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Science. 2018 Sep 21;361(6408):1259-1262. doi: 10.1126/science.aas9129. Epub 2018 Aug 30.

Engineered CRISPR-Cas9 nuclease with expanded targeting space.

Author information

1
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.
2
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
3
McGovern Institute for Brain Research, Cambridge, MA 02139, USA.
4
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
5
Institute for Advanced Biosciences, Keio University, 14-1 Baba-cho, Tsuruoka, Yamagata 997-0035, Japan.
6
Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan.
7
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
8
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
9
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
10
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
11
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
12
Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

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 nucleobase is compensated by newly introduced non-base-specific interactions, thereby 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.

PMID:
30166441
PMCID:
PMC6368452
DOI:
10.1126/science.aas9129
[Indexed for MEDLINE]
Free PMC Article

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