PAM-Adjacent DNA Flexibility Tunes CRISPR-Cas12a Off-Target Binding

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 were generated, and library was subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The work adds support to the notion that physical property of the DNA modulates Cas12a target discrimination and has implications in Cas12a-based applications.