Academic Journal
Conditional dCas12-Mediated Gene Knockdown and Complementation in Chlamydia.
| Τίτλος: | Conditional dCas12-Mediated Gene Knockdown and Complementation in Chlamydia. |
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| Συγγραφείς: | Wang Y; Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, 08820, USA., Fan H; Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, 08820, USA. fanhu@rwjms.rutgers.edu. |
| Πηγή: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2027; Vol. 3073, pp. 243-258. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| Imprint Name(s): | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press |
| Ιατρικοί όροι (MeSH): | Gene Knockdown Techniques*/methods , Chlamydia trachomatis*/genetics , Genetic Complementation Test*/methods , CRISPR-Associated Proteins*/genetics , CRISPR-Associated Proteins*/metabolism , CRISPR-Cas Systems*, RNA, Guide, CRISPR-Cas Systems/genetics ; Humans |
| Περίληψη: | We describe an oligonucleotide RNA-guided, DNase-dead CRISPR-associated protein 12 (dCas12)-mediated transcriptional interference (CRISPRi) system optimized for gene knockdown in Chlamydia trachomatis. In this system, while guide RNAs are expressed constitutively, dCas12 expression is conditional and depends on the presence of the small molecule anhydrotetracycline (ATC). This design enables temporal control of gene repression, allowing the functional analysis of a wide range of genes, including essential genes, without permanent genetic disruption. Due to its T-rich protospacer adjacent motif (PAM) requirement, dCas12 is particularly well-suited for targeting genes in the highly A/T-rich genomes of pathogenic chlamydiae. We further detail strategies for genetic complementation through co-expression of dCas12-resistant alleles, thereby permitting validation of knockdown phenotypes. (© 2027. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Competing Interests: | Conflict of Interest: The authors have no conflicts of interest to declare that are relevant to the content of this chapter. |
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| Contributed Indexing: | Keywords: Chlamydia; CRISPR interference; CRISPRi; Conditional knockdown; Essential genes; Gene knockdown; Gene silencing; Inducible knockdown |
| Substance Nomenclature: | 0 (RNA, Guide, CRISPR-Cas Systems) 0 (CRISPR-Associated Proteins) |
| Entry Date(s): | Date Created: 20260922 Date Completed: 20260922 Latest Revision: 20260922 |
| Update Code: | 20260923 |
| DOI: | 10.1007/978-1-0716-5531-3_18 |
| PMID: | 42771155 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1940-6029 |
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| DOI: | 10.1007/978-1-0716-5531-3_18 |