Extended Data Fig. 9: RIP-seq analysis of RNAs bound to Cas9 from Lactobacillus rhamnosus GG. LrhCas9 with or without a 3xFLAG affinity was expressed from a plasmid, and the lysate was subjected to RIP-seq analysis.
From: Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA
LrhCas9 with or without a 3xFLAG affinity was expressed from a plasmid, and the lysate was subjected to RIP-seq analysis. a, Western blotting analysis of samples for RIP-seq using LrhCas9 in Lactobacillus rhamnosus GG. Western blotting confirmed enrichment of LrhCas9-FLAG. Co-immunoprecipitated RNAs were isolated and subjected to next-generation sequencing. Results from duplicate independent experiments are shown on the left and right. b, Distribution of RNA classes based on total mapped reads. hkRNAs: house-keeping RNAs. ncRNAs: non-coding RNAs. c, Mapped reads for the CRISPR locus with the genome of L. rhamnosus GG (NC_013198.1). The scale above the plot indicates the location in the genome. The CRISPR locus is encoded on the negative strand. Positional coverage for total reads and reads aligning with a reference length ≤ 50 nts was normalized based on the total number of aligned reads in each sample. The maximum read length for the NGS run was 76 nts, explaining the drop in unfiltered read counts shortly downstream of the transcriptional start site. See Supplementary Table 1 for statistics from the RIP-seq analyses. Results in b and c are representative of duplicate independent experiments. d, Plasmid clearance by the CRISPR-Cas9 system in L. rhamnosus GG. The corresponding target of the ecrRNA or crRNA1 was encoded within the transformed plasmid. L.O.D.: limit of detection. There was no detectable ecrRNA-directed plasmid clearance. Values represent the geometric mean and standard deviation from three independent experiments starting from separate colonies. **: P < 0.01. n.s.: P > 0.05. Statistical tests were performed using a two-tailed Student’s t-test with unequal variance, n = 3.
