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...

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Veröffentlicht in:Innovation (New York, NY) NY), 2022-07, Vol.3 (4), p.100264-100264, Article 100264
Hauptverfasser: Chen, Yangcan, Hu, Yanping, Wang, Xinge, Luo, Shengqiu, Yang, Ning, Chen, Yi, Li, Zhikun, Zhou, Qi, Li, Wei
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Sprache:eng
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Zusammenfassung: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, 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. [Display omitted] •Improving Editing Activity by Synergistic Engineering (MIDAS) of Cas nucleases•MIDAS can improve the activity of Cas12i, Cas12b, and CasX•Engineering high-efficiency Cas12iMax and high-specificity Cas12iHiFi
ISSN:2666-6758
2666-6758
DOI:10.1016/j.xinn.2022.100264