Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

Abstract

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we show that simultaneous injections of three distinct dgRNPs per gene into one-cell stage embryos dramatically enhanced the efficiency of biallelic gene disruptions with greater consistency than any single dgRNP injections tested on 4 different genes. Importantly, this mutagenesis method is capable of disrupting genes encoding distinct types of proteins, including secreted and membrane-bound forms, and the generated F0 animals fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, we provide evidence that this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Therefore, our approach offers a highly efficient genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.