Journal
SCIENCE
Volume 361, Issue 6408, Pages 1259-1262Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.aas9129
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Funding
- JST [JPMJPR13L8, 10814]
- JSPS [26291010, 15H01463, 16J06287, 17H01394, 18H02428]
- AMED [JP18gm5010001, 17g6110007h0002, 16am0301002h0003]
- Takeda Science Foundation
- NICHD [P01HD087157, R01HD088412]
- Bill & Melinda Gates Foundation [OPP1160866]
- NEDO (Genome Editing Program)
- NIMH [5DP1-MH100706, 1R01-MH110049]
- NIDDK [5R01DK097768-03]
- New York Stem Cell foundation
- Simons foundation
- Paul G. Allen Family foundation
- Vallee foundation
- CSTI, SIP
- Grants-in-Aid for Scientific Research [26291010, 17H01394, 16J06287, 15H01463, 18H02428] Funding Source: KAKEN
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The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genomeediting 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.
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