Fig. 4. Drug discovery techniques combining drug-to-target and target-to-drug approaches. A) Target-based whole-cell phenotypic screening. In this technique, a compound library is screened against cells over-expressing a target protein (from a mycobacterial vector). Assessing cell viability at a fixed drug concentration enables the comparison of drug sensitivities between strains with and without the over-expression of the target protein and can reveal inhibitor-target engagement. B) Target protein knockdown by CRISPRi. In this approach, target protein expression is reduced by interfering with the transcription of mRNA. Anhydrotetracycline (ATc) regulates the transcription of a small guide RNA (sgRNA) and a catalytically dead endonuclease, dCas9. The sgRNA contains a short complementary sequence to the target gene and a region for dCas9 recognition. A proto-spacer adjacent motif (PAM) is essential for dCas9 binding. The sgRNA–dCas9 complex anneals to the target sequence, adjacent to PAM, blocking transcription by RNA polymerase, and depleting the expression of the target protein. C) Target protein knockdown by protease degradation allows the reduction of a specific protein through targeted degradation. In this specific approach, a degradation tag (DAS) and a barcode are integrated onto the 3′ end of the target gene. ATc drives the expression of SspB, which recognises the DAS tag and shuttles the protein to a Clp protease. In a drug screen, a strain will exhibit increased inhibitor sensitivity if the degraded protein is the drug target. The specific strain can be detected from a mixed culture of hypomorphs through quantitative sequencing of the barcode.