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

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

Author information

  • 1Department 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.
  • 2Broad 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.
  • 3Broad 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.
  • 4Biomolecular Characterization Team and CREST/JST, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
  • 5Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032, Japan.
  • 6Broad 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.
  • 7Department 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.

Copyright © 2014 Elsevier Inc. All rights reserved.

PMID:
24529477
[PubMed - indexed for MEDLINE]
PMCID:
PMC4139937
Free PMC Article
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