Abstract Number: OC 74.2
Meeting: ISTH 2022 Congress
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.
To cite this abstract in AMA style:Dhinoja S, De Maria A, Jagadeeswaran P. Generation of zebrafish f9like mutant by CRISPR/Cas9 technology [abstract]. https://abstracts.isth.org/abstract/generation-of-zebrafish-f9like-mutant-by-crispr-cas9-technology/. Accessed September 26, 2022.
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