Genome Editing of Factor V in Zebrafish Embryos Results in a Severe Hemostatic Defect without Spontaneous Hemorrhage

Abstract

The cessation of bleeding following trauma is a crucial element in vertebrate survival.Factor V (F5) serves as an essential cofactor in the penultimate step of coagulation, the conversion of prothrombin to thrombin. In humans, genetic deficiency of F5 is rare and clinically diverse, with presentations ranging from neonatal intracranial hemorrhage to mild bleeding later in life. This patient variability suggests the presence of modifiers, unlinked genes inherited separately from the F5 locus. Complete loss through gene targeting of mouse F5 results in embryonic and neonatal lethality, interfering with further detailed studies. Zebrafish possess many distinct advantages for the study of coagulation and modifier genes, including high fecundity, optical clarity, external development, as well as extensive homology of the mammalian hemostatic system. Here we report the role of F5 in zebrafish development using genome editing mediated targeted mutagenesis. The f5 locus was identified through a BLAST search of zebrafish genomic sequence, identifying a protein with 48% amino acid identity and 66% similarity to human F5. In situ hybridization revealed expression of f5 mRNA localized to the developing liver at 5 days post fertilization (dpf). CRISPR RNA guided nucleases were designed to target exon 4 of F5 and injected into several hundred wild type zebrafish one cell stage embryos to produce adult fish (F0) with a panel of insertion/deletion mutations in f5. Adult fish (F1) heterozygous for a 49 base pair deletion were identified and incrossed to generate groups of offspring for analysis. This deletion was initiated at amino acid 171, within the A1 domain of F5. Genotyping was performed after phenotypic analysis so that observers were blinded during data collection. Homozygous mutants demonstrated significantly decreased survival compared to their heterozygous and wild type siblings, with die off beginning between 2 and 4 weeks post fertilization and 100% mortality by 7 months of age. Visual observation of development and circulation revealed that homozygous mutant embryos and larvae were indistinguishable from wild type siblings, with no signs of hemorrhage. Since there was no observable bleeding, we used induced occlusive thrombus formation by laser mediated endothelial ablation of the posterior cardinal vein at 3 dpf to assess hemostasis. We found that the ability to produce occlusive thrombi was absent in f5-/- mutants, a bleeding phenotype, while wild type and heterozygous siblings were phenotypically normal. This bleeding phenotype was rescued in 73% of embryos (p=0.0006) at 3 dpf after injection of zebrafish f5 cDNA at the one cell stage. In summary, we have demonstrated strong conservation of zebrafish and mammalian F5, including site of synthesis and requirement for hemostasis. Surprisingly, embryos and larvae, as well as young adults, tolerate what is a severe and lethal defect in mammals, allowing accessibility not easily achieved in murine models. This suggests the possibility of species-specific factors enabling survival in fish. Identification of these factors, combined with small molecule screens, could lead to novel therapeutic modalities for patients with bleeding disorders. DisclosuresNo relevant conflicts of interest to declare.