High‐throughput functional genomics: A (myco)bacterial perspective

Abstract

Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high‐throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high‐throughput functional genomics, focusing primarily on transposon (Tn)‐based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.

A spotlight on the tuberculosis epidemic in South Africa

“…In an exciting new discovery, the tight, PcaA-dependent packing of tubercle bacilli growing in cords was shown to protect M. tuberculosis against antibiotic-mediated clearance by allowing the bacilli to remain transcriptionally active under drug exposure and to re-grow after drug removal 8 . High-throughput functional genomic analyses by transposon mutagenesis and inducible CRISPR interference have enabled mycobacterial gene function to be probed at scale 36 . This approach has provided a new metric—the vulnerability index—for comparing, selecting and prioritising new TB drug targets 37 .…”