Structure of the miniature type V-F CRISPR-Cas effector enzyme

RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and...

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Bibliographic Details
Published in:Molecular cell Vol. 81; no. 3; p. 558
Main Authors: Takeda, Satoru N, Nakagawa, Ryoya, Okazaki, Sae, Hirano, Hisato, Kobayashi, Kan, Kusakizako, Tsukasa, Nishizawa, Tomohiro, Yamashita, Keitaro, Nishimasu, Hiroshi, Nureki, Osamu
Format: Journal Article
Language:English
Published: United States 04.02.2021
Subjects:
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins - genetics
CRISPR-Associated Proteins - metabolism
CRISPR-Associated Proteins - ultrastructure
CRISPR-Cas Systems
Cryoelectron Microscopy
DNA - genetics
DNA - metabolism
DNA - ultrastructure
Gene Editing
Models, Molecular
Nucleotide Motifs
Protein Binding
Protein Interaction Domains and Motifs
Protein Multimerization
Protein Subunits
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
RNA, Guide, CRISPR-Cas Systems - ultrastructure
Structure-Activity Relationship
Cas12f
CRISPR-Cas
genome editing
Cryo-EM
ISSN:1097-4164, 1097-4164
Online Access:Get more information
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Summary:RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
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ISSN:1097-4164
1097-4164
DOI:10.1016/j.molcel.2020.11.035