Engineered CRISPR-Cas9 nuclease with expanded targeting space

Engineered 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 repor...

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Podrobná bibliografie
Hlavní autoři: 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
Další autoři: Massachusetts Institute of Technology. Department of Biological Engineering
Médium: Článek
Jazyk:English
Vydáno: American Association for the Advancement of Science (AAAS) 2020
On-line přístup:https://hdl.handle.net/1721.1/126396
Popis
Shrnutí: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.

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