Genome-wide CRISPRi screens reveal the essentialome and determinants for susceptibility to dalbavancin inStaphylococcus aureus

Abstract

Antibiotic resistance and tolerance remain a major problem for treatment of staphylococcal infections. Knowing genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for >Staphylococcus aureus, demonstrated its use by quantifying the essentialome in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we found genes previously reported to be involved in antibiotic susceptibility, but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional knock-out approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, while knockdown of the Shikimate pathway surprisingly has the opposite effect. The results presented here demonstrate the potential of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.

The function of CozE proteins is linked to lipoteichoic acid biosynthesis inStaphylococcus aureus

“…Similar to what was reported previously (26), we were unable to obtain a double Δ cozEa Δ cozEb mutant in NCTC8325-4 by allelic replacement using the pMAD-vector (29). We therefore used an established two-plasmid CRISPR interference (CRISPRi) system for knockdown of gene expression (26, 30). In this system, dCas9 is expressed from an IPTG-inducible promoter on one plasmid, and the gene-specific sgRNA is constitutively expressed from the other.…”

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin resistantStaphylococcus aureus

“…The CRISPRi plasmids used in this study are described in Liu et al, (60). Transformation in E. coli IM08B and S. aureus JE2 was done as described herein (60).…”

“…The CRISPRi plasmids used in this study are described in Liu et al, (60). Transformation in E. coli IM08B and S. aureus JE2 was done as described herein (60). Uptake of the pLOW-dCas9_aad9 plasmid was confirmed by PCR with primers pLOW_aad9_F and pLOW_aad9_R.…”

“…Uptake of the pLOW-dCas9_aad9 plasmid was confirmed by PCR with primers pLOW_aad9_F and pLOW_aad9_R. The No-target sgRNA and sgRNA specific for pbp4 and spx were cloned into vector pVL2336 using Golden Gate cloning as described in Liu et al (60). The purified pVL2336-sgRNA plasmids were transformed into S. aureus JE2 and successful plasmid transformation was confirmed by PCR using the primers pVL2336_5517bp_F and pVL2336_339bp_R.…”

Rationally Designed Pooled CRISPRi-Seq Uncovers an Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

“…CRISPRi can be applied to high throughput levels to create tunable 6,7 and reversible 11 CRISPRi mutant libraries. Several pooled [12][13][14][15][16][17][18][19] and arrayed 7,[20][21][22] CRISPRi-based mutant libraries have been developed to facilitate the study of bacterial essential pathways 12,[22][23][24][25] , identification of antibiotic susceptibility determinants 18,20,[26][27][28][29][30] and novel antimicrobial MOA 7 . Pooled libraries, which typically involve disrupting essential genes en masse, offer streamlined construction.…”