title: Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.
abstract: Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Enbo
authors.initials: E
authors.lastname: Ma
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Harrington
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: O'Connell
authors.affiliation: Howard Hughes Medical Institute.
authors.forename: Kaihong
authors.initials: K
authors.lastname: Zhou
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D012329:RNA, Bacterial; D004720:Endonucleases