PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.

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

SUBMITTER: Allen A 

PROVIDER: S-EPMC11811290 | biostudies-literature | 2025 Feb

REPOSITORIES: biostudies-literature