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

Stefano Stella, Pablo Alcón, Guillermo Montoya

Research output: Contribution to journalLetterResearchpeer-review

157 Citations (Scopus)

Abstract

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
JournalNature
Volume546
Issue number7659
Pages (from-to)559-563
Number of pages5
ISSN0028-0836
DOIs
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

Keywords

  • 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

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