The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited
by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence.
Here we have aimed to loosen these PAM constraints and have generated new PAM mutant
variants of the four Cas12a orthologs that are active in mammalian and plant cells,
by combining the mutations of their corresponding RR and RVR variants with altered
PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an
in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based
assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also
includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a)
was found to further increase the activity of that variant in a PAM-dependent manner.
Due to the overlapping but still different PAM preferences of impLbCas12a and the
recently reported enAsCas12a variant, they complement each other to provide increased
efficiency for genome editing and transcriptome modulating applications.
