Showcase: Zebrafish Verified-Clutch Service - Two phased project for getting GFP and loxP insertions at native loci
Zebrafish are a powerful organism for drug discovery and are starting to secure a role in personalized medicine discovery.1 Understanding Knudra Transgenics uses a CRISPR approach using Cas9 complexes and long-homology arms 2-4 to create clutches of embryos testing positive for a high degree of mutagenesis.
Verified Clutch Service. The Verified Clutch Service is a zebrafish project done in two phases. In the first phase, we screen a set of CRISPR sgRNAs to identify efficient cutters of the genome. Next, in the second phase, we use the most efficient cutter along with a donor homology specific to the cut site. By utilizing a verified cutter and a donor homology built specifically for that sgRNA we see our success for germline integrations increase significantly and GFP insertions in zebrafish are now becoming routine in the clutches supplied to our researcher customers. The collaboration of having us design the edit, make the injection mix, inject, and verify editing in a portion of the injected clutch is working well for most of our researchers. They can then use their facilities (at a lower cost) to identify germline edits.
Floxed Conditionals. We are currently exploring the creation of floxed conditional alleles in fish. We are using the two-phased approach. In the standard first phase approach, we screen a set of CRISPR sgRNAs to find out which ones are efficient cutters of the genome. Next, in a modified second phase approach, we use a pair of good cutters, each with high efficiency for cutting pre and post of the start and stop codons of a gene. We are currently screening this new type of projects to see if this can get the bi-allele conversion on the same strand. It may be that getting double loxP insertion on one chromosome is a rare and elusive event. Nevertheless, we will know that for whichever line has a single loxP site inserted, the other site is just a simple phase II repeat effort to floxed allele transgenesis.
For ordering information click here: http://www.knudra.com/fish-crispr
2. Kawahara A, Hisano Y, Ota S, Taimatsu K. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish. Int J Mol Sci. 2016 May 13;17(5). pii: E727. doi: 10.3390/ijms17050727.
4. Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H, Yamamoto T, Kawahara A. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci Rep. 2015 Mar 5;5:8841. doi: 10.1038/srep08841.