title: PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.
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.
authors.affiliation: Structurel Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: yangh3@mskcc.org.
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: Pu
authors.initials: P
authors.lastname: Gao
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; NE-CAT, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60349, USA.
authors.forename: Kanagalaghatta R
authors.initials: KR
authors.lastname: Rajashankar
authors.affiliation: Structurel Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pateld@mskcc.org.
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D056450:Alicyclobacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D022661:RNA, Untranslated / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D000099260:Empty Spiracles Homeobox Proteins
chemical_list: D004706:Endodeoxyribonucleases; D018398:Homeodomain Proteins; D022661:RNA, Untranslated; D014157:Transcription Factors; D000099260:Empty Spiracles Homeobox Proteins
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