Knockout of von Willebrand factor in Zebrafish by <scp>CRISPR</scp>/Cas9 mutagenesis

Iyer, Neha; Tcheuyap, Vanina Toffessi; Schneider, Sara Jane; Marshall, Vanessa; Jagadeeswaran, Pudur

doi:10.1111/bjh.15910

Cited by 12 publications

(10 citation statements)

References 11 publications

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“…[15][16][17][18][19] In addition, targeted mutations in a number of coagulation-related proteins, including fibrinogen, 20,21 have demonstrated the zebrafish's utility in modeling factor deficiencies and for assessing the impact of patient-derived polymorphisms. [22][23][24][25] Zebrafish have nucleated thrombocytes, 26 which are considered cellular equivalents to platelets in hemostasis. They participate in blood clotting in a platelet-like manner, rapidly binding and aggregating at injury sites.…”

Section: Introductionmentioning

confidence: 99%

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

et al. 2020

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Plasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

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Section: Introductionmentioning

confidence: 99%

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

et al. 2020

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“…The model’s major strength is a combination of high fecundity, accessibility of near transparent larval tissues and blood vessels that can be observed and manipulated in real time, and robust tools to make genetic modifications [ 24 ]. This has been demonstrated in a number of recent models of hemostasis and coagulation factor deficiencies that recapitulate phenotypes found in patients and enable screening of potential pathogenic variants [ 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders

Fish

Freire

Sanza

et al. 2021

IJMS

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Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga−/− larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/− larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19–56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19–56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga−/− mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19–56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

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“…This model is attractive for studying human hemostatic disorders because of the availability of laser thrombosis assay using transparent larvae and the availability of knockdown methods [18][19][20] and robust genome editing tools for knockouts of desired genes. 21,22 Here, we aim to identify the F9 ortholog in zebrafish, which would facilitate the creation of hemophilia B model by knockout of this ortholog. Moreover, such a model will be an asset to identify the mechanism of increased FIX production postpuberty in hemophilia B Leyden patients by replacing the zebrafish f9 promoter with human hemophilia B Leyden promoter by homologous recombination.…”

Section: Introductionmentioning

confidence: 99%

“…Zebrafish is a widely used model for studying hemostasis and coagulation 15‐17 because it exhibits tremendous homology with the mammalian coagulation system. This model is attractive for studying human hemostatic disorders because of the availability of laser thrombosis assay using transparent larvae and the availability of knockdown methods 18‐20 and robust genome editing tools for knockouts of desired genes 21,22 . Here, we aim to identify the F9 ortholog in zebrafish, which would facilitate the creation of hemophilia B model by knockout of this ortholog.…”

Section: Introductionmentioning

confidence: 99%

Identification of zebrafish ortholog for human coagulation factor IX and its age‐dependent expression

Iyer

Qaryoute

Kacham

et al. 2021

Journal of Thrombosis and Haemostasis

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Background: Coagulation factor IX (FIX) is a serine protease zymogen involved in the intrinsic blood coagulation pathway, and its deficiency causes hemophilia B. Zebrafish has three f9 genes, and the ortholog to human F9 is unknown.Objective: To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology.Methods: Gene and protein sequence analysis for three f9 genes, f9a, f9b, and f9l, present in the zebrafish genome was performed. In vivo and in vitro assays after knockdown of each gene and immunodepletion using specific antibodies were carried out.Results: Sequence analysis revealed that f9a and f9b are similar to human F9, whereas f9l is similar to human F10. RNA analysis showed an age-dependent increase in expression of all three genes. Zebrafish f9a gene knockdown and Fixa immunodepletion prolonged kinetic partial thromboplastin time (kPTT), whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kinetic prothrombin time, and kinetic Russell viper venom activation time. Laser-assisted venous thrombosis increased time to occlusion after f9a and f9l knockdown and antibody inhibition of Fixa and Fixl. Further, analysis of plasma proteins by mass spectrometry and immunohistochemistry detected all three proteins. Conclusions: Our findings suggest that zebrafish f9a has functional activity similar to human F9. Fixl is functionally similar to Fx. The age-dependent increases of these factors are comparable to those observed in mice and humans. Thus, the zebrafish model could be used to study factors involved in increasing f9a expression during aging. It could also be used to test whether normal human Factor IX and Factor IX Leyden promoter work in zebrafish background.

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Knockout of von Willebrand factor in Zebrafish by CRISPR/Cas9 mutagenesis

Cited by 12 publications

References 11 publications

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders

Identification of zebrafish ortholog for human coagulation factor IX and its age‐dependent expression

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