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Nat Biotechnol. 2015 Dec;33(12):1293-1298. doi: 10.1038/nbt.3404. Epub 2015 Nov 2.

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

Kleinstiver BP1,2,3,4, Prew MS1,2,3, Tsai SQ1,2,3,4, Nguyen NT1,2,3, Topkar VV1,2,3, Zheng Z5, Joung JK1,2,3,4.

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Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.


CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

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