Precision correction of the GJB2 c.235delC mutation by prime editing in vitro.

Hereditary hearing loss, predominantly attributed to mutations in the GJB2 gene, constitutes a significant global health issue, with the c.235delC mutation being the most prevalent pathogenic allele in East Asian populations. Existing interventions, such as hearing aids and cochlear implants, mitigate symptoms but fail to correct the underlying genetic defects, highlighting the need for precise therapeutic approaches. In this study, we developed a cellular disease model (293T-GJB2mut-EGFP) incorporating the c.235delC mutation and employed prime editing (PE) to restore the GJB2 functionality. We systematically optimized the pegRNA architecture and identified that the optimal pegRNA (PBS 9 bp, RTT 16 bp) achieved 37.15% correction with PE2. Incorporating Csy4 or evopreQ1motifs at the pegRNA 3' end resulted in comparable efficiencies (~37%). The introduction of two synonymous mutations (pegRNAMMD2) increased the efficiency to 48.94%. Among the seven prime editor variants, PEmax showed the highest intrinsic activity (49.14%). The PE3b nicking strategy with a + 1 nick sgRNA further improved the efficiency to 58.05%. To address adeno-associated virus (AAV) packaging limitations, a split-intein dual-AAV system for PEmax (bisected between residues 1153-1154) was developed. After nicking optimization, it showed comparable correction efficiency to a previously reported split site (residues 1024-1025), with no statistically significant difference. Off-target deep sequencing of the top four predicted loci revealed no significant editing above the background. These findings establish a comprehensively optimized prime editing toolkit for GJB2 c.235delC correction and provide a foundation for future AAV-mediated in vivo gene therapy for hereditary hearing loss.