ArticleInfection and immunity2021
CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis.
Article in Infection and immunity, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 57 papers.
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Who cites it
57 citing papers in PubMed, 82 citations in OpenAlex.
- Analysis of Chlamydia trachomatis Gene Function via Controlled Up- or Downregulation.Methods in molecular biology (Clifton, N.J.) · 2027Article
- Chromosome Editing Using the Lambda Red Recombinase System in Chlamydia trachomatis and Chlamydia muridarum.Methods in molecular biology (Clifton, N.J.) · 2027Article
- Disruption of Chlamydia trachomatis Essential Genes Using DOPE (Dependence on Plasmid-Mediated Expression).Methods in molecular biology (Clifton, N.J.) · 2027Article
- A Conditional Knockout Mutant Approach to Study Essential Genes in Chlamydia in Cell Culture and In Vivo.Methods in molecular biology (Clifton, N.J.) · 2027Article
- Conditional dCas12-Mediated Gene Knockdown and Complementation in Chlamydia.Methods in molecular biology (Clifton, N.J.) · 2027Article
- Chlamydia trachomatis orchestrates multifaceted host cell manipulation for immune evasion.Communications biology · 2026Review
- CRISPRi and beyond: studying essential gene function in the obligate intracellular bacteriumJournal of bacteriology · 2026Review
- Minute-scale control of ubiquitin-mediated degradation reveals dynamics of bacterial secreted effector-functions.Nature communications · 2026Article
- Characterization of the cell division-associated peptidoglycan amidase AmiA ofJournal of bacteriology · 2026Article
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- Pathogenicity and virulence ofVirulence · 2025Review
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- The second messenger signaling molecule cyclic di-AMP drives developmental cycle progression ineLife · 2025Article
- ThebioRxiv : the preprint server for biology · 2025Article
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- A platform supporting generation and isolation of random transposon mutants inJournal of bacteriology · 2025Article
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- Effect of tryptophan starvation on inclusion membrane composition and chlamydial-host interactions.Infection and immunity · 2025Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
The ability to inducibly repress gene expression is critical to the study of organisms, like Chlamydia, with reduced genomes in which the majority of genes are likely to be essential. We recently described the feasibility of a CRISPR interference (CRISPRi) system to inducibly repress gene expression in Chlamydia trachomatis. However, the initial system suffered from some drawbacks, primarily leaky expression of the anhydrotetracycline (aTc)-inducible dCas9 ortholog and plasmid instability, which prevented population-wide studies (e.g., transcript analyses) of the effects of knockdown. Here, we describe various modifications to the original system that have allowed us to measure gene expression changes within a transformed population of C. trachomatis serovar L2. These modifications include (i) a change in the vector backbone, (ii) the introduction of a weaker ribosome binding site driving dCas9 translation, and (iii) the addition of a degradation tag to dCas9 itself. With these changes, we demonstrate the ability to inducibly repress a target gene sequence, as measured by the absence of protein by immunofluorescence analysis and by decreased transcript levels. Importantly, the expression of dCas9 alone (i.e., without a guide RNA [gRNA]) had minimal impact on chlamydial growth or development. We also describe complementation of the knockdown effect by introducing a transcriptional fusion of the target gene 3' to dCas9. Finally, we demonstrate the functionality of a second CRISPRi system based on a dCas12 system that expands the number of potential chromosomal targets. These tools should provide the ability to study essential gene function in Chlamydia.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.