Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis

Due to lipid-rich cell wall, slow growth and pathogenic nature, it is difficult to manipulate Mycobacterium tuberculosis (Mtb) genome by conventional tools. Recently we have introduced a novel CRISPRi approach for repression of genes in mycobacteria. Although the existing CRISPRi plasmid is proven useful for silencing individual targets, disruption of multiple ORFs remains challenging in mycobacteria. Herein, we report construction of the guide sequence expressing plasmid, pGrna to facilitate cloning and expression of multiple guide sequence cassettes targeting a versatile set of Mtb genes from a single plasmid. Using the modified plasmid, pGrna2, it was shown that expression of all the 10 extracellular sigma factor-encoding genes together with sigB and sigF can be efficiently repressed in Mtb expressing dCas9. In vitro growth analysis indicates that simultaneous knockdown of these non-essential transcriptional regulators is lethal for growth. Importantly, the Δ12sig strain exhibits sensitivity to transcriptional inhibitor rifampicin and oxidative stress diamide, further implying involvement of these genes in controlling bacterial stress response. To the best of my knowledge, this is the first report wherein 12 genes have been efficiently silenced together in a single recombinant strain of Mtb. The modified pGrna2 plasmid offers a powerful tool to decipher the functioning of genes that are redundant or regulate a particular metabolic pathway and can be useful in identification of novel anti-tuberculosis drug targets.