA sequences and annotation.
View all literature mentionsAmerican chemical, life science and biotechnology company owned by Merck KGaA. Merger of Sigma Chemical Company and Aldrich Chemical Company. Provides organic and inorganic chemicals, building blocks, reagents, advanced materials and stable isotopes for chemical synthesis, medicinal chemistry and materials science, antibiotics, buffers, carbohydrates, enzymes, forensic tools, hematology and histology, nucleotides, proteins, peptides, amino acids and their derivatives.
View all literature mentionsCell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsMus musculus with name C57BL/6J from IMSR.
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