FlexiAsCas12a and other Cas12a variants enhance their applicability in prime editing

Amongst the genome manipulation tools based on the versatile CRISPR-Cas system, prime editing is the most prominent one as a method that enables precise insertions, deletions, and substitutions without inducing double-strand breaks. Cas12a nucleases are widely used for genome editing and nucleic acid detection, owing to their unique properties; however, their relatively long PAM requirements limit their applicability. Our aim was to develop PAM-flexible Cas12a variants capable of functioning effectively within mammalian cells, thereby enabling the cleavage of targets previously inaccessible to Cas12a nucleases. Amongst the Lb-, As-, Mb-, and FnCas12a variants we have developed, flexiAsCas12a (AsCas12a with flexible PAM recognition) has proved to be the most effective, expanding the range of recognized PAM sequences by Cas12a variants to include TCCG, TCCT, GTCG, GTCA, GATT, and AATT sequences. Thereby, it joins the repertoire of Cas12a PAM variants, enabling access to an increasing number of target sequences by Cas12a nucleases. Using the currently available Cas12a variants with relaxed PAM recognition (impLbCas12a, flexiAsCas12a, and enAsCas12a), we have developed circular RNA-guided split prime editors and have validated their functionality on non-canonical PAM sequences.