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

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

Author information

1
Structurel Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: yangh3@mskcc.org.
2
Structurel Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
3
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; NE-CAT, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60349, USA.
4
Structurel Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pateld@mskcc.org.

Abstract

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

PMID:
27984729
PMCID:
PMC5278635
DOI:
10.1016/j.cell.2016.11.053
[Indexed for MEDLINE]
Free PMC Article

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