A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes
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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2023-12-01
|
| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/86903 |
| _version_ | 1797391386692026368 |
|---|---|
| author | Nicole A Ellis Kevin S Myers Jessica Tung Anne Davidson Ward Kathryn Johnston Katherine E Bonnington Timothy J Donohue Matthias P Machner |
| author_facet | Nicole A Ellis Kevin S Myers Jessica Tung Anne Davidson Ward Kathryn Johnston Katherine E Bonnington Timothy J Donohue Matthias P Machner |
| author_sort | Nicole A Ellis |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T23:32:13Z |
| format | Article |
| id | doaj.art-f1393647dd45428abfca48191a9b0da7 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-08T23:32:13Z |
| publishDate | 2023-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-f1393647dd45428abfca48191a9b0da72023-12-14T11:56:46ZengeLife Sciences Publications LtdeLife2050-084X2023-12-011210.7554/eLife.86903A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genesNicole A Ellis0https://orcid.org/0000-0001-9592-8415Kevin S Myers1https://orcid.org/0000-0003-3302-3877Jessica Tung2Anne Davidson Ward3Kathryn Johnston4Katherine E Bonnington5Timothy J Donohue6https://orcid.org/0000-0001-8738-2467Matthias P Machner7https://orcid.org/0000-0002-6971-7451Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesGreat Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, United States; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, United StatesEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesGreat Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, United States; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, United States; Department of Bacteriology, University of Wisconsin-Madison, Madison, United StatesEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United StatesIdentifying 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.https://elifesciences.org/articles/86903Legionella pneumophilaintracellular pathogentranslocated effectors |
| spellingShingle | Nicole A Ellis Kevin S Myers Jessica Tung Anne Davidson Ward Kathryn Johnston Katherine E Bonnington Timothy J Donohue Matthias P Machner A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes eLife Legionella pneumophila intracellular pathogen translocated effectors |
| title | A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes |
| title_full | A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes |
| title_fullStr | A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes |
| title_full_unstemmed | A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes |
| title_short | A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes |
| title_sort | randomized multiplex crispri seq approach for the identification of critical combinations of genes |
| topic | Legionella pneumophila intracellular pathogen translocated effectors |
| url | https://elifesciences.org/articles/86903 |
| work_keys_str_mv | AT nicoleaellis arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT kevinsmyers arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT jessicatung arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT annedavidsonward arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT kathrynjohnston arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT katherineebonnington arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT timothyjdonohue arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT matthiaspmachner arandomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT nicoleaellis randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT kevinsmyers randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT jessicatung randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT annedavidsonward randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT kathrynjohnston randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT katherineebonnington randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT timothyjdonohue randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes AT matthiaspmachner randomizedmultiplexcrispriseqapproachfortheidentificationofcriticalcombinationsofgenes |