DNA Interrogation by the CRISPR RNA-guided Endonuclease Cas9

Nature. 2014 Mar 6;507(7490):62-7. doi: 10.1038/nature13011. Epub 2014 Jan 29.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Publication types

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

MeSH terms

  • Apoenzymes / metabolism
  • Base Pairing*
  • Base Sequence
  • Biocatalysis
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • DNA Cleavage*
  • Diffusion
  • Endonucleases / metabolism*
  • Enzyme Activation
  • Genetic Engineering / methods
  • Genome / genetics
  • Nucleic Acid Denaturation
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / genetics
  • Nucleic Acid Heteroduplexes / metabolism
  • Nucleotide Motifs
  • RNA / chemistry
  • RNA / genetics*
  • RNA / metabolism
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / immunology
  • Substrate Specificity
  • Thermodynamics

Substances

  • Apoenzymes
  • CRISPR-Associated Proteins
  • Nucleic Acid Heteroduplexes
  • RNA
  • DNA
  • Endonucleases