CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish
| Autor: | João Amorim, José Bessa, Renata Bordeira-Carriço, Ana Gali-Macedo, Chiara Perrod |
|---|---|
| Přispěvatelé: | Instituto de Investigação e Inovação em Saúde |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
animal structures
Microinjections Gene Editing / methods ved/biology.organism_classification_rank.species Zebrafish / genetics Sequence Deletion / genetics Mutagenesis (molecular biology technique) Genomics Computational biology General Biochemistry Genetics and Molecular Biology Genome engineering 03 medical and health sciences 0302 clinical medicine Model Organisms Protocol Genetics CRISPR Animals RNA Guide / genetics lcsh:Science (General) Model organism Microinjections / methods Zebrafish Sequence Deletion 030304 developmental biology Gene Editing Cloning 0303 health sciences Genome General Immunology and Microbiology biology ved/biology Cas9 General Neuroscience CRISPR-Cas Systems / physiology biology.organism_classification 3. Good health Mutagenesis Site-Directed / methods Mutagenesis Site-Directed CRISPR-Cas Systems Genetic Engineering Genetic Engineering / methods 030217 neurology & neurosurgery RNA Guide Kinetoplastida lcsh:Q1-390 |
| Zdroj: | STAR Protocols STAR Protocols, Vol 1, Iss 3, Pp 100208-(2020) |
| Popis: | Summary Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020). Graphical Abstract Highlights • Step-by-step details from design to production of target-specific sgRNAs • Microinjection of sgRNAs/Cas9 complexes into one-cell-stage zebrafish embryos • PCR-based genotyping of in vivo deletions • Cost- and time-effective pipeline for establishment of a zebrafish stable mutant line Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. |
| Databáze: | OpenAIRE |
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