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Fig. 6 Genome engineering of a large deletion in vwf. (A) The structure of the vwf gene in zebrafish, with deletion indicated. (B) Sequencing of vwf–/– fish shows a large deletion (73.7 kb) between exons 5 and 51. (C) Genomic DNA amplification of vwf mutants. (D) Laser-mediated endothelial injury was performed on larvae at 3 dpf (venous) and 5 dpf (arterial). The TTO was not significantly different among vwf mutant clutchmates (Mann-Whitney U, P = .5 for venous and P = .04 for arterial, only values <.03 are considered significant after Bonferroni correction). Circles represent individual larvae. Horizontal bars represent the median TTO. (E) Laser-mediated injury at 3 dpf from an incross of vwf+/−;at3+/− shows no change in the at3–/– DIC bleeding phenotype with loss of Vwf (Mann-Whitney U, P = .12). (F) Spontaneous fibrin deposition observed in 5 dpf larvae from vwf+/−;at3+/− incrosses. Bar graph represents percentage of larvae in each category: score 0 having no GFP-labeled fibrin deposits in the PCV, score 1 <5 occurrences, score 2 with 5 to 25 occurrences, and score 3 with widespread continuous threads of fibrin in the PCV and/or surrounding regions. Statistical significance was determined by Kruskal-Wallis testing and pairwise comparisons among vwf+/+;at3–/–, vwf+/−;at3–/–, and vwf–/–;at3–/– (P = .3). Larvae were confirmed to express the fgb-egfp transgene in the liver prior to scoring. (G) pzf8 injected into 1-cell stage embryos collected from vwf14-28Δ/Δ;at3+/− incrosses and evaluated for spontaneous thrombi formation via fibrin deposition scoring at 5 dpf. pzf8 injected zebrafish larvae exhibited significant fibrin deposition compared to uninjected controls in all genotypes. Statistical significance was determined by ordinal logistic regression followed by Dunnett adjustments (∗∗∗P < .001; ∗P < .05). ns, nonsignificant.