PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease

• Hui Yang ^[1] ; Pu Gao ; Kanagalaghatta R. Rajashankar
  1. [1] Memorial Sloan Kettering Cancer Center

     Memorial Sloan Kettering Cancer Center

     Estados Unidos

     
• Localización: Cell, ISSN 0092-8674, Vol. 167, Nº. 7, 2016, págs. 1814-1828
• Idioma: inglés
• Texto completo no disponible (Saber más ...)
• Resumen

  • 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.