Engineered CRISPR-Cas9 nuclease with expanded targeting space

Hiroshi Nishimasu(The University of Tokyo), Xi Shi(Broad Institute), Soh Ishiguro(Keio University), Linyi Gao(Broad Institute), Seiichi Hirano(The University of Tokyo), Sae Okazaki(The University of Tokyo), Taichi Noda(The University of Osaka), Omar O. Abudayyeh(Broad Institute), Jonathan S. Gootenberg(Broad Institute), Hideto Mori(Keio University), Seiya Oura(The University of Osaka), Benjamin Holmes(Broad Institute), Mamoru Tanaka(The University of Tokyo), M. Seki(The University of Tokyo), Hisato Hirano(The University of Tokyo), Hiroyuki Aburatani(The University of Tokyo), Ryuichiro Ishitani(The University of Tokyo), Masahito Ikawa(Systems Biology Institute), Nozomu Yachie(Keio University), Feng Zhang(Broad Institute), Osamu Nureki(The University of Tokyo)
Science
August 30, 2018
10.1126/science.aas9129
Cited by 1,126

Abstract

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.

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