TY - JOUR

T1 - Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

AU - Stella, Stefano

AU - Alcón, Pablo

AU - Montoya, Guillermo

N1 - Erratum: Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage Nature 546, 559–563 (2017); doi:10.1038/nature22398

PY - 2017/6/22

Y1 - 2017/6/22

N2 - 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.

AB - 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.

KW - Acidaminococcus

KW - Adenosine Triphosphate

KW - Base Pairing

KW - Crystallography, X-Ray

KW - DNA

KW - DNA Cleavage

KW - Endonucleases

KW - Francisella

KW - Gene Editing

KW - Gram-Positive Bacteria

KW - Lysine

KW - Models, Molecular

KW - Protein Domains

KW - Protein Engineering

KW - RNA, Guide

KW - Substrate Specificity

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1038/nature22398

DO - 10.1038/nature22398

M3 - Letter

C2 - 28562584

VL - 546

SP - 559

EP - 563

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7659

ER -