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Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
Published on: February 5, 2014
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A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting
Éric Samarut1,2, Alexandra Lissouba3,4, Pierre Drapeau3,5
1Department of Neurosciences, Research Center of the University of Montreal Hospital Center (CRCHUM), Université de Montréal, Montréal, QC, Canada. eric.samarut@umontreal.ca.
BMC Genomics
|August 6, 2016
Summary
This study presents a rapid method for identifying CRISPR-induced mutations in zebrafish. The optimized technique combines HotSHOT DNA extraction with Evagreen PCR and high-resolution melting analysis for fast, cost-effective genotyping.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 is a widely used genome editing tool.
- Validating guide RNA targets in model organisms is limited by time and cost for indel identification.
Purpose of the Study:
- To develop a rapid and cost-effective method for identifying CRISPR-induced indels.
- To optimize the process for high-throughput genotyping in zebrafish.
Main Methods:
- Combined HotSHOT genomic DNA extraction with two-step Evagreen PCR and high-resolution melting (HRM) assay.
- Utilized fin-clips for adult zebrafish DNA extraction and embryo injections for early-stage analysis.
- Performed mutagenesis kinetic assays to determine the earliest time point for indel observation.
Main Results:
- Genotyped adult zebrafish from fin-clips in under 2 hours.
- Achieved early readout of guide RNA effectiveness within 4 hours post-embryo injection.
- Identified the 2-cell stage as the earliest time point for observing indels.
Conclusions:
- The combined approach offers rapid, high-throughput genotyping for zebrafish and other model organisms.
- This method is more sensitive than standard PCR, enabling early detection of CRISPR-induced indels.
- The optimized technique reduces time and cost associated with CRISPR/Cas9 validation.
