World Aquaculture Society Meetings

Add To Calendar 24/02/2016 11:45:0024/02/2016 12:05:00America/ChicagoAquaculture 2016USE OF GENOME-EDITING NUCLEASES, PARTICULARLY CRISPR/CAS9 FOR HIGH THROUGHPUT MUTAGENESIS IN ZEBRAFISH Vendome BThe World Aquaculture Societyjohnc@was.orgfalseanrl65yqlzh3g1q0dme13067DD/MM/YYYY


Raman Sood*, Blake Carrington, Kevin Bishop, Gaurav Varshney, MaryPat Jones, Shawn Burgess and Paul Liu
National Human Genome Research Institute,
National Institutes of Health,
Bethesda, MD, USA

Recent advances in genome editing by zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and clustered regularly interspersed palindromic repeat-Cas9 nucleases (CRISPR-Cas9) have revolutionized the use of zebrafish in disease modeling. The nucleases create a double strand break in DNA at the target site leading to the activation of DNA repair pathways. Random insertions and deletions (indels) due to improper repair by non homologous end joining lead to gene knockout mutations whereas homology-directed repair can be favored by providing a template for knock-in of specific mutations or transgenes. We have developed a pipeline for high throughput targeted mutagenesis and generated heritable genetic mutations in >150 genes using ZFNs, TALENs and mostly CRISPR/Cas9. Here, we will discuss our approach for high throughput mutagenesis, including detailed protocols for somatic activity analysis, efficient founder screening strategies, and genotyping of the mutant fish. Since we have data from all 3 platforms, we will also present a comparison of their mutagenesis efficiencies and their mutation signatures. To save time and reduce fish husbandry costs, we developed CRISPR-STAT (Somatic Tissue Analysis Test) as a pre-screening tool to identify highly active single-guide RNAs (sgRNAs). Furthermore, we have targeted multiple genes (upto 8) by pooling of sgRNAs and will provide data on its efficiency and impact on cost of founder screening and fish husbandry. We will also present data on the performance of various homology-directed repair mediated knock-in strategies for modeling of disease-associated gain-of-function variants and insertion of transgenes.

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