Abstract
Cas12a is a class 2 type V CRISPR-associated nuclease that uses an effector complex comprised of a single protein activated by a CRISPR-encoded small RNA to cleave double-stranded DNA at specific sites. Cas12a processes unique features as compared to other CRISPR effector nucleases such as Cas9, and has been demonstrated as an effective tool for manipulating complex genomes. Prior studies have indicated that DNA flexibility at the region adjacent to the protospacer-adjacent-motif (PAM) contributes to Cas12a target recognition. Here, we adapted a SELEX-seq approach to further examine the connection between PAM-adjacent DNA flexibility and off-target binding by Cas12a. A DNA library containing DNA-DNA mismatches at PAM + 1 to + 6 positions was generated and subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The bound and unbound populations were sequenced to determine the propensity for off-target binding for each of the individual sequences. Analyzing the position and nucleotide dependency of the DNA-DNA mismatches showed that PAM-dependent Cas12a off-target binding requires unpairing of the protospacer at PAM + 1 and increases with unpairing at PAM + 2 and + 3. This revealed that PAM-adjacent DNA flexibility can tune Cas12a off-target binding. The work adds support to the notion that physical properties of the DNA modulate Cas12a target discrimination, and has implications on Cas12a-based applications.