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| Status |
Public on Jun 27, 2023 |
| Title |
CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron |
| Organism |
Bacteroides thetaiotaomicron |
| Experiment type |
Other
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| Summary |
Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bac-teroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different pro-tospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules for and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the previously uncharacterized sRNA BTnc167 to increase susceptibility to bile salts, likely through the regulation of genes involved in Bacteroides cell surface structure. Our work lays the groundwork for systematically uncovering hidden functions of bacterial sRNAs under a variety of con-ditions and, generally, to unlock the genetic potential of these major human gut mutualists.
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| Overall design |
CRISPRi screen in B. thetaiotaomicron. These samples consist of sequenced PCR products of the genomic region containing the gRNA/arrays. The "library" samples are the steady-state CRISPRi library, the "tyg" samples are derived from B. thetaiotaomicron grown in rich medium, the "bile" samples are derived from B. thetaiotaomicron grown in rich medium + bile salts.
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| Web link |
https://www.pnas.org/doi/abs/10.1073/pnas.2311323121
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| Contributor(s) |
Prezza G, Liao C, Reichardt S, Beisel CL, Westermann AJ |
| Citation(s) |
38294941 |
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| Submission date |
Jun 22, 2023 |
| Last update date |
Feb 08, 2024 |
| Contact name |
Alexander J Westermann |
| Organization name |
Helmholtz Institute for RNA-based Infection Research (HIRI)
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| Street address |
Josef Schneider Strasse , D15
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| City |
Wuerzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platforms (2) |
| GPL28094 |
Illumina NextSeq 500 (Bacteroides thetaiotaomicron) |
| GPL33520 |
NextSeq 2000 (Bacteroides thetaiotaomicron) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA986833 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE235620_RAW.tar |
50.0 Kb |
(http)(custom) |
TAR (of TXT) |
| GSE235620_reference_all.fasta.gz |
9.9 Kb |
(ftp)(http) |
FASTA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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