Browsing by Author "Clements, Thomas P"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    A Rice CRISPy Treat: Improving CRISPR-Cas9 gene editing in the zebrafish to facilitate analysis of genes implicated in neural angiogenesis in an F0 screen
    (2018-04-16) Clements, Thomas P; Wagner, Daniel S
    Scientists are eager for novel mutants to study gene function, evolutionary relationships, and even perform drug screens. Zebrafish are a well-established model for scientific research (Kimmel et al., 1995) and have been a premiere model for both forward and reverse genetic research. However, each current method to produce novel gene knockouts (KO) is not without their drawbacks. In order to address this need, I have developed a novel Cas9 fusion (ExoCas9) to enhance the efficiency of CRISPR-Cas based gene knockouts in the zebrafish. This fusion increases the efficiency of gene KOs as well as the average size of deletions produced. I used this fusion to screen for F0 neural angiogenic specific defects (brain hemorrhages) in genes implicated in TGF-β pathway response. I tested 70 single guide (sgRNAs) representing 26 individual genes using ExoCas9 and confirmed most targeted genes produced an observable brain hemorrhage phenotype greater than 10% for at least one sgRNA. I also looked for malformations in zebrafish vasculature hallmarks in the Casper KDR transgenic line, which has GFP-labeled vasculature, on a subset of genes from the ExoCas9 F0 screen. I prioritized additional experiments in genes also implicated in WNT neural angiogenesis (Hupe et al., 2017). I hypothesize that TGF-β and Wnt- pathways coordinately regulate a set of genes essential for brain angiogenesis and blood brain barrier (BBB) formation.