SNAP annotations: PDB entry 8sfn

PDB-id

8sfn; DSSR-derived features in text and JSON formats; DNAproDB

Class

DNA binding protein-DNA-RNA

Method

cryo-EM (3.3 Å)

Summary

Wt crispr-cas12a with a 16bp r-loop and nontarget strand in the ruvc active site.

Reference

Strohkendl I, Saha A, Moy C, Nguyen AH, Ahsan M, Russell R, Palermo G, Taylor DW (2024): "Cas12a domain flexibility guides R-loop formation and forces RuvC resetting." Mol.Cell, 84, 2717-2731.e6. doi: 10.1016/j.molcel.2024.06.007.

Abstract

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.