Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing  

作　　者：Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen Zhikun Li Qi Zhou Wei Li 

机构地区：[1]State Key Laboratory of Stem Cell and Reproductive Biology,Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China [2]University of Chinese Academy of Sciences,Beijing 100049,China [3]Institute for Stem Cell and Regenerative Medicine,Chinese Academy of Sciences,Beijing 100101,China [4]Bejing Institute for Stem Cell and Regenerative Medicine,Beijing 100101,China

出　　处：《The Innovation》2022年第4期74-82,共9页创新（英文）

基　　金：supported by the National Key Research and Development Program(2019YFA0110800 and 2020YFA0707900 to W.L.and 2018YFA0108400 and 2019YFA0903800 to Q.Z.);the Strategic Priority Research Programof the Chinese Academy of Sciences(XDA16030403 to W.L.);the National Natural Science Foundation of China(31621004 to Q.Z.and W.L.);and the CAS Project for Young Scientists in Basic Research(YSBR-012 to W.L.).

摘　　要：The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools.However,the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells,which significantly limits their utility.Here,we have developed a method termed Improving Editing Activity by Synergistic Engineering(MIDAS).This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions of Cas nuclease with the protospacer adjacent motif(PAM)and the single-stranded DNA(ssDNA)substrate in the catalytic pocket simultaneously.MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i,Cas12b,and CasX in human cells.Notably,a Cas12i variant,Cas12iMax,exhibited robust activity with a very broad PAM range(NTNN,NNTN,NAAN,and NCAN)and higher efficiency than the current widely used Cas nucleases.A high-fidelity version of Cas12iMax(Cas12iHiFi)has been further engineered to minimize off-target effects.Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.

关 键 词：EDITING engineering MAMMALIAN 

分 类 号：Q78[生物学—分子生物学]