Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

Research output: Contribution to journalLetterResearchpeer-review

Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

Original languageEnglish
Issue number7659
Pages (from-to)559-563
Number of pages5
Publication statusPublished - 22 Jun 2017

Bibliographical note

Erratum: Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage
Nature 546, 559–563 (2017); doi:10.1038/nature22398

    Research areas

  • Acidaminococcus, Adenosine Triphosphate, Base Pairing, Crystallography, X-Ray, DNA, DNA Cleavage, Endonucleases, Francisella, Gene Editing, Gram-Positive Bacteria, Lysine, Models, Molecular, Protein Domains, Protein Engineering, RNA, Guide, Substrate Specificity, Journal Article, Research Support, Non-U.S. Gov't

ID: 184290061