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

B群链球菌（Group B Streptococcus，GBS）菌株CNCTC 10/84经基因工程改造，在cas9基因位点引入三类特异性突变：一是通过氯霉素抗性标记进行等位交换替换以实现cas9基因敲除（Cas9 knockout，Cas9敲除株）；二是引入D10A与H845A突变，获得催化失活的dCas9；三是引入R1339A与R1441A突变，得到无法识别原间隔序列邻近基序（protospacer adjacent motifs，PAM）的sCas9。野生型（Wild type，WT）菌株与各突变菌株均设置三份生物学重复样本，分别在两个生长时间点（OD₆₀₀=0.6与OD₆₀₀=1.2）收集样本用于RNA纯化。此外，将CNCTC 10/84 dCas9与A909 dCas9分别转化携带p3015b表达质粒的菌株，该质粒搭载靶向下调CNCTC 10/84 dCas9中cyl操纵子、以及A909 dCas9中covR/covS双组分系统的单向导RNA（single guide RNA，sgRNA）表达盒。同时设置携带"sham" sgRNA质粒的对照转化株进行平行培养，并在OD₆₀₀=1.2时收集样本纯化RNA。所有RNA样本均用于RNA测序（RNA-seq）及后续生物信息学分析。实验整体设计：针对CNCTC 10/84的四类Cas9变体（Cas9敲除株、dCas9、sCas9及野生型），在两个生长阶段（OD₆₀₀=0.6与OD₆₀₀=1.2）内及跨阶段开展比较转录组学分析；进一步在OD₆₀₀=1.2条件下，比较分别靶向下调cyl基因（CNCTC 10/84 dCas9）与covR基因（A909 dCas9）的菌株，及其对应的"sham" sgRNA对照株（该sgRNA不靶向任何染色体基因）的转录组差异。