Highlights

•Postmitotic m-3j-gRNA1 treatment mediates predominant 1-bp deletion events

•m-3j-gRNA1 partially restores frameshift in Pcdh15^av−3j mutant gene

•m-3j-gRNA1 partially restores PCDH15 function in Pcdh15^av−3j mutant hair cells

•m-3j-gRNA1 partially restores hearing and balance of Pcdh15^av−3j mutant mice

Although frameshift mutations lead to 22% of inherited Mendelian disorders in humans, there is no efficient in vivo gene therapy strategy available to date, particularly in nondividing cells. Here, we show that nonhomologous end-joining (NHEJ)-mediated nonrandom editing profiles compensate the frameshift mutation in the Pcdh15 gene and restore the lost mechanotransduction function in postmitotic hair cells of Pcdh15^av−3J mice, an animal model of human nonsyndromic deafness DFNB23. Identified by an ex vivo evaluation system in cultured cochlear explants, the selected guide RNA restores reading frame in approximately 50% of indel products and recovers mechanotransduction in more than 70% of targeted hair cells. In vivo treatment shows that half of the animals gain improvements in auditory responses, and balance function is restored in the majority of injected mutant mice. These results demonstrate that NHEJ-mediated reading-frame restoration is a simple and efficient strategy in postmitotic systems.