A CRISPRi-based genetic resource to study essentialStaphylococcus aureusgenes

Abstract

We have optimized a CRISPR interference system to facilitate gene knockdown in the gram-positive bacterial pathogen Staphylococcus aureus. For this, we used a CRISPRi system derived from Streptococcus pyogenes which requires the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In the system described in this work, dcas9 is expressed from a single copy in the chromosome of methicillin resistant S. aureus (MRSA) strains COL or JE2, under the control of a tightly regulated promoter inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables high-efficiency, inducible, knockdown of both essential and nonessential genes and was used for the construction of the Lisbon CRISPRi mutant library (LCML) of 241 strains, in the background of JE2, containing sgRNAs targeting 209 essential genes/operons. This library allows the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library which consists of nearly 2000 strains, each containing a transposon insertion within a non-essential gene. Together the two libraries should facilitate the study of S. aureus pathogenesis and biology.

Processive movement ofStaphylococcus aureusessential septal peptidoglycan synthases is independent of FtsZ treadmilling and drives cell constriction

“…Strains ectopically producing HT-DivIB, RodA-HT, FtsW-HT, FtsZ-HT, EzrA-HT and iST-PBP1 were constructed using pBCB13 and pBCB43 vectors 43,44 , which are derivatives of pMAD that allow gene expression from the ectopic spa locus under the control of (IPTG-inducible) spac promoter and (Atc-inducible) xyl-tetO promoter, respectively. Briefly, a codon-optimized halo-tag sequence (Supplementary Information) was amplified with the primer pairs 6713/6714 and 6715/6716, digested with SmaI and cloned into equally digested pBCB13, to generate plasmids pBCB13-Nht and pBCB13-htC, respectively.…”

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

“…Thus, CRISPRi will also inhibit expression of other genes co-transcribed with the target gene. CRISPRi has proven highly useful for functional studies of essential genes in a diversity of bacteria, including S. aureus (8)(9)(10)(11)(12). The relative simplicity and programmability of CRISPRi has also allowed upscaling to genome-wide CRISPRi libraries in bacterial species such as Escherichia coli (13)(14)(15), Streptococcus pneumoniae (16), Streptococcus salivarius (17), Bacillus subtilis (18), Mycobacterium tuberculosis (19), Acinetobacter baumanii (20) and Vibrio natriegens (21).…”