Discovering and self-processing functional screening of CRISPR-Cas systems by foundation models

Our CHOOSER framework has enabled the identification of new homologues within the Cas12Lambda family. We focused on one particular candidate, EphcCasLambda, to assess its DNA cleavage activity using a PAM depletion assay. To this end, we engineered a plasmid library with 8 randomized nucleotides located immediately upstream of the 5' end of the target sequence. We then subjected these plasmids to in vitro cleavage with purified EphcCasLambda. Post-cleavage, the remaining uncleaved plasmids were isolated and amplified for subsequent sequencing analysis. The sequencing results from the PAM depletion assay are expected to reveal the prefered PAM motifs specificity of EphcCasLambda.