Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease

Nature. 2014 Sep 25;513(7519):569-73. doi: 10.1038/nature13579. Epub 2014 Jul 27.

Abstract

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arginine / genetics
  • Arginine / metabolism
  • Base Pairing*
  • Base Sequence
  • CRISPR-Associated Proteins / metabolism*
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism*
  • Endonucleases / metabolism*
  • Models, Molecular
  • Nucleic Acid Denaturation
  • Nucleotide Motifs*
  • Protein Conformation
  • RNA, Guide / chemistry
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Streptococcus pyogenes / enzymology*
  • Substrate Specificity

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • DNA
  • Arginine
  • Endonucleases

Associated data

  • PDB/4UN3
  • PDB/4UN4
  • PDB/4UN5