Figure 4

Knocking out mib1 generates a hemorrhagic phenotype. (A) Genomic region of chromosome 2 containing mib1 gene. Two patient mutations G596 and R906 are indicated. gRNAs (magenta) were designed to target both regions. (B) T7 endonuclease activity against gRNA target amplicons. Uninjected DNA pool (?) produces one fragmentation pattern, mib1-injected DNA pool (+) produces extra bands (filled arrowheads) caused by heteroduplex DNA formation due to editing and subsequent digestion by T7 endonuclease. (C) Brightfield macroscopic image of representative series of uninjected larvae at 3 dpf. Scale bar: 1 mm. (D) Similar image of mib1 knockout fish. Note severe body curvature defects. (E) Uninjected 54 hpf fish, dorsal view. Scale bar: 250 µm. (F) Similar mib1 knockout fish exhibiting cranial hemorrhage (filled arrowhead). (G) Brightfield image of 50 hpf mib1 knockout fish with aortic hemorrhage (filled arrowhead, scale bar: 500 µm). (H) Quantification of hemorrhagic phenotype. Mib1 knockout fish exhibit significantly more hemorrhagic events (uninj: 1%, mib1: 31%, chi-square test, p < 0.001).