Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

Science. 2016 Feb 19;351(6275):867-71. doi: 10.1126/science.aad8282. Epub 2016 Jan 14.

Abstract

Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Catalytic Domain
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA Cleavage*
  • Endonucleases / chemistry*
  • Endonucleases / ultrastructure
  • Genetic Engineering
  • Genome
  • Nucleic Acid Conformation
  • Protein Conformation
  • RNA / chemistry
  • RNA, Guide
  • Streptococcus pyogenes / enzymology*

Substances

  • RNA, Guide
  • RNA
  • DNA
  • Endonucleases

Associated data

  • PDB/5F9R