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Fig. 11 Investigation of genetic compensation by inactivating upf3 paralogs in stable knockouts of hearing genes. (A) The msrb3 F0 knockouts exhibited a reduced AEBR compared to either Cas9-injected or upf3a;upf3b F0 knockout larvae. Similarly, msrb3 homozygous (Hom) knockouts showed a reduced AEBR compared to either WT or heterozygous (Het) larvae. RT-qPCR showed a significant reduction in tmprss3a, whereas tmprss3b was slightly reduced in both tmprss3a;tmprss3b heterozygous and homozygous knockouts. (B) The tmprss3a F0, tmprss3b F0, tmprss3a;tmprss3b F0, tmprss3aHom;tmprss3bHom and tmprss3aHom;tmprss3bHom coinjected with upf3a and upf3b gRNAs (tmprss3aHom;tmprss3bHom;upf3a;upf3b F0) showed a reduced AEBR compared to Cas9-injected larvae. Furthermore, tmprss3a;tmprss3b F0, tmprss3aHom;tmprss3bHom, and tmprss3aHom;tmprss3bHom;upf3a;upf3b F0 larvae exhibited a similar degree of reduction in AEBR assay. RT-qPCR showed a significant reduction in tmprss3a, whereas tmprss3b was slightly reduced in both tmprss3a;tmprss3b heterozygous and homozygous knockouts. (C-F) Comparisons of the AEBR assay for control versus F0, stable knockout, and stable knockout coinjected with upf3a and upf3b gRNAs in coch (C), tbc1d24 (D), pou4f3 (E), and gipc3 (F) genes showed no difference. RT-qPCR analysis was performed to determine the gene expression levels in each knockout, and the results are shown in the right panel. Each group was compared with every other group. The percentage of responses to 12 sound stimuli for AEBR were calculated for each larva. Statistical significance was calculated by Brown–Forsthye and Welch's ANOVA with Dunnett's T3 multiple comparisons test: not significant (ns) P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.