CRISPR-Cas systems comprise a CRISPR RNA (crRNA)-guided CRISPR-associated (Cas) nuclease for providing immune protection. The complementary base pairing between crRNA and the invader genome leads to the formation of an "R-loop", which triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. This molecular mechanism has been repurposed for genome applications using Cas9 and Cas12a. Cas12a has several favorable features for applications including its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage. Gene editing with these Cas proteins, however, has some setbacks due to off-target and non-specific DNA cleavages. To increase the specificity in DNA cleavage, we introduced proline/alanine substitutions at different positions along a conserved arginine/lysine-rich "bridge helix" (BH) of Cas12a that plays an integral role in mediating conformational changes needed for DNA cleavage. Cleavage kinetics analyses reveal that enhanced helical integrity of the BH of Francisella novicida Cas12a provided by alanine substitutions increases DNA cleavage efficiency, while reducing the ability of the variants to discriminate DNA mismatches. Proline substitutions demonstrate an opposite effect by reducing the efficiency of cleaving on-target DNA, but almost completely abolishing linearization of a target with a mismatch in the middle of the R-loop. These results parallel those reported for Cas9 and show that balancing the helicity of BH through rational amino acid substitutions can finetune Cas12a's off-target profiles. This may provide a strategy for enhancing specificity of Cas12a in genome manipulation.
