(A) Schematic of zebrafish gucy1a1 gene presenting all the exons in box forms and introns in the form of connecting lines, a’ showing sequencing result comparison of zebrafish wild-type allele (WT) vs. mutant (Mut) at gRNA target site on gucy1a1 gene exon 1, a” (in the green box) highlights 4 base pair deletion (highlighted as red dashes) at the mRNA level in the knockout zebrafish compared to the wild types, and a” (in the yellow box) shows the deletion of 4 base pair leading to premature stop codon at the amino acid level after 16 amino acids. (B) Schematic of gRNA + spCas9 mRNA in one-cell stage zebrafish embryos leading to identification of F0 founder fish, which were out crossed with AB wild-types following which heterozygotes were identified and in crossed. (C) mRNA relative expression of gucy1a1 gene was observed significantly reduced in the gucy1a1^+/– (±50%) and gucy1a1^{– /–} (±90%), error bars represent ±SD. (D) Schematic represents the conserved genomic positioning of both gucy1a1 and gucy1b1 genes in human and zebrafish genome, screen shots taken from Ensemble genome browser.

Blood flow parameters measured from AB-wild types, heterozygote and homozygote for gucy1a1 gene in zebrafish larvae. Blood flow (nL/sec), arterial pulse (beats per minute), linear velocity (μm/sec), and vessel diameter (μm) were measured from gucy1a1^+/– (n = 12), gucy1a1^{– /–} (n = 12), and AB-wild-type (n = 10) larvae per group. Data are presented as mean ± standard deviation, significance was set at p < 0.05, and stars denote the significant differences.

(A) Graphs represent mRNA downregulation of gucy1a1, gucy1b1, and cct7 genes using morpholino (MO) in zebrafish. No cross reactivity of morpholinos was observed between the two subunits of gucy gene in the mRNA expression. (B) Graphs represent comparison of blood flow parameters measured from gucy1a1 (n = 12), gucy1b1 (n = 12), gucy1a1+gucy1b1 (n = 12), and control-MO (n = 10). (C) Graphs presenting comparison of blood flow parameters measured from AB-wild-type (n = 10), cct7-MO (n = 12), gucy1a1^+/– (n = 12), and gucy1a1^+/– + cct7-MO (n = 12). Data are presented as mean ± standard deviation, significance was set at p < 0.05, and stars denote the significant differences.

(A) Graph presents cGMP levels (p mol per larvae) measured from AB-wild-type larvae at 4 dpf treated with H₂O, 1, 5, and 10 μM BAY41-2272 concentration. (B) cGMP levels measured and compared from gucy1a1^+/– treated with H₂O, 1, 5, and 10 μM BAY41-2272 concentration. (C) cGMP levels measured and compared between AB-wild types, morpholino injected controls, gucy1a1-MO, gucy1b1-MO, and gucy1a1+gucy1b1-MO. Data are presented as mean ± standard deviation from n = 30 larvae per group, significance was set at p < 0.05, and stars denote the significant differences.