Crystal structure of Cas9 in complex with guide RNA and target DNA

Hiroshi Nishimasu; F Ann Ran; Patrick D Hsu; Silvana Konermann; Soraya I Shehata; Naoshi Dohmae; Ryuichiro Ishitani; Feng Zhang; Osamu Nureki

doi:10.1016/j.cell.2014.02.001

Crystal structure of Cas9 in complex with guide RNA and target DNA

Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13.

Authors

Hiroshi Nishimasu¹, F Ann Ran², Patrick D Hsu², Silvana Konermann³, Soraya I Shehata³, Naoshi Dohmae⁴, Ryuichiro Ishitani⁵, Feng Zhang⁶, Osamu Nureki⁷

Affiliations

¹ Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan.
² Broad Institute of MIT and Harvard, Cambridge, MA 02141, 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 Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02141, 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.
⁴ Biomolecular Characterization Team and CREST/JST, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
⁵ Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA 02141, 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. Electronic address: zhang@broadinstitute.org.
⁷ Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan. Electronic address: nureki@biochem.s.u-tokyo.ac.jp.

Abstract

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Bacteria / enzymology
CRISPR-Associated Proteins / chemistry*
CRISPR-Associated Proteins / metabolism
Crystallography, X-Ray*
DNA, Bacterial / chemistry
DNA, Bacterial / metabolism
Endonucleases / chemistry*
Endonucleases / metabolism
Models, Molecular
Molecular Sequence Data
Protein Structure, Tertiary
RNA, Bacterial / chemistry*
RNA, Bacterial / metabolism
RNA, Small Untranslated
Sequence Alignment
Streptococcus pyogenes / chemistry*
Streptococcus pyogenes / enzymology
Streptococcus pyogenes / metabolism

Substances

CRISPR-Associated Proteins
DNA, Bacterial
RNA, Bacterial
Endonucleases
RNA, Small Untranslated

Associated data

PDB/4OO8

Abstract

Publication types

MeSH terms

Substances

Associated data

Grants and funding