CRISPR/Cas9 mediated generation of zebrafish f9a mutant as a model for hemophilia B.

Abstract

Aim: This study aimed to develop a zebrafish model for hemophilia B by creating a f9a knockout, as f9a has previously demonstrated functional similarity to human Factor IX.

Methods: Using CRISPR/Cas9 technology, two gRNAs targeting exon 8 of the f9a gene, were injected along with Cas9 protein into single-cell zebrafish wild-type embryos. DNA was harvested from the tail tips of the resulting adult zebrafish and screened for mutations using PCR. The founder mutant was crossed with wild-type fish to confirm heritability and subsequently reared to homozygosity. Homozygous mutants were analyzed through quantitative RT-PCR and Western blot to assess f9a RNA and F9a protein levels, respectively. Functional assays like kinetic partial thromboplastin time (kPTT), bleeding assay in adult mutants, and venous laser injury on mutant larvae were performed to assess the hemostatic role.

Results: Around 61 adults from the CRISPR/Cas9 knockouts were screened, which resulted in a mutant line with a 72 bp deletion in the exon 8 encoding catalytic domain. Quantitative RT-PCR and Western Blot analysis showed reduced levels of f9a RNA and F9a protein in the homozygous mutants compared to wild-type siblings. At five dpf, f9a homozygous mutant larvae demonstrated prolonged venous occlusion times in a laser injury assay. Additionally, plasma from the mutants displayed delayed fibrin formation in kPTT assays and exhibited increased bleeding after mechanical injury.

Conclusion: This study created a zebrafish f9a knockout model that mimics the bleeding phenotype observed in hemophilia B patients, which will be valuable for evaluating novel therapeutic approaches for hemophilia B.