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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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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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.
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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 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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DOI: https://doi.org/10.1038/nprot.2016.141
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