清脆的
基因组编辑
基因组工程
计算生物学
Cas9
合成生物学
引导RNA
质粒
计算机科学
细菌基因组大小
基因组
生物
遗传学
DNA
基因
作者
Yaojun Tong,Tue Sparholt Jørgensen,Christopher M. Whitford,Tilmann Weber,Sang Yup Lee
标识
DOI:10.1038/s41467-021-25541-3
摘要
Abstract 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.
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