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A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish

Nature Protocols volume 11, pages 2357–2375 (2016)Cite this article

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Abstract

The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.

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Figure 1
Figure 2: Schematic of sgRNA synthesis and three-primer PCR strategies for fluorescence PCR or next-gen sequencing.
Figure 3: Agarose gel electrophoresis of Cas9 mRNA, target assembly and sgRNA.
Figure 4: Schematic using the diameter of a sphere to calculate the dose for injection into the embryos.
Figure 5: Evaluation of sgRNA activity by pigmentation phenotype and CRISPR-STAT plots.
Figure 6: Fluorescence PCR plots of F1 embryos from an injected founder fish crossed with a WT fish.
Figure 7: Fin amputation of an adult zebrafish caudal fin for genomic DNA extraction.
Figure 8: Chromatograms of a WT and a heterozygous sample.

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Acknowledgements

We thank J. Fekecs and U. Harper for help with figures, and D. Prebilic, H. Mahon and other staff of the National Institutes of Health zebrafish facility for excellent animal care. This research is funded by the Intramural Research Program of the National Human Genome Research Institute; National Institutes of Health (S.M.B. and R.S.).

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Author notes

  1. Matthew C LaFave

    Present address: Present address: Synthetic Genomics, San Diego, USA.,

  2. Gaurav K Varshney and Blake Carrington: These authors contributed equally to this work.

Authors and Affiliations

  1. Developmental Genomics Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Gaurav K Varshney, Wuhong Pei, Zelin Chen, Lisha Xu, Matthew C LaFave & Shawn M Burgess

  2. Functional & Chemical Genomics Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA

    Gaurav K Varshney

  3. Zebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Blake Carrington, Kevin Bishop & Raman Sood

  4. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai, China

    Chunxin Fan

  5. Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA

    Marypat Jones

  6. Department of Organismal Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

    Johan Ledin

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Contributions

G.K.V., B.C., R.S. and S.M.B. developed the protocol and wrote the manuscript, with inputs from all other authors; G.K.V., B.C., W.P., K.B., C.F., L.X., M.P.J. and J.L. performed the experiments. M.C.L. and Z.C. wrote and tested the bioinformatics pipeline, ampliconDIVider.

Corresponding author

Correspondence to Shawn M Burgess.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Screen capture of the zebrafish CRISPR/Cas9 track hub.

This is a screen capture of the first exon of zebrafish FGF2. Most of the ZebrafishGenomics tracks are displayed. The top track shows all computationally predicted SpCas9 target sites. They are color coded by target length (gray=20, blue=19, orange=18), with darker colors having fewer predicted off-target sequences. The sequences can be seen in the left column and the predicted off-target number can be seen at the end of the sequence. The “GA CRISPR/Cas9 Targets” track shows targets that can be synthesized by Sp6 and the “GG CRISPR/Cas9 Targets” are guides that can be synthesized by T7. The black bars in the “NHGRI-1 Invariant” track indicate where the genomic sequence of the NHGRI-1 strain matches the reference sequence. Gaps represent variation. The “NHGRI-1 Variants” track shows possible SNV’s at the given position. Researchers should avoid regions with gaps or SNV’s. Green arrowhead indicates what we would consider the best target for this region.

Supplementary information

Supplementary Text and Figures (PDF 465 kb)

Supplementary Tables 1–3

Supplementary Table 1. sgRNA target sequences, primer sequences and amplicon sizes used to generate the data in Figures 5 and 6. Supplementary Table 2. Module manager settings for a 3130xl or 3730 capillary sequencer using a 36-cm or 50-cm array. Injection times marked in red are the variable settings between the CRISPRSTAT and fragment analysis methods. Supplementary Table 3. Barcoded M13 primer sequences. (PDF 115 kb)

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Varshney, G., Carrington, B., Pei, W. et al. A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish. Nat Protoc 11, 2357–2375 (2016). https://doi.org/10.1038/nprot.2016.141

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