A porcine congenital deafness model with unconditional knockout of GJB2 generated by CRISPR/Cas9 genomic editing.

GJB2, the primary gene responsible for DFNB1, the most prevalent non-syndromic hearing loss (NSHL), has variants that account for over 50% of all prelingual hearing loss (HL). Mice are the main model for congenital hearing loss (CHL) research, but they have delayed auditory maturation postnatally, and unconditional Gjb2 knockout in mice causes embryonic lethality. Pigs have similar inner-ear anatomy to humans and, like humans, have matured auditory function and fully differentiated cochlea at birth. Currently, there is no GJB2 unconditional knockout animal model for GJB2-related CHLs research, and whether unconditional GJB2 deletion causes embryonic lethality in pigs or if GJB2-deficient pigs can recapitulate typical clinical pathological characteristics remains unclear. In this study, we employed CRISPR/Cas9 to establish the first unconditional GJB2 knockout pig model. The mutant GJB2 alleles in the founder pig were stably germline-transmitted to subsequent generations. Homozygous GJB2 knockout pigs exhibited no embryonic lethality and showed profound hearing loss, cochlear hair cell depletion and impaired Organ of Corti's development. This GJB2 unconditional knockout pig model has not been reported before and demonstrates GJB2 mutation pathological characteristics consistent with clinical patients, validating its potential in investigating the pathogenic mechanisms and therapeutic interventions of GJB2-deficient CHLs.