biostudies-literature00530Nishimasu HNICHD NIH HHSNIDDK NIH HHSNIMH NIH HHSNHLBI NIH HHSNHGRI NIH HHS1259-1262https://www.ebi.ac.uk/biostudies/studies/S-EPMC6368452biostudies-literatureUnknown361(6408)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.Science (New York, N.Y.)Engineered CRISPR-Cas9 nuclease with expanded targeting space.PMC6368452R01 DK097768DP1 MH100706DP1 HL141201R01 HD088412P01 HD087157R01 MH110049RM1 HG006193R01 HG009761Zhang FAburatani HIshiguro SYachie NHirano HOkazaki SShi XHirano SIshitani RNishimasu HGao LNoda TMori HNureki OGootenberg JSHolmes BAbudayyeh OOOura SIkawa MTanaka MSeki M53falseEngineered CRISPR-Cas9 nuclease with expanded targeting space.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.2018-01-01T00:00:00Z2018 Sep2020-11-09T09:04:43Z2019-08-04T07:14:33ZS-EPMC63684523016644110.1126/science.aas9129