CRISPR RNA-dependent binding and cleavage of endogenous RNAs by the Campylobacter jejuni Cas9

This study investigates the RNA targets and cleavage sites of endogenous Cas9 in the food-borne pathogen Campylobacter jejuni. Direct RNA binding targets of Cas9 in C. jejuni strain NCTC11168 were determined using RIP-seq. The Cleavage sites were then predicted in the RNA targets by comparing total transcriptome data from WT and deletion (cas9, crRNA3, tracrRNA, CRISPR-tracrRNA) strains. PAMs for the CjeCas9 were enriched using the PAM-SCANR platform, which operates through a GFP reporter gene. Upon GFP (and thus functional PAM) enrichment, fluorescing cells were isolated using FACS and prepared plasmid DNA was amplified and prepared for sequencing. Overall design: Identification of Cas9 binding and cleavage sites in C. jejuni strain NCTC11168. Four total files are included for the PAM-SCANR dataset. Raw reads are available for both biological replicates, both pre- and post-FACS. After extracting the variable PAM region from the reads, enrichment was calculated by comparing the pre-FACS PAM counts to the post-FACS counts. These calculations are presented in the included processed data file.