Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems

Mari Yamada; Yuto Watanabe; Jonathan S Gootenberg; Hisato Hirano; F Ann Ran; Takanori Nakane; Ryuichiro Ishitani; Feng Zhang; Hiroshi Nishimasu; Osamu Nureki

doi:10.1016/j.molcel.2017.02.007

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems

Mol Cell. 2017 Mar 16;65(6):1109-1121.e3. doi: 10.1016/j.molcel.2017.02.007.

Authors

Affiliations

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Society of Fellows, Harvard University, Cambridge, MA 02138, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁵ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address: nisimasu@bs.s.u-tokyo.ac.jp.
⁶ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address: nureki@bs.s.u-tokyo.ac.jp.

PMID: 28306506
DOI: 10.1016/j.molcel.2017.02.007

Abstract

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.

Keywords: CRISPR-Cas system; Cas9; RNA triplex; crystal structure; protospacer adjacent motif.

Publication types

Comparative Study

MeSH terms

Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
Binding Sites
CRISPR-Associated Proteins / chemistry
CRISPR-Associated Proteins / metabolism*
CRISPR-Cas Systems*
Campylobacter jejuni / enzymology*
DNA, Bacterial / chemistry
DNA, Bacterial / metabolism
Endonucleases / chemistry
Endonucleases / metabolism*
Models, Molecular
Nucleic Acid Conformation
Protein Binding
Protein Conformation
RNA, Bacterial / chemistry
RNA, Bacterial / metabolism
RNA, Guide, CRISPR-Cas Systems / chemistry
RNA, Guide, CRISPR-Cas Systems / metabolism
Structure-Activity Relationship
Substrate Specificity

Substances

Bacterial Proteins
CRISPR-Associated Proteins
DNA, Bacterial
RNA, Bacterial
RNA, Guide, CRISPR-Cas Systems
Endonucleases