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 Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineer...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) Vol. 361; no. 6408; p. 1259
Main Authors: 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, Tanaka, Mamoru, Seki, Motoaki, Hirano, Hisato, Aburatani, Hiroyuki, Ishitani, Ryuichiro, Ikawa, Masahito, Yachie, Nozomu, Zhang, Feng, Nureki, Osamu
Format: Journal Article
Language:English
Published: United States 21.09.2018
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Crystallography, X-Ray
Endonucleases - chemistry
Endonucleases - genetics
Gene Editing
HEK293 Cells
Humans
Protein Engineering
ISSN:1095-9203, 1095-9203
Online Access:Get more information
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Summary:The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used 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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ISSN:1095-9203
1095-9203
DOI:10.1126/science.aas9129