Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA

RIP-seq analysis to identify SpyCas9 and LrhCas9 bound RNAs using co-immunoprecipitation and sequencing. SpyCas9, tracrRNA, and CRIPSR array encoding loci of S. pyogenes SF370 were cloned into a low copy plasmid. DNA encoding 3XFLAG tag was inserted upstream of the stop codon of the SpyCas9 gene. Mutations were introduced into the leader region through Q5 mutagenesis. The resulting plasmids were transferred into a E. coli BW25113 strain with the endogenous CRISPR system removed. Co-immunoprecipitations were conducted to extract the SpyCas9 bound RNA for RIP-seq using the untagged SpyCas9 as control. For Lactobacillus rhamnosus, the plasmid harboring the LrhCas9 encoding locus and 3XFLAG tag was transferred into Lactobacillus rhamnosus and used for Co-immunoprecipitation and extraction of RNA for RIP-seq. The wild type Lactobacillus rhamnosus was used as control. R1 and R2 represent two biological replicates from coIP experiments used for RIP-seq.