@article{Winkler2023,
abstract = {Molecular Microbiology. 2023;00:1-18. | 1 wileyonlinelibrary.com/journal/mmi 1 | INTRODUC TI ON Two decades ago, publication of the complete genome sequence of Mycobacterium tuberculosis brought into sharp focus a problem that continues to pervade much of (micro)biology, affecting even highly studied model organisms (Kustatscher et al., 2022; Wicke et al., 2023): although the sequences of most M. tuberculosis genes (or, at least, ORFs) could be identified, the functions of {\~{}}60{\%} remained (and still remain) imprecisely predicted or unknown, with experimental validation wanting (Cole et al., 1998). 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.},
author = {Winkler, Kristy R and Mizrahi, Valerie and Warner, Digby F and {De Wet}, Timothy J},
doi = {10.1111/MMI.15103},
file = {:C$\backslash$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Winkler et al. - 2023 - High-throughput functional genomics a (myco)bacterial perspective.pdf:pdf},
issn = {1365-2958},
journal = {Molecular Microbiology},
keywords = {CRISPRi,Mycobacterium tuberculosis,OA,Tn,TraSH,fund{\_}not{\_}ack,review,seq,transposon mutagenesis},
mendeley-tags = {OA,fund{\_}not{\_}ack,review},
month = {jun},
pages = {10.1111/mmi.15103},
pmid = {37278255},
publisher = {John Wiley {\&} Sons, Ltd},
title = {{High-throughput functional genomics: a (myco)bacterial perspective}},
url = {https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.15103 https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.15103 https://onlinelibrary.wiley.com/doi/10.1111/mmi.15103},
year = {2023}
}

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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. 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