PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

Cell. 2016 Dec 15;167(7):1814-1828.e12. doi: 10.1016/j.cell.2016.11.053.


C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

Keywords: C2c1; RuvC catalytic pocket; binary complex with sgRNA; genome editing tool; sequence-specific PAM recognition; structure; ternary complex with added DNA; type V CRISPR-Cas endonuclease.

MeSH terms

  • Alicyclobacillus / classification
  • Alicyclobacillus / enzymology*
  • Alicyclobacillus / genetics
  • Alicyclobacillus / metabolism
  • CRISPR-Cas Systems*
  • Crystallography, X-Ray
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Gene Editing
  • Homeodomain Proteins / genetics
  • Humans
  • Models, Molecular
  • RNA, Untranslated / metabolism
  • Transcription Factors / genetics


  • Homeodomain Proteins
  • RNA, Untranslated
  • Transcription Factors
  • empty spiracles homeobox proteins
  • Endodeoxyribonucleases