C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism

Liang Liu; Peng Chen; Min Wang; Xueyan Li; Jiuyu Wang; Maolu Yin; Yanli Wang

doi:10.1016/j.molcel.2016.11.040

C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism

Mol Cell. 2017 Jan 19;65(2):310-322. doi: 10.1016/j.molcel.2016.11.040. Epub 2016 Dec 15.

Authors

Liang Liu¹, Peng Chen², Min Wang¹, Xueyan Li², Jiuyu Wang¹, Maolu Yin², Yanli Wang³

Affiliations

¹ Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
² Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
³ Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Collaborative Innovation Center of Genetics and Development, Shanghai 200438, China. Electronic address: [email protected].

PMID: 27989439
DOI: 10.1016/j.molcel.2016.11.040

Abstract

C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.

Keywords: C2c1; CRISPR-Cas system; DNA endonuclease; crystal structure; genome-editing.

MeSH terms

Alicyclobacillus / enzymology*
Alicyclobacillus / genetics
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
CRISPR-Associated Proteins / chemistry
CRISPR-Associated Proteins / genetics
CRISPR-Associated Proteins / metabolism*
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Breaks, Double-Stranded*
DNA, Bacterial / chemistry
DNA, Bacterial / genetics
DNA, Bacterial / metabolism*
Endodeoxyribonucleases / chemistry
Endodeoxyribonucleases / genetics
Endodeoxyribonucleases / metabolism*
Models, Molecular
Nucleic Acid Conformation
Nucleic Acid Heteroduplexes / chemistry
Nucleic Acid Heteroduplexes / genetics
Nucleic Acid Heteroduplexes / metabolism*
Protein Binding
Protein Interaction Domains and Motifs
RNA, Bacterial / chemistry
RNA, Bacterial / genetics
RNA, Bacterial / metabolism*
RNA, Guide, CRISPR-Cas Systems / chemistry
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism*
Structure-Activity Relationship

Substances

Bacterial Proteins
CRISPR-Associated Proteins
DNA, Bacterial
Nucleic Acid Heteroduplexes
RNA, Bacterial
RNA, Guide, CRISPR-Cas Systems
Endodeoxyribonucleases