O14 Next-generation sequencing precision: on- and off-target analysis of gene editing in recessive dystrophic epidermolysis bullosa

Abstract Introduction and aims Base editors (BEs) and prime editors (PEs) offer distinct advantages over CRISPR-Cas9 for correcting pathogenic variants. Previously, our group achieved 94.6% efficiency in correcting the COL7A1 c.5047 C>T nonsense pathogenic variant in fibroblasts, associated with recessive dystrophic epidermolysis bullosa (RDEB), using adenine base editor 8e (ABE8e). However, the safety and cell type-specific efficiencies of these techniques remain unclear. This study aims to use a PE to correct the c.5047 C>T pathogenic variant in primary patient fibroblasts and epidermal stem cells to promote wound healing and compare the efficiency and off-target effects of BE and PE using next-generation sequencing (NGS). Methods Primary fibroblasts carrying a heterozygous COL7A1 c.5047 C>T pathogenic variant were transfected with ABE8e mRNA and the PE5bmax system using electroporation and lipid nanoparticles (LNPs). Sanger sequencing and NGS were used to confirm pathogenic variant correction and assess editing efficiencies. Whole-genome sequencing (WGS) was conducted for genome-wide off-target analysis, with pairwise variant calling performed to identify somatic variants in edited vs. unedited samples. Targeted amplicon sequencing was employed to evaluate editing accuracy at the target site. Results ABE8e delivered via LNPs corrected the c.5047 C>T pathogenic variant in fibroblasts with 84% efficiency using lipofectamine, as confirmed by Sanger sequencing and NGS. Prime Editor PE5bmax achieved 43% overall editing efficiency, with 32% variant correction confirmed by Sanger sequencing. Preliminary WGS analysis revealed increased indels and mismatches across the genome in edited samples, although further variant characterization is needed for a comprehensive safety profile. Conclusions ABE8e and PE5bmax successfully corrected the c.5047 C>T mutation in fibroblasts, with respective on-target efficiencies of 84% and 32%. These strategies are being extended to epidermal stem cells for potential wound healing therapies. Comprehensive WGS and targeted sequencing will aid in optimizing these techniques for clinical applications.