CRISPRi-Mediated Gene Silencing in Biofilm Cycle and Quorum Sensing

Biofilm is an assembly of architecturally, bacterial mono-species with different functional attributes but with similar genetical makeup. It is generally formed when multicellularity is induced to cope up with the survival of their population density at threshold level. BfC (biofilm cycle) undergoes four different phases, namely, attachment on substratum (both biotic as well as abiotic), nonmotile interactive growth phase simulated by QS (quorum sensing) factors, biosynthesis of EPS (exopolymeric substances) matrix and its mediated maturation, and fourth disassembly factors inducing detachment. These phases are regulated by the activation of number of genes (csrA, fliA, flhD, luxS, lsrK, lsrR, qseB, mqsR, pfs, qseC, motA etc) that governs virulence factors. bcsA operon, csgD, fimB, hha, pgaC, etc. are reported to be activated only during the BfC while quorum sensing is achieved through autoinducers. N-acyl-homoserine lactones and autoinducing peptides which form AHL system of Gram-negative bacteria and AIP system of Gram-positive bacteria, respectively, serve as major signaling molecules in quorum sensing. Prokaryotes possess multiple pathways to repair endogenous DNA and double-strand breaks via bacterial genomes deploying CRISPR-Cas is menacing alien DNA from varied sources. Therefore, CRISPR interference (CRISPRi) inhibits biofilm formation by changing gene expression of quorum sensing cassettes. In CRISPRi, small guide RNA activates the inactive dCas9 proteins catalytically to adhere with corresponding promoter region and in turn silencing the QS gene expression by sterically hindering transcription initiation or elongation. CRISPRi strategy also goes with silencing of sensor kinase genes reproducing phenotypic flaws in the biofilm formation and surface motility. Decreased motility, swarming movements, lack of adhesins, and derangement of biofilm assist in decreasing virulence and pathogenicity.