PAM screening of OpbCas12e
The PAM screening using in vitro systems is adapted from a previous study with modifications. The N5 plasmid library was incubated with Cas12e protein and sgRNA at a ratio of 10:15:1 (Cas12e:sgRNA:plasmid) at 37C for 30 min in a cleavage buffer (20 mM TrisHCl pH 7.5 at 37C, 50/150/300 mM NaCl, 10 mM MgCl2, 1 mM DTT). The reaction was stopped by adding 15 mM EDTA with 1 mg/mL proteinase K and was incubated at 50C for 30 min. Linearized plasmid was extracted and end-repaired by T4 DNA polymerase (Thermo Fisher Scientific) with 1 mM dNTP (Sangon Biotech). A dA was added to the 3 end of the products by Dreamtaq polymerase (Thermo Fisher Scientific) with 1 mM dATP (Sangon Biotech). Next, the adapters with a 3 dT overhang was ligated to the purified products with the same mass concentration by fast T4 DNA ligase (Beyotime). A fragment was amplified from the region containing the N5 PAMs using two primers, one pairing to the upstream of the PAM, the other pairing to the adapter. Next the purified PCR products were applied to the TIANSeq Fast DNA Library Prep Kit (TIANGEN) for Illumina Novaseq PE150 next-generation sequencing (NGS). As a control, N5 plasmid library was incubated in the blank cleavage buffer at 37C for 30 min. Then a fragment was PCR amplified by two primers paired to the upstream and downstream of the PAM before applied to NGS library preparation using kits. The depletion fold-change for each PAM were analyzed using the number of matched reads in Cas12e and control groups normalized with total reads.