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A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA.;Oklahoma Med Res Fdn, Funct & Chem Genom Program, 825 NE 13th St, Oklahoma City, OK 73104 USA..
NHGRI, Zebrafish Core, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
NHGRI, Zebrafish Core, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
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2016 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 11, no 12, p. 2357-2375Article in journal (Refereed) Published
Abstract [en]

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 F-1 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.

Place, publisher, year, edition, pages
2016. Vol. 11, no 12, p. 2357-2375
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-308888DOI: 10.1038/nprot.2016.141ISI: 000386976800004PubMedID: 27809318OAI: oai:DiVA.org:uu-308888DiVA, id: diva2:1051355
Funder
NIH (National Institute of Health)Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2018-01-13Bibliographically approved

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Ledin, Johan

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