RNA-seq transcriptomic analyses of group B Streptococcus Cas9 variants and CRISPRi knockdown strains

Group B Streptococcus (GBS) strain CNCTC 10/84 was modified to have specific mutations to the cas9 gene: allelic exchange replacement with a chloramphenicol resistance marker (Cas9 knockout), D10A and H845A (catalytically inactive dCas9), or R1339A and R1441A (sCas9 unable to scan for protospacer adjacent motifs). Wild type (WT) and mutant strains were grown in biological triplicate samples and used for RNA purification at two growth timepoints: OD600=0.6 and OD600=1.2. Additionally, CNCTC 10/84 dCas9 and A909 dCas9 were transformed with p3015b expression plasmids expressing sgRNA cassettes designed to downregulate the cyl operon in CNCTC 10/84 dCas9 and the covR/covS two-component system in A909 dCas9. Triplicate samples were grown alongside control transformants bearing "sham" sgRNA plasmid. RNA was purified at OD600=1.2. All RNA samples were used for RNA-seq and subsequent bioinformatic analyses. Overall design: Comparative transcriptomics of CNCTC 10/84 Cas9 variants (KO, dCas9, sCas9, and WT) between and across two growth phases (OD 0.6 and OD 1.2). Further comparison of CNCTC 10/84 dCas9 and A909 dCas9 at OD 1.2 in which specific genes (cyl and CovR, respectively) have been downregulated; these comparisons are made to "sham" controls in which the sgRNA does not target any chromosomal genes.