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

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