A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes
- National Institutes of Health (NIH), Bethesda, MD (United States). Eunice Kennedy Shriver National Institute of Child Health and Human Development; Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
- Univ. of Wisconsin, Madison, WI (United States)
- National Institutes of Health (NIH), Bethesda, MD (United States). Eunice Kennedy Shriver National Institute of Child Health and Human Development
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 multiplex, randomized CRISPR interference sequencing (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.
- Research Organization:
- Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Contributing Organization:
- NIH NICHD Molecular Genomics Core
- Grant/Contract Number:
- SC0018409
- OSTI ID:
- 1992293
- 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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A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes
Journal Article
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Wed Dec 13 19:00:00 EST 2023
· eLife
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OSTI ID:2229935