A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

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CRISPR prime editing enables double-strand break free engineering of the genome. Here the authors present a toolkit for prime editing in E. coli. CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.
Publisher
NATURE PORTFOLIO
Issue Date
2021-09
Language
English
Article Type
Article
Citation

NATURE COMMUNICATIONS, v.12, no.1

ISSN
2041-1723
DOI
10.1038/s41467-021-25541-3
URI
http://hdl.handle.net/10203/287875
Appears in Collection
CBE-Journal Papers(저널논문)
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