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

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 between 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, Cas12i(Max), 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 Cas12i(Max) (Cas12i(HiFi)) 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.