Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage
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Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. / Stella, Stefano; Alcón, Pablo; Montoya, Guillermo.
I: Nature, Bind 546, Nr. 7659, 22.06.2017, s. 559-563.Publikation: Bidrag til tidsskrift › Letter › Forskning › fagfællebedømt
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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 -
ID: 184290061