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.