Engineered CRISPR-Cas9 nuclease with expanded targeting space
Name
nihms-1004927.pdf
Description
Accepted version
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1.1 MB
Format
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Checksum (MD5)
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Author(s)
Nishimasu, Hiroshi
Shi, Xi
Ishiguro, Soh
Gao, Linyi
Hirano, Seiichi
Okazaki, Sae
Noda, Taichi
Abudayyeh, Omar O.
Gootenberg, Jonathan S
Mori, Hideto
Date Issued
August 2018
Journal
Science
Publisher
American Association for the Advancement of Science (AAAS)
Citation
Nishimasu, Hiroshi et al. "Engineered CRISPR-Cas9 nuclease with expanded targeting space." Science 361, 6408 (August 2018): 1259-1262 © The Authors
Version
Author's final manuscript
Abstract
The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third G is compensated by newly introduced non-base-specific interactions, enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
Broad Institute of MIT and Harvard
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1126/science.aas9129
