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

Publication Type:

Journal Article


Cell, Volume 167, Issue 7, p.1814-1828.e12 (2016)


Alicyclobacillus, CRISPR-Cas Systems, Crystallography, X-Ray, Endodeoxyribonucleases, Gene Editing, Homeodomain Proteins, Humans, Models, Molecular, RNA, Untranslated, Transcription Factors


<p>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&nbsp;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&nbsp;inserted tryptophan, facilitating zippering up of&nbsp;20-bp guide RNA:target DNA heteroduplex on ternary complex&nbsp;formation. Notably, the PAM-interacting cleft adopts&nbsp;a &quot;locked&quot; 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.</p>

PDB: 5U34 (AacC2c1-sgRNA binary complex), PDB: 5U31 (AacC2c1-sgRNA-DNA ternary complex containing excess 8-mer non-target DNA strand), PDB: 5U30 (AacC2c1-sgRNA-extended target DNA ternary complex), and PDB: 5U33 (AacC2c1-sgRNA-extended non-target DNA ternary complex)
AacC2c1-sgRNA-DNA Ternary Complex