Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

Ziying Hu; Chengdong Zhang; Shuai Wang; Siqi Gao; Jingjing Wei; Miaomiao Li; Linghui Hou; Huilin Mao; Yanyan Wei; Tao Qi; Hongmao Liu; Dong Liu; Feng Lan; Daru Lu; Hongyan Wang; Jixi Li; Yongming Wang

doi:10.1093/nar/gkab148

GeneticsNucleic Acids ResearchFeb 23, 2021

Peer-reviewed journal article

Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

Ziying Hu iD, Chengdong Zhang iD, Shuai Wang iD, Siqi Gao iD, Jingjing Wei iD, Miaomiao Li iD, Linghui Hou iD, Huilin Mao, Yanyan Wei iD, Tao Qi, Hongmao Liu iD, Dong Liu iD, Feng Lan iD, Daru Lu iD, Hongyan Wang iD, Jixi Li iD, Yongming Wang iD

Abstract

The compact CRISPR/Cas9 system, which can be delivered by adeno-associated virus (AAV), is a promising platform for therapeutic applications. However, current compact Cas9 nucleases have limited activity, targeting scope and specificity. Here, we identified three compact SaCas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Interestingly, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. We further generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We lastly engineered SlugCas9 with mutations to generate a high fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

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