College of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, People's Republic of China.
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.
Open Biol. 2022 Jan;12(1):210241. doi: 10.1098/rsob.210241. Epub 2022 Jan 12.
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 nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 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.
新型基于 CRISPR 的基因组编辑技术不断推动生命科学的进步，但主要源自于 II 型 CRISPR-Cas9 和 V 型 CRISPR-Cas12a 这两种用于真核生物的系统。在此，我们报告了一种新型基于 I-F 型系统的 CRISPR-n（切口酶）Cas3 基因组编辑工具。我们证明，通过将 Cas3 解旋酶结构域中的任何催化残基突变为丙氨酸，可以产生 nCas3 变体。虽然 nCas3 通过质粒的过表达会导致严重的细胞毒性，但 nCas3 可以引入靶向双链断裂，在不造成明显细胞杀伤的情况下促进基因组编辑。通过利用这种 CRISPR-nCas3 基因插入技术，可以一致地完成基因或基因组 DNA 片段的核苷酸取代和缺失，包括同时去除两个大的基因组片段。我们的工作描述了第一个基于 CRISPR-nCas3 的基因组编辑技术的建立，从而提供了一种简单而有用的方法，将自然中最丰富的 I 型系统转化为先进的基因组编辑工具，以促进高通量的原核工程。
