ZFIN ID: ZDB-PUB-161105-21
A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
Varshney, G.K., Carrington, B., Pei, W., Bishop, K., Chen, Z., Fan, C., Xu, L., Jones, M., LaFave, M.C., Ledin, J., Sood, R., Burgess, S.M.
Date: 2016
Source: Nature Protocols   11: 2357-2375 (Journal)
Registered Authors: Burgess, Shawn, Ledin, Johan, Pei, Wuhong, Sood, Raman, Varshney, Gaurav, Xu, Lisha
Keywords: CRISPR-Cas9, genome editing, Genetic engineering, Mutagenesis, Zebrafish
MeSH Terms:
  • Animals
  • CRISPR-Cas Systems/genetics*
  • Genomics/methods*
  • High-Throughput Nucleotide Sequencing/methods*
  • Mutagenesis*
  • Zebrafish/genetics*
PubMed: 27809318 Full text @ Nat. Protoc.
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.