A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Ellis, Nicole A.; Myers, Kevin S.; Tung, Jessica; Davidson Ward, Anne; Johnston, Kathryn; Bonnington, Katherine E.; Donohue, Timothy J.; Machner, Matthias P.

doi:10.7554/eLife.86903

Title: A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Journal Article · 13 December 2023 · eLife

DOI: https://doi.org/10.7554/eLife.86903 · OSTI ID:2229938

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; Tung, Jessica; Davidson Ward, Anne; Johnston, Kathryn; Bonnington, Katherine E.;

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Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0018409

OSTI ID:: 2229938

Alternate ID(s):: OSTI ID: 2472372; OSTI ID: 2229940

Journal Information:: eLife, Journal Name: eLife Vol. 12; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.Copyright Statement

Country of Publication:: United States

Language:: English

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