Genome-Scale CRISPRi Platform Used to Quantify Gene Vulnerability in TB

A research team from the Rockefeller University has quantified vulnerability in a pathogen, producing an index that ranks almost every essential gene in Mycobacterium tuberculosis by the amount of inhibition necessary to disable the gene and cripple the cell. Their findings were published in Cell. 

The team developed a technique to look at the whole genome of the pathogen at once, and rank each essential gene based on how much of it would need to be inhibited in order to kill the bacteria.  “We developed a system that can be tuned, from no inhibition to nearly 100 percent inhibition,” says researcher Barbara Bosch. “This allowed us to determine whether the bacteria were having serious fitness costs, or whether they were still alive and kicking.” 

The resulting index, which relates inhibition percentages to bacterial fitness, suggests that vulnerability is a key factor in determining whether antibiotics succeed. Two of the most vulnerable genes, for example, happen to also be the targets of the two most potent TB drugs on the market. Conversely, two of the least vulnerable genes, coaA and def, were once-promising drug targets before antibiotics tailored to inhibit those genes failed to kill the bacteria. The invulnerability of those targets may be one reason that these therapies flopped.  

The index also identified several new targets that are essential, highly vulnerable, and as-yet unexplored by drug developers. Some of those targets are even more vulnerable than the current first-line TB therapies and influence surprisingly diverse activities in the cell. 

“We expected that genes involved in the central dogma would be vulnerable—to replicate, you need to be able to turn DNA into RNA into a protein,” lead researcher Jeremy Rock says. “But some of the most vulnerable genes were involved in protein folding and secretion. We wouldn’t necessarily have predicted that.” In future studies, Rock and his team hope to test their index on TB that has infected a living organism; their current index is based solely on how TB reacts to inhibition in a Petri dish. “The next big step is investigating how vulnerability changes in the context of the host-pathogen relationship,” Rock says. “In vivo studies will give us a more complete picture of vulnerability.”