OC 74.2 - Generation of zebrafish f9like mutant by CRISPR/Cas9 technology

Background: Coagulation factor IX participates in the intrinsic pathway of coagulation. Previously we have shown that zebrafish have three factor IX genes: f9a, f9b, and f9like, and found that f9like is functionally similar to human coagulation factor X. At present, there are no knockout models for these genes to corroborate previous findings.

Aims: To create and characterize f9like knockout mutant zebrafish using CRISPR Cas 9 technology.

Methods: Three different gRNAs targeting exon 8 of the f9like gene were synthesized, and they were microinjected along with Cas9 protein into the yolk sac of the single-cell wild-type zebrafish embryos. The embryos were reared, and at 2.5 months post-fertilization, DNA was isolated from tail clips and amplified by PCR. The PCR products were run on polyacrylamide gels and the gel-purified PCR products were sequenced by the Sanger method.

Results: We injected approximately 300 zebrafish embryos and screened 60 adult fish for insertions or deletions in exon 8 using PAGE. Exon 8 was targeted as it encompassed the catalytic domain of the gene. Out of 60 fish, 9 fish showed two bands, one corresponding to the wild-type and the other either had deletion or insertion. These fish individually were crossed with wild-type fish, and only one of them transmitted the mutation to the next generation. The DNA bands from this homozygous mutant were sequenced, and the chromatograms revealed that there was a 14 bp deletion that generated a frameshift mutation causing premature termination and thus, yielded an f9like founder mutant line. We are currently characterizing this mutant with coagulation and bleeding assays.

Conclusion(s): We successfully created an f9like mutant that has 14bp deletion in exon 8 by CRISPR/Cas9 method. This f9like mutant should be useful in the creation of double mutants with the already known fx mutant to test whether it shows embryonic and developmental lethality.