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.
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.).
