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High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
Gaurav K Varshney1, Wuhong Pei1, Matthew C LaFave1
1Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;
Genome Research
|June 7, 2015
Summary
We developed a high-throughput CRISPR/Cas9 pipeline for zebrafish genome editing, enabling saturation mutagenesis and efficient large-scale phenotyping. This method significantly outperforms older techniques, reducing time and resources for genetic studies.
Area of Science:
- Genetics
- Molecular Biology
- Zebrafish Model Organisms
Background:
- CRISPR/Cas9 has revolutionized genome editing in model organisms.
- Targeted mutagenesis and large-scale phenotyping are crucial for genetic research.
Purpose of the Study:
- To establish a high-throughput CRISPR/Cas9 pipeline for zebrafish.
- To enable saturation mutagenesis and efficient phenotyping.
- To compare CRISPR/Cas9 efficiency with TALENs and ZFNs.
Main Methods:
- Cloning-free single-guide RNA (sgRNA) synthesis.
- Fluorescent PCR and multiplexed, high-throughput sequencing for mutant identification.
- Inbreeding F1 generation for rapid phenotyping.
Main Results:
- 99% mutation generation success rate and 28% average germline transmission rate across 162 loci.
- Verified 678 unique alleles from 58 genes.
- CRISPR/Cas9 demonstrated sixfold higher efficiency than TALENs and ZFNs.
- Identified a key sequence motif (GG/GA at 5' end) for predicting sgRNA efficiency.
Conclusions:
- The developed pipeline is highly efficient for zebrafish mutagenesis and phenotyping.
- Early phenotyping in F1 generation reduces animal husbandry and time.
- CRISPR/Cas9 is a superior genome-editing tool compared to TALENs and ZFNs in zebrafish.
