Cas12a is one of the most commonly used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its target among numerous nonspecific and off-target sites. Cas12a utilizes one-dimensional diffusion along the contour of dsDNA to efficiently search for its target. However, due to a lack of structural information of the transient diffusing complex, the residues mediating the one-dimensional diffusion of Cas12a are unknown. Here, combining single-molecule and ensemble assays, we found that nonspecific interactions between Cas12a and dsDNA at the PAM downstream cause asymmetric target search regions of Cas12a flanking the PAM site, which guided us to identify a positive-charge-enriched alpha helix in the REC2 domain serving as a conserved element to facilitate one-dimensional diffusion-driven target search of AsCas12a, LbCas12a and FnCas12a. In addition, this alpha helix assists the target cleavage process of AsCas12a via stabilizing the cleavage states. Thus, neutralizing positive charges within this helix not only significantly slows target search but also enhances the specificity of AsCas12a both in vitro and in living cells. Similar behaviors are detected when residues mediating diffusion of SpCas9 are mutated. Thus, engineering residues mediating diffusion on dsDNA is a new avenue to optimize and enrich the versatile CRISPR-Cas toolbox.
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