Targeted disruption of at3 using genome editing nucleases results in adult lethality. (A) ZFN-induced deletions in somatic cells. Of 16 pooled morphologically normal embryos from 2 individual experiments, 3 of 192 sequences were identified with Δ5 (2x) and Δ9 (1x) in exon 5. The top line is the at3 reference genome sequence from an unrelated individual. The blue and green arrows indicate 5′ and 3′ ZFN-binding sites, respectively. Deletions are indicated by red dashes. (B) Sequences of ZFN-induced mutations transmitted through the germline. Red dashes and letters indicate deletions and insertions, respectively. (C) Survival curve of a total of 3 clutches derived from at3^Δ90 heterozygous incrosses shows loss of homozygotes. Offspring were genotyped at 2 to 4 months of age and tracked over 1 year. The results demonstrate a highly significant difference in the survival of homozygous mutants by log-rank (Mantel-Cox) analysis (P < .0001). (D) Externally visible secondary hemorrhage in an at3^Δ90 homozygous mutant is indicated by an arrow. This fish was identified while alive, anesthetized, and the image was captured using a Leica MZ16FA microscope and a Canon EOS60D camera. (E-F) RT-PCR analysis of at3^Δ90 mutants demonstrates reduction of at3 expression in heterozygotes and absence of homozygous mutant mRNA. (E) Top: Total mRNA was prepared from adult fish (4 mpf) followed by qualitative RT-PCR. Bottom: Genotyping of at3^Δ90 mutants. (F) Total mRNA was prepared from pooled larvae (3 dpf, n = 15 for each genotype) followed by quantitative real-time PCR. Error bars represent standard deviation and statistical significance was determined by t test. (G) at3^Δ22 homozygous mutants were injected with a ubi regulated at3 cDNA expression vector and followed for 1 year.