Randomized PAM depletion of AsCas12a

We report the PAMs of AsCas12a using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and AsCas12a-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of AsCas12a. Overall design: Uncleaved DNA from the randomized PAM library was amplified using Illumina Nextera Indices