Related Experiment Video
Updated: Aug 23, 2025
11:27
Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio
Published on: August 28, 2018
22.2K
CRISPR-Cas9-induced gene knockout in zebrafish.
Raghavender Medishetti1, Keerthana Balamurugan1, Krishnaveni Yadavalli2
1Dr. Reddy's Institute of Life Sciences, Hyderabad, Telangana, India; Doctoral Student Registered with Manipal Academy of Higher Education, Manipal, Karnataka, India.
STAR Protocols
|November 1, 2022
Summary
This protocol details CRISPR-Cas9 gene knockout in zebrafish for studying human diseases. It focuses on the PHLPP1 gene, offering a broadly applicable method for genotype-phenotype research.
Area of Science:
- Genetics and Genomics
- Developmental Biology
- Molecular Biology
Background:
- CRISPR technology revolutionizes genotype-phenotype studies in human disease models.
- Zebrafish are a valuable model organism for genetic research due to their rapid development and high fecundity.
Purpose of the Study:
- To describe a protocol for CRISPR-Cas9-induced gene knockout in zebrafish.
- To target the PHLPP1 gene for genotype-phenotype analysis in a specific zebrafish strain.
- To provide a broadly applicable method for gene editing in zebrafish.
Main Methods:
- Utilizing purified Cas9 protein and in vitro-transcribed sgRNA for CRISPR-Cas9 gene editing.
- Implementing the protocol in an Indian wild-caught zebrafish strain.
- Focusing on the PHLPP1 gene for targeted knockout.
Main Results:
- Successful CRISPR-Cas9-induced gene knockout in zebrafish.
- Demonstration of a protocol applicable to various genes and zebrafish strains.
- Identification of key factors influencing protocol success, including sgRNA efficiency and mutant viability.
Conclusions:
- The described CRISPR-Cas9 protocol provides an effective method for gene knockout in zebrafish.
- This approach facilitates genotype-phenotype studies of human disease models using zebrafish.
- Optimization of factors such as zebrafish health, sgRNA characteristics, and mutant viability is crucial for successful gene editing.
