Engineered CRISPR-Cas9 nuclease with expanded targeting space

By H. Nishimasu, Xi Shi, Soh Ishiguro, L. Gao, S. Hirano, S. Okazaki, Taichi Noda, Omar Abudayyeh, Jonathan Gootenberg, Hideto Mori and 11 more. Science, 2018

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Expanding the targeting space of Cas9 CRISPR-Cas9 associates with a guide RNA to target and cleave a specific DNA site next to a protospacer adjacent motif (PAM). Streptococcus pyogenes Cas9 (SpCas9), the one most often used for genome editing, only recognizes the NGG sequence (where N is any nucleobase) as the PAM, which restricts regions in the genome that can be targeted. To address this limitation, Nishimasu et al. created a SpCas9 variant that recognizes NG rather than NGG. The SpCas9-NG variant increased the targeting range, had a specificity similar to that of the wild-type enzyme, and could be used with a base editor. Thus, SpCas9-NG is a powerful addition to the CRISPR-Cas9 genome engineering toolbox and will be useful in a broad range of applications, from basic research to clinical therapeutics. Science, this issue p. 1259 An engineered CRISPR-Cas9 nuclease increases the range of genomic sequences that can be targeted in Cas9-mediated genome engineering. 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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/21b4e61c76070106cb73f9b73bd9a29d10a40c27

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