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| Title | Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation. |
|---|---|
| Journal, issue, pages | Nat Commun, Year 2026 |
| Publish date | Jan 28, 2026 |
 Authors | Chhandosee Ganguly / Swarmistha Devi Aribam / Alberto Monteiro Dos Santos / Lindsie Martin / Leonard M Thomas / Yihan Shao / Rakhi Rajan /  |
| PubMed Abstract | 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 ...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 FnoCas12a that 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. |
 External links | Nat Commun / PubMed:41605928 |
| Methods | EM (single particle) |
| Resolution | 3.21 - 4.0 Å |
| Structure data | EMDB-48337, PDB-9mkt: EMDB-48338, PDB-9mku: EMDB-48339, PDB-9mkv: EMDB-48340, PDB-9mkw: EMDB-48341, PDB-9mkx: |
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 Keywords | DNA BINDING PROTEIN / Cas12a / bridge helix / loop-to-helical transition / conformational cascade / allostery / off-target DNA cleavage |
francisella tularensis subsp. novicida u112 (bacteria)