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.

Keywords: Mycobacterium tuberculosis; CRISPRi; Tn-seq; TraSH; transposon mutagenesis.

FIGURE 1

High‐throughput functional genomic approaches for bacteria. High‐throughput approaches begin with creation of either pooled or arrayed mutant libraries. If barcoded, arrayed mutant libraries can be pooled and utilized in downstream assays. Similarly, pooled mutant libraries can be arrayed, sequenced, and deconvoluted. The identified mutants are utilized to create non‐redundant mutant libraries. Pooled mutant libraries are largely dependent on sequence‐based readouts, generally reflecting relative abundance of mutants, although alternative features can be assayed when combined with cell sorting. Various single‐cell RNA‐sequencing techniques have been developed for bacteria (Blattman et al., ; Dixit et al., ; Imdahl et al., ; Kuchina et al., ; Ma et al., 2023) but have yet to be used to probe pooled mutant libraries. Arrayed mutant libraries are extremely flexible, though complicated to construct, and can potentially be applied to any assay modality that is scalable, including transcriptomic, metabolomic, growth, and imaging.

FIGURE 2

High‐throughput functional genomic approaches applied to mycobacteria. Most high‐throughput functional genomic approaches applied to mycobacteria have utilized biomass‐dependent TraSH, Tn‐seq, or CRISPRi. These have been applied in (conditional) gene essentiality screens, to identify genes required for infection, to elucidate gene–gene interactions, and in chemical‐genetic interaction studies. As an example of the latter, a recent chemical‐genetic interaction study utilized pooled collection of CRISPRi mutants (Poulton et al., 2022). An increasing number of studies are applying alternative outputs to biomass, including two screens that utilized cell sorting (Baranowski et al., ; Rego et al., 2017) and two that employ morphological profiling (De Wet et al., ; Smith et al., 2020).