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A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
Gaurav K Varshney1,2, Blake Carrington3, Wuhong Pei1
1Developmental Genomics Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.
Nature Protocols
|November 4, 2016
Summary
This study presents a high-throughput CRISPR/Cas9 workflow for zebrafish, enabling rapid gene targeting and mutant identification. The scalable protocol allows researchers to establish stable lines efficiently, accelerating functional genomic studies.
Area of Science:
- Genetics and Genomics
- Developmental Biology
- Zebrafish Models
Background:
- Zebrafish are crucial model organisms for studying development and disease.
- CRISPR/Cas9 gene targeting is effective in zebrafish but often lacks scalability.
- Existing methods for guide RNA synthesis and mutant identification are not high-throughput.
Purpose of the Study:
- To present a complete, scalable workflow for high-throughput CRISPR/Cas9 mutagenesis and phenotyping in zebrafish.
- To detail methods for efficient target selection, sgRNA synthesis, and mutant identification.
- To enable rapid establishment of genetically modified zebrafish lines for functional genomics.
Main Methods:
- Cloning-free single-guide RNA (sgRNA) synthesis and microinjection.
- Validation of sgRNA target activity and founder screening using fluorescence PCR.
- Lesion determination via Sanger or next-generation sequencing and subsequent genotyping.
- A comprehensive workflow from target selection to stable line establishment.
Main Results:
- sgRNAs can be evaluated within 3 days.
- Germline-transmitting mutations identified within 3 months.
- Stable zebrafish lines established within 6 months.
- The protocol allows targeting of tens to hundreds of genes per researcher per year.
Conclusions:
- The presented workflow significantly enhances the throughput of CRISPR/Cas9 gene editing in zebrafish.
- This method accelerates functional genomic studies by enabling rapid generation of mutant lines.
- The protocol is practical for researchers aiming to study numerous genes in zebrafish models.
