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 in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis‐ and trans‐cleavages and show its conserved role in target discrimination among Cas nucleases.

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