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Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity inStreptococcus pyogenes(Spy) Cas9. Here, we examined a BH variant of Cas12a fromFrancisella novicida(FnoCas12aKD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12aKD2Paccumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2Palso possess reducedtrans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in bothcis‐andtrans‐cleavages and show its conserved role in target discrimination among Cas nucleases.
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This content will become publicly available on January 10, 2026
Helical transition of the bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation
ABSTRACT CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed aFrancisella novicidaCas12a variant (FnoCas12aKD2P) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH-mediated activation of Cas12a. We captured five structures of FnoCas12aKD2Pat different states of conformational activation. Comparison with wild-type (FnoCas12aWT) structures unravels a mechanism where BH acts as a trigger that allosterically activates REC lobe movements by tracking the number of base pairs in the growing RNA-DNA hybrid to undergo a loop-to-helical transition and bending to latch onto the hybrid. The transition of the BH is coupled to the previously reported loop-to-helix transition of the “lid”, essential for opening RuvC endonuclease, through direct interactions of residues of the BH and the lid. We also observe structural details of cooperativity of BH and “helix-1” of RuvC for activation, a previously proposed interaction. Overall, our study enables development of high-fidelity Cas12a and Cas9 variants by BH-modifications.
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- Award ID(s):
- 2424888
- PAR ID:
- 10615862
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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