PAM depeletion of LesCas12e
PAM depletion assays were conducted as previously described with modification. Ampicillin-resistant plasmid libraries containing randomized N5-PAM sequences were transformed into E. coli DH5, which had been pre-transformed with a streptomycin-resistant plasmid encoding Cas12e proteins and the cognate sgRNA targeting the N5 PAM plasmid. In the control group, the pre-transformed plasmids encoded the Cas12e protein and a non-targeting cognate sgRNA. After transformation, 1 mL LB medium was added into the cells for 1-hour culturing at 37C. Then, the cells were plated on selective medium containing streptomycin (50 mg/mL) and Ampicillin (100 mg/mL) for 12 h at 37C. Plasmid DNA was extracted and the PAM-including region was amplified with adapters for Illumina sequencing. The 5-nucleotide PAM sequences were extracted, and their frequencies were calculated. PAM sequences depleted by more than 5-fold were identified and used to generate sequence logos via WebLogo3.