Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Arturo Carabias; Anders Fuglsang; Piero Temperini; Tillmann Pape; Nicholas Sofos; Stefano Stella; Simon Erlendsson; Guillermo Montoya

doi:10.1038/s41467-021-24707-3

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Arturo Carabias, Anders Fuglsang, Piero Temperini, Tillmann Pape, Nicholas Sofos, Stefano Stella, Simon Erlendsson, Guillermo Montoya^*

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

27 Citations (Scopus)

Abstract

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

Original language	English
Article number	4476
Journal	Nature Communications
Volume	12
Issue number	1
ISSN	2041-1723
DOIs	https://doi.org/10.1038/s41467-021-24707-3
Publication status	Published - 1 Dec 2021

Bibliographical note

Funding Information:
We thank the Danish Cryo-EM National Facility in CFIM at the University of Copenhagen for support during cryo-EM data collection supported by grant NNF0024386. Data processing has been performed at the Computerome, the Danish National Computer for Life Sciences. G.M. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. SE is funded by the Novo Nordisk Foundation (Grant NNF17OC0030788). G.M. is part of the Novo Nordisk Foundation Center for Protein Research (CPR), which is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). This work was also supported by the cryoNET (grant NNF17SA0030214), and Distinguished Investigator (NNF18OC0055061) grants to G.M.

Publisher Copyright:
© 2021, The Author(s).

Access to Document

10.1038/s41467-021-24707-3

Cite this

@article{d76560671755408aa6503cc4139c7d46,

title = "Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3",

abstract = "CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.",

author = "Arturo Carabias and Anders Fuglsang and Piero Temperini and Tillmann Pape and Nicholas Sofos and Stefano Stella and Simon Erlendsson and Guillermo Montoya",

note = "Funding Information: We thank the Danish Cryo-EM National Facility in CFIM at the University of Copenhagen for support during cryo-EM data collection supported by grant NNF0024386. Data processing has been performed at the Computerome, the Danish National Computer for Life Sciences. G.M. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. SE is funded by the Novo Nordisk Foundation (Grant NNF17OC0030788). G.M. is part of the Novo Nordisk Foundation Center for Protein Research (CPR), which is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). This work was also supported by the cryoNET (grant NNF17SA0030214), and Distinguished Investigator (NNF18OC0055061) grants to G.M. Publisher Copyright: {\textcopyright} 2021, The Author(s).",

year = "2021",

month = dec,

day = "1",

doi = "10.1038/s41467-021-24707-3",

language = "English",

volume = "12",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "nature publishing group",

number = "1",

}

TY - JOUR

T1 - Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

AU - Carabias, Arturo

AU - Fuglsang, Anders

AU - Temperini, Piero

AU - Pape, Tillmann

AU - Sofos, Nicholas

AU - Stella, Stefano

AU - Erlendsson, Simon

AU - Montoya, Guillermo

N1 - Funding Information: We thank the Danish Cryo-EM National Facility in CFIM at the University of Copenhagen for support during cryo-EM data collection supported by grant NNF0024386. Data processing has been performed at the Computerome, the Danish National Computer for Life Sciences. G.M. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. SE is funded by the Novo Nordisk Foundation (Grant NNF17OC0030788). G.M. is part of the Novo Nordisk Foundation Center for Protein Research (CPR), which is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). This work was also supported by the cryoNET (grant NNF17SA0030214), and Distinguished Investigator (NNF18OC0055061) grants to G.M. Publisher Copyright: © 2021, The Author(s).

PY - 2021/12/1

Y1 - 2021/12/1

N2 - CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

AB - CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

UR - http://www.scopus.com/inward/record.url?scp=85111127933&partnerID=8YFLogxK

U2 - 10.1038/s41467-021-24707-3

DO - 10.1038/s41467-021-24707-3

M3 - Journal article

C2 - 34294706

AN - SCOPUS:85111127933

VL - 12

JO - Nature Communications

JF - Nature Communications

SN - 2041-1723

IS - 1

M1 - 4476

ER -

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Abstract

Bibliographical note

Access to Document

Other files and links

Cite this