Characterization of RNA cleavage and PFS recognition by CRISPR-Cas12a3 and Cas12a4 nucleases
To investigate protospacer-flanking sequence (PFS) recognition preferences of CRISPR-Cas12a3 and Cas12a4 nucleases, we screened a library of possible PFS sequences in Escherichia coli using plasmid interference assays. To determine RNA substrate preferences, we performed nanopore direct RNA sequencing of cell-free transcription–translation reactions and complementary in vitro assays.