Combined Computational–Experimental Approach to Explore the Molecular Mechanism of SaCas9 with a Broadened DNA Targeting Range
Despite accelerating development of CRISPR technology, there remains high demand for further interrogation of its fundamental biology. This is particularly fascinating as new improved CRISPR tools were artificially engineered to harbor beneficial features but often lack mechanistic explanation. SaCa...
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Published in | Journal of the American Chemical Society Vol. 141; no. 16; pp. 6545 - 6552 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
24.04.2019
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Subjects | |
Online Access | Get full text |
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Summary: | Despite accelerating development of CRISPR technology, there remains high demand for further interrogation of its fundamental biology. This is particularly fascinating as new improved CRISPR tools were artificially engineered to harbor beneficial features but often lack mechanistic explanation. SaCas9, a minimal Cas9 ideal for in vivo applications, suffers from long protospacer adjacent motif (PAM), which prompted effort on mutant KKH SaCas9 with relaxed PAM requirement. Leveraging structure-based molecular dynamics simulation, free-energy perturbation, and targeted experimentation, we developed a workflow for probing SaCas9 and a series of its variants, revealing intriguing dynamics of PAM recognition and the molecular mechanism of KKH mutations. Furthermore, we deployed this approach to design and validate new mutant SaCas9, SaCas9-NR and SaCas9-RL, with enhanced targeting range distinctive from the KKH mutant and improved activity in mammalian cells. Overall, our approach provides a dynamic understanding of SaCas9 PAM recognition and a new gateway to enlighten rationally designed Cas9 variants harboring novel properties. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0002-7863 1520-5126 |
DOI: | 10.1021/jacs.8b13144 |