A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish.
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Varshney GK, Carrington B, Pei W, Bishop K, Chen Z, Fan C, Xu L, Jones M, LaFave MC, Ledin J, Sood R, Burgess SM
Nat Protoc 11 (12) 2357-2375 [2016-12-00; online 2016-10-27]
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
PubMed 27809318
DOI 10.1038/nprot.2016.141
Crossref 10.1038/nprot.2016.141
pii: nprot.2016.141
pmc: PMC5630457
mid: NIHMS905110