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bioRxiv · 10.1101/2021.07.12.451994

Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Hao, Y.·Wang, Q.·Li, J.·Yang, S.·Ma, L.·Zheng, Y.·Peng, W.
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Abstract

New CRISPR-based genome editing technologies are developed to continuedly 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 an endogenous Type I system of Zymomonas mobilis. 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, 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, easy, yet useful approach to convert many endogenous Type I systems into advanced genome editing tools. We envision that many CRISPR-nCas3-based toolkits would be soon available for various industrially important non-model bacteria that carry active Type I systems to facilitate high-throughput prokaryotic engineering.

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Hao, Y., Wang, Q., Li, J., Yang, S., Ma, L., Zheng, Y., Peng, W.. 2021-07-12. Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering. https://doi.org/10.1101/2021.07.12.451994

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