Molecular Cell
Volume 81, Issue 3, 4 February 2021, Pages 558-570.e3
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Article
Structure of the miniature type V-F CRISPR-Cas effector enzyme

https://doi.org/10.1016/j.molcel.2020.11.035Get rights and content
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Highlights

  • Cryo-EM structure of Cas12f in complex with a guide RNA and its target DNA

  • Type V-F effector complex consisting of a Cas12f dimer and a single guide RNA

  • Molecular mechanism of the RNA-guided DNA cleavage by the miniature Cas12f

  • Evolutionary insights into a possible origin of the type V CRISPR-Cas enzymes

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.

Keywords

CRISPR-Cas
Cas12f
Cryo-EM
genome editing

Cited by (0)

2

Present address: Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

3

Present address: Peptidream Inc., 3-25-23 Tonomachi, Kawasaki-ku, Kawasaki City, Kanagawa Prefecture, 210-0821, Japan

4

Present address: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

5

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