Fig. 6

Co-mutation in tp53 gene rescues female-to-male sex reversal of fancp homozygous knockouts.

(A) RT-PCR confirmed the absence of any aberrant splice variants around tp53 gene mutation. Sanger sequencing confirmed the presence of CRISPR-Cas9 introduced 7bp deletion mutation. The products were resolved on 2% agarose gel. (B) Reporter assay to check the expression of tp53 frameshift mutant. Representative images at 1 dpf of embryos co-injected with the specified reporter mRNA and TagRFP are shown as RFP (left panel), GFP (middle panel) and merged (right panel). (C) Western blot analysis shows absence of Tp53 protein in the knockout embryo extracts. Twenty-four hpf embryos collected from tp53^hg91/hg91 homozygous knockout fish incross were used to check the loss of expression of Tp53 protein. Embryos obtained from TAB5 incross were used as WT controls. Expression of β-actin was used as loading control. (D) Progenies from fancp^hg67/+;tp53^hg91/+ and fancp^hg67/+;tp53^hg91/hg91 breeding were genotyped around 4 mpf and the sex was determined. Number of male and female fish in each genotype category is presented. The genotypes are marked on the X-axis. Among the fancp homozygous knockouts, only males were observed with fancp^hg67/hg67;tp53^hg91/+ genotype, whereas both males and females were observed with fancp^hg67/hg67;tp53^hg91/hg91 genotype.