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Engineered CRISPR-Cas9 nuclease with expanded targeting space

Author(s)
Nishimasu, Hiroshi; Shi, Xi; Ishiguro, Soh; Gao, Linyi; Hirano, Seiichi; Okazaki, Sae; Noda, Taichi; Abudayyeh, Omar O.; Gootenberg, Jonathan S; Mori, Hideto; Oura, Seiya; Holmes, Benjamin Ray; Tanaka, Mamoru; Seki, Motoaki; Hirano, Hisato; Aburatani, Hiroyuki; Ishitani, Ryuichiro; Ikawa, Masahito; Yachie, Nozomu; Zhang, Feng; Nureki, Osamu; Zhang, Feng; ... Show more Show less
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Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/
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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.
Date issued
2018-08
URI
https://hdl.handle.net/1721.1/126396
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
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
ISSN
0036-8075
1095-9203

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