CRISPRi-seq identified genetic factors modulating susceptibility to β-lactams in S. aureus.

The CRISPRi library, targeting over 97% of the genomic features of S. aureus NCTC8325–4 [59], was transformed into a NCTC8325–4 strain where dCas9 expression is controlled by a tetracycline-inducible promoter on the chromosome [60]. The induced library was grown in the presence and absence of 0.008 µg/mL penicillin G for ~20 generations at 37ºC in milk. Plasmids were subsequently isolated, and the sgRNA abundances were determined by Illumina sequencing, as previously described [58]. Log2 fold-changes (Log2FC) between penicillin G treated (PenG) and non-treated (minPenG) samples are presented for each sgRNA, along with the corresponding p-values (Padj). Explanation of columns headings: sgRNA; sgRNA identification number, Locus tag; locus tag of the gene in S. aureus NCTC8325–4 targeted by the sgRNA, name; gene name, minPenG essentiality; whether the targeted gene is classified as essential, neutral or costly for growth without penicillin G added, PenG essentiality; whether the targeted gene is classified as essential, neutral or costly for growth with 0.008 µg/mL penicillin G added, Log2FC; log2 fold-change in sgRNA abundance between conditions with or without penicillin G; Padj; adjusted P-value, Interactions; whether (yes or no) there were significant (Padj 2FC between conditions with or without penicillin G. Transcriptional units (sgRNAs) whose knockdown were found to significantly alter sensitivity to penicillin are labelled with colors, where blue indicates increased sensitivity and red indicates reduced sensitivity. SAOUHSC_00659 (cxaR) is shown in bold. (XLSX)