## 2 Genomics
### Target Identification
The authors showed that production and export of the complex lipid, phthalocerol dimycoceroste (PDIM), was required for the secretion of ESX-1 substrates and permeabilization of the phagosome, revealing key virulence determinants, alongside defining pathways that are essential to the metabolism of intracellular bacilli, identifying potential targets for drug discovery. Plainly, Tn mutant libraries will only identify genes that are essential in the model under investigation, successful drug targets are likely required to be essential across several different conditions to mimic the variety of microenvironments encountered by _M.tb_ during natural infection. Gene deletion or inactivation results in the absence of gene product, this may not be reflective of drug action, where complete inhibition of protein function may not occur [18]. Conditional expression strategies that reduce rather than abrogate protein function may better represent drug action, and crucially allows essential gene targets to be investigated in the laboratory. Conditional expression using inducible promoter systems (for example, Tet ON/OFF or Pip ON/OFF) allow the expression of essential genes to be increased or reduced to understand gene function, model drug target inhibition and genetically-validate drug targets. The utility of such an inducible gene expression system was highlighted by [PERSON] et al. where _M.tb_ mutants depleted for 474 essential genes (termed hypomorphs) were screened against a large pool of potential inhibitors, allowing for >8.5 million chemical-genetic interactions to take place [19]. An episomaly-encoded _sspB_ gene was introduced to control protein degradation via a carboxyl-terminal fused DAS-tag. In addition, a 20 nucleotide \"barcode\" was introduced to facilitate enumeration by sequencing the barcoded PCR products derived from the mutants when pooled. The expression of SspB was controlled by a TetON promoter that was induced in response to anhydrotetracycline; by using TetON promoters with varying strengths, the level of gene product targeted for degradation by the SspB gene could be titrated. Primary screening using this hypomorph methodology identified over 10-fold more hits than whole cell screening wild type _M.tb_. As expected, well known antimicrobial drug classes showed interactions with specific hypomorphs, such as fluoroquinolones with their target GyrA, as well as rifampicin with the \(\beta\) subunit of RNA polymerase. This screening approach identified 39 inhibitors that targeted cellular components that are already clinically-validated antimicrobial drug targets; DNA gyrase, mycolic acid synthesis, and folate biosynthesis, targeted by the fluoroquinolones, isoniazid, and para-amino-salicylic acid, respectively [20]. The 39 compounds were either novel chemical entities or known compounds with re-purposed activity, such as the plant alkaloid tryptophan. In addition to finding novel inhibitors for well-validated targets, hypomorph screening also identified inhibitors against novel cellular targets. [PERSON] et al. demonstrated that strains hypomorphic for the putative efflux pump EfpA were inhibited by the compound BRD-8000 (MIC 6 \(\upmu\)M) but this compound showed no activity against wild type _M.tb_ (MIC \(\geq\) 50 \(\upmu\)M). Subsequent chemical optimization improved activity of BRD-8000 against wild type _M.tb_ to an MIC of 800 nM, an increase of \(\geq\)63-fold activity. _M.tb_ spontaneous mutants resistant to this modified BRD-8000 compound showed a single point mutation in _eprA_, demonstrating that chemical modification of BRD-8000 had not altered target specificity. Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) has the potential to revolutionize the field, enabling precise gene silencing to identify and validate drug targets. A nuclease, dCas9 containing two mutations that eradicate its nuclease activity, is targeted to a mycobacterial gene of interest by a single guide RNA (sgRNA).