Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

Enbo Ma; Lucas B Harrington; Mitchell R O'Connell; Kaihong Zhou; Jennifer A Doudna

doi:10.1016/j.molcel.2015.10.030

Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

Mol Cell. 2015 Nov 5;60(3):398-407. doi: 10.1016/j.molcel.2015.10.030.

Authors

Enbo Ma¹, Lucas B Harrington¹, Mitchell R O'Connell², Kaihong Zhou³, Jennifer A Doudna⁴

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² 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.
³ Howard Hughes Medical Institute.
⁴ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
CRISPR-Cas Systems / physiology*
DNA, Single-Stranded / genetics
DNA, Single-Stranded / metabolism*
Endonucleases / genetics
Endonucleases / metabolism*
RNA, Bacterial / genetics
RNA, Bacterial / metabolism
Streptococcus pyogenes / enzymology*
Streptococcus pyogenes / genetics

Substances

Bacterial Proteins
DNA, Single-Stranded
RNA, Bacterial
Endonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding