title: Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.
abstract: 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.
authors.affiliation: 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.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.affiliation: 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.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Prew
authors.affiliation: 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.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.affiliation: 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.
authors.forename: Nhu T
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: 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.
authors.forename: Ved V
authors.initials: VV
authors.lastname: Topkar
authors.affiliation: Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.
authors.forename: Zongli
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: 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.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung