Randomized PAM depletion of phylogenetically-diverse Cas12a nucleases

We report the PAMs of phylogenetically-diverse Cas12a nucleases using a cell-free TXTL-based PAM screen. By adding a 5N randomized PAM library and Cas12a-gRNA in vitro, recognized PAM sequences were cleaved, while non-recognized PAMs remained. By amplifying the non-cleaved DNA, we used next-generation sequencing to analyze the depletion of functional PAMs of these Cas12a nucleases. Overall design: Uncleaved DNA from the randomized PAM library was amplified using Illumina Nextera Indices