Nature communicationsJan 28, 2026

The bridge helix of Cas12a controls DNA binding and cutting activity

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Feb 1, 2026

Abstract

Five structures of a modified Cas12a variant reveal key conformational changes associated with DNA cleavage.

Conformational activation of the Cas12a variant involves a loop-to-helical transition and bending of the bridge helix.
The changes in the bridge helix act as an allosteric trigger for the propagation of RNA-DNA hybrids.
The remodeling of the bridge helix and helix 1 of the RuvC motif-II is coupled and necessary for hybrid accommodation.
The transition of the bridge helix is crucial for opening the RuvC active site pocket, allowing DNA entry and cleavage.
Structural comparisons among Cas12 and Cas9 families highlight the diversity of bridge helix organization in these enzymes.

Simplified

CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12athat are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes. KD2P KD2P WT

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Competing interests

Competing interests: R.R. is an inventor on US patents related to FnoCas12a bridge helix variants (US11459552B2 and US12163166B2; status = granted) that were filed by the University of Oklahoma. These patents describe how amino acid substitutions in the bridge helix of Cas12a increase DNA cleavage selectivity. This manuscript presents the structure and mechanism of one of the bridge helix variants (FnoCas12a-K969P/D970P, abbreviated as FnoCas12aKD2P) that was described in the patents. All other authors declare no competing interests.

PubMed

The bridge helix of Cas12a controls DNA binding and cutting activity

Abstract

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