Figure 4 - figure supplement 3

Figure 4 - figure supplement 3 Gross ocular phenotype analyses of ntn1-deficient zebrafish.

(a) Gene-editing strategy using a single sgRNA targeting the first exon of zebrafish ntn1a. CRISPR/Cas9 was used to generate heterozygous (ntn1^+/-; G0) founders. These were crossed to generate homozygous G1 embryos (ntn1^-/-). (b) Panels showing the coloboma microphthalmia and coloboma (arrow) phenotypes in gene-edited ntn1^-/- embryos compared to wild-type. (c) Sanger sequencing confirmed the homozygous gene-edited ntn1a allele in 100% of phenotypic G1 embryos. (d) In silico translation of encoded mutant allele aligned to wild-type (first 153 amino acids shown of 603 aa ntn1a protein are shown). The gene edited mutation encodes a frame-shift in the first exon resulting in a truncated ntn1a of 105 amino acids (p.Cys90Ala.fs15). (e) Morpholino experiments produced bilateral coloboma in 100% of embryos injected with ntn1a translation-blocking MO, with no ocular phenotypes observed in control MO injected embryos. The optic fissures are indicated by arrows. (f) Tables with penetrance of colobomas in gene-edited embryos and MO embryos compared to controls.