PAM depletion assay of Cas12 hacker-1
100 ng of the PAM library plasmids were electroporated into the E. coli DH5 electrocompetent cells expressing the CRISPR-Cas12 hacker system. The transformants were plated on the LB agar containing 50 g/mL apramycin and 50 g/mL carbenicillin. Colonies were harvested for plasmid extraction. The amplicon sequencing library was constructed by a two-step amplification method. The PCR product of PAM randomized region was purified using VAHTS DNA Clean Beads (Vazyme Biotech). Then, the amplicon sequencing library was prepared using NEBNext Ultra II DNA Library Prep Kit (Vazyme Biotech) and sequenced on an Illumina Novaseq 6000 platform at HaploX Genomics Center (Jiangxi, China). For data analysis, the number of each PAM sequence in the randomized regions was counted. Subsequently, the proportion of each PAM in the experimental group (targeted by Cas12 hacker) and the control group (non-targeted) was calculated.