Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering |
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Authors: | Yile Hao Qinhua Wang Jie Li Shihui Yang Yanli Zheng Wenfang Peng |
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Affiliation: | 1. College of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, People''s Republic of China ; 2. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Engineering Research Center for Bio-enzyme Catalysis, Environmental Microbial Technology Center of Hubei Province, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, School of Life Sciences, Hubei University, Wuhan 430062, People''s Republic of China |
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Abstract: | New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering. |
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Keywords: | Cas3 nickase genome editing high-efficiency CRISPR-Cas large genomic fragments deletion |
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