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A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening
Ylenia D'Agostino1, Annamaria Locascio1, Filomena Ristoratore1
1Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
Molecular Biotechnology
|December 18, 2015
Summary
We developed a fast, affordable genotyping method for detecting gene knock-outs in zebrafish. This approach simplifies screening of CRISPR/Cas9 edited animals, reducing time and cost for researchers.
Area of Science:
- Molecular Biology
- Genetics
- Animal Models
Background:
- Genome editing tools like CRISPR/Cas9 enable gene knock-outs in animal models.
- Screening founder (F0) and carrier (F1) animals is time-consuming and costly.
- High-resolution melting (HRM) analysis is effective for detecting insertions/deletions (InDels) but requires expensive equipment.
Purpose of the Study:
- To develop a rapid, cost-effective, high-throughput genotyping method for genome-edited animals.
- To provide an efficient alternative to HRM analysis for large-scale InDel detection.
- To streamline the screening process for CRISPR/Cas9 modified zebrafish.
Main Methods:
- Utilized a ViiA™7 real-time PCR system.
- Optimized melting curve analysis parameters.
- Discriminated between mutant and wild-type melting curves.
Main Results:
- Achieved genotyping efficiency comparable to HRM analysis.
- Demonstrated the ability to differentiate mutant and wild-type samples via melting curves.
- Validated a simple, rapid, and inexpensive method for massive screening.
Conclusions:
- The proposed method offers a cost-effective, high-throughput solution for preliminary genotyping.
- This approach reduces the number of embryos requiring subsequent sequencing.
- The versatile method is adaptable for various genome editing strategies and animal models.
