Inducible CRISPRi/dCas9 platforms in Lactococcus lactis; application in essential gene silencing

Plasmid- and genome-based inducible CRISPRi systems were developed for Lactococcus lactis. The nisin-inducible promoter PnisA was used to drive the expression of dCas9 from Streptococcus pyogenes, while the strong constitutive L. lactis promoter Pusp45 was used to express single-guide RNA. In addition, a superfolder fluorescence protein was fused to the C-terminal of dCas9 to visualize the induction level of the fusion protein. Both plasmid systems were successfully used to inhibit the expression of the firefly luciferase reporter gene. The CRISPRi system was tested by silencing the genes encoding the major autolysin AcmA, tubulin FtsZ and penicillin-binding protein Pbp2b, the absence of which resulted in the expected morphological phenotypes. However, the plasmid-based systems were leaky, and mutant phenotypes were already observed without the addition of the inducer nisin. An improved CRISPRi system was obtained by inserting dcas9 gene variants into the L. lactis chromosome to dcrease their copy number. The chromosome-based (cb) CRISPRi system lowered the expression of dCas9-sfGFP appr. 20 times compared to the plasmid-based system and the cells maintained their normal morphology in the absence of nisin. The tight cbCRISPRi system was used to silence the essential L. lactis gene ybeY, which resulted in a dramatic growth defect. Introducing a synonymous mutation of amino acid codon at the sgRNA recognition site on ybeY can recover the phenotype. RNA sequencing of the ybeY-silenced strain indicated that decreasing the expression of ybeY downregulated the expression of proteins that are part of the 30S and 50S subunits. In addition, the expression of large numbers of genes encoding membrane proteins, including ATP synthase subunits, sugar/amino acid permeases, and multiple types of transporters, was downregulated upon the silencing of ybeY. The CRISPRi systems contrusted in this study will be a valuable genetic tool box for future research of L. lactis.