A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish.
Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving r...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2025-02-01
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| Series: | PLoS Genetics |
| Online Access: | https://doi.org/10.1371/journal.pgen.1011594 |
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| author | Thomas Juan Tonatiuh Molina Lihan Xie Sofia Papadopoulou Bárbara Cardoso Shivam Govind Jha Didier Y R Stainier |
| author_facet | Thomas Juan Tonatiuh Molina Lihan Xie Sofia Papadopoulou Bárbara Cardoso Shivam Govind Jha Didier Y R Stainier |
| author_sort | Thomas Juan |
| collection | DOAJ |
| description | Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an in vivo reporter strategy, we first show that inserting the T3H48 self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the T3H48 ribozyme in the second intron of the albino gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the T3H48 ribozyme, called RiboFlip, to control the mRNA levels of the albino gene. RiboFlip activation induced mRNA knockdown and also recapitulated the albino mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies. |
| format | Article |
| id | doaj-art-684db51b73984ceab42f9c3c8b60a949 |
| institution | OA Journals |
| issn | 1553-7390 1553-7404 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS Genetics |
| spelling | doaj-art-684db51b73984ceab42f9c3c8b60a9492025-08-20T02:16:17ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042025-02-01212e101159410.1371/journal.pgen.1011594A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish.Thomas JuanTonatiuh MolinaLihan XieSofia PapadopoulouBárbara CardosoShivam Govind JhaDidier Y R StainierPrecise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an in vivo reporter strategy, we first show that inserting the T3H48 self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the T3H48 ribozyme in the second intron of the albino gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the T3H48 ribozyme, called RiboFlip, to control the mRNA levels of the albino gene. RiboFlip activation induced mRNA knockdown and also recapitulated the albino mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies.https://doi.org/10.1371/journal.pgen.1011594 |
| spellingShingle | Thomas Juan Tonatiuh Molina Lihan Xie Sofia Papadopoulou Bárbara Cardoso Shivam Govind Jha Didier Y R Stainier A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. PLoS Genetics |
| title | A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. |
| title_full | A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. |
| title_fullStr | A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. |
| title_full_unstemmed | A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. |
| title_short | A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. |
| title_sort | recombinase activated ribozyme to knock down endogenous gene expression in zebrafish |
| url | https://doi.org/10.1371/journal.pgen.1011594 |
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