FIG 4.

The CRISPRi system can simultaneously target multiple genes or sites. (A) Knockdown of hla and spa simultaneously by CRISPRi. Both genes are efficiently silenced in the double-knockdown strain. (B) Enhanced knockdown of hla by expression of both sgRNA1 and sgRNA2. The hla knockdown levels in the single- and double-knockdown strains were determined by alpha hemolysis and qRT-PCR. The alpha hemolysis data are representative of three independent experiments. The error bars indicate the standard errors of the means of three biological replicates. *, P < 0.05; **, P < 0.01. Abbreviations: NSD0, NCTC 8325 carrying plasmid pSD1; NSD1, NCTC 8325 expressing the hla sgRNA1; NSD2, NCTC 8325 expressing the hla sgRNA2; NSD7, NCTC 8325 expressing the spa sgRNA1; NSD8, NCTC 8325 expressing both the hla sgRNA1 and the hla sgRNA2; NSD9, NCTC 8325 expressing both the hla sgRNA2 and the spa sgRNA1.