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Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

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Structure of the type V-C CRISPR-Cas effector enzyme

Nina Kurihara1, Ryoya Nakagawa1, Hisato Hirano1

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Molecular Cell
|April 2, 2022
PubMed

Insights

This study reveals the structure of Cas12c2, a CRISPR-Cas enzyme, showing how it binds DNA and RNA. It also uncovers a unique RNA processing mechanism essential for genome engineering tools.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • RNA-guided CRISPR-Cas nucleases are essential genome-engineering tools.
  • Type V Cas12 family enzymes exhibit functional diversity.
  • Cas12c2 utilizes CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) for DNA targeting with a TN PAM.

Purpose of the Study:

  • To elucidate the structural basis of Cas12c2 function.
  • To understand the mechanism of guide RNA binding and DNA recognition.
  • To investigate the unique RNA processing activity of Cas12c2.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) for structural determination.
  • Biochemical assays to analyze enzyme activity and interactions.

Main Results:

  • Determined cryo-EM structures of Cas12c2-guide RNA and Cas12c2-guide RNA-target DNA complexes.
  • Revealed an unexpected X-junction architecture formed by crRNA and tracrRNA.
  • Identified specific hydrogen-bonding interactions between Cas12c2 and the TN PAM, recognizing a single T nucleotide.
  • Demonstrated a unique crRNA processing mechanism mediated by the RuvC catalytic site.

Conclusions:

  • The findings provide detailed structural insights into the Cas12c2 CRISPR-Cas effector.
  • This work enhances the mechanistic understanding of diverse type V CRISPR-Cas systems.
  • The unique features of Cas12c2 may offer new possibilities for genome engineering applications.
Keywords:
CRISPRCas12ccryo-EM

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