PAM Identification Assay for Cas12a Orthologs

The in vitro PAM identification assay was performed as described previously¹⁸ . Briefly, whole cell lysate from HEK293T cells, overexpressing one of the Cas12a orthologs was prepared with lysis buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol, 0.1% Triton X-100) supplemented with EDTA-free cOmplete Protease Inhibitor Cocktail (Roche). CrRNA with corresponding direct repeat sequences were transcribed in vitro using custom oligonucleotides and HiScribe T7 in vitro Transcription Kit (NEB) according to the manufacturer’s recommended protocol for small RNA transcripts. The PAM library consisted of a pUC19 plasmid carrying a degenerate 8-bp sequence 5’ of a 33-bp target site^{7 (link)}. The library was pre-cleaved with XmnI and column purified prior to use (Qiagen). Each in vitro cleavage reaction consisted of 1 ul 10x CutSmart buffer (NEB), 200 ng PAM library, 500 ng in vitro transcribed crRNA, 10 ul cell lysate and water for a total volume of 20 ul. Reactions were incubated at 37°C for one hour and stopped by adding 500 ul buffer PB (Qiagen) followed by column purification. Purified DNA was amplified and sequenced using a MiSeq (Illumina) with a single-end 150-cycle kit. Sequencing results were entered into the PAM discovery pipeline^{7 (link)}.