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CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish
João Pedro Amorim1,2, Renata Bordeira-Carriço1,2, Ana Gali-Macedo1,2
1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
STAR Protocols
|December 30, 2020
Summary
This study details a CRISPR-Cas9 workflow for creating targeted genomic deletions in zebrafish. This efficient method facilitates the rapid generation of zebrafish models for functional genetics research.
Area of Science:
- Genomics
- Molecular Biology
- Zebrafish Genetics
Background:
- The CRISPR-Cas9 system has transformed genome engineering since its introduction.
- Zebrafish are a valuable model organism for genetic studies due to their rapid development and optical transparency.
Purpose of the Study:
- To present a validated workflow for generating targeted genomic deletions in zebrafish using CRISPR-Cas9.
- To establish a reliable method for creating zebrafish mutant lines for functional genetics.
Main Methods:
- Design, cloning, and synthesis of single-guide RNAs (sgRNAs) and Cas9 mRNA.
- Microinjection of CRISPR-Cas9 components into zebrafish embryos.
- Genotype screening to identify and establish mutant zebrafish lines.
Main Results:
- A validated and efficient pipeline for generating targeted genomic deletions in zebrafish was established.
- The workflow enables the creation of specific zebrafish models for further research.
Conclusions:
- The presented CRISPR-Cas9 pipeline is versatile and efficient for generating zebrafish models.
- This approach is widely applicable in functional genetics research for creating custom mutant lines.