Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

Autor:	Strohkendl I; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA., Saha A; Department of Bioengineering, University of California, Riverside, Riverside, CA 92521, USA., Moy C; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA., Nguyen AH; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA., Ahsan M; Department of Bioengineering, University of California, Riverside, Riverside, CA 92521, USA., Russell R; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX 78712, USA., Palermo G; Department of Bioengineering, University of California, Riverside, Riverside, CA 92521, USA; Department of Chemistry, University of California, Riverside, Riverside, CA 92521, USA., Taylor DW; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; LIVESTRONG Cancer Institute, Dell Medical School, University of Texas at Austin, Austin, TX 78712, USA. Electronic address: dtaylor@utexas.edu.
Jazyk:	angličtina
Zdroj:	Molecular cell [Mol Cell] 2024 Jul 25; Vol. 84 (14), pp. 2717-2731.e6. Date of Electronic Publication: 2024 Jul 01.
DOI:	10.1016/j.molcel.2024.06.007
Abstrakt:	The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors. Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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