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.
Keywords: Mycobacterium tuberculosis; CRISPRi; Tn-seq; TraSH; transposon mutagenesis.
© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.