Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish.
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| Natura: | Artículo científico |
| Lingua: | en |
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Journal of visualized experiments : JoVE
2025
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| _version_ | 1868266167298162690 |
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| author | Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming |
| author_facet | Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming |
| collection | PubMed - marine biology |
| contents | Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish. Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming Animals Zebrafish Female Gene Knock-In Techniques CRISPR-Cas Systems Animals, Genetically Modified RNA-Binding Proteins Zebrafish Proteins Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model. |
| format | Artículo científico |
| id | pubmed_40788907 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | Journal of visualized experiments : JoVE |
| record_format | pubmed |
| spellingShingle | Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish. Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming Animals Zebrafish Female Gene Knock-In Techniques CRISPR-Cas Systems Animals, Genetically Modified RNA-Binding Proteins Zebrafish Proteins Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish. Zhang, Yizhuang Fu, Ziping Yang, Boya Wang, Jiasheng Lu, Tong Shi, De-Li Shao, Ming Animals Zebrafish Female Gene Knock-In Techniques CRISPR-Cas Systems Animals, Genetically Modified RNA-Binding Proteins Zebrafish Proteins Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model. |
| title | Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish. |
| topic | Animals Zebrafish Female Gene Knock-In Techniques CRISPR-Cas Systems Animals, Genetically Modified RNA-Binding Proteins Zebrafish Proteins |
| url | https://pubmed.ncbi.nlm.nih.gov/40788907/ |