Editing of the SLURP1 gene in Mal de Meleda (MDM) disease using prime editing technology

Mal de Meleda (MDM) is a rare form of inflammatory palmoplantar keratoderma caused by mutations in the SLURP1 gene, which encodes the secreted mammalian Ly-6/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1) protein located on chromosome 8q24.3, which regulates epidermal homeostasis and skin inflammation. Prime editing, an improved version of CRISPR-Cas9, was described in 2019 and is an effective genome editing technology that can perform precise base editing. In this thesis, we aimed to correct the mutation using Prime editing in an in vitro model belonging to an individual with a c.286C>T mutation in the SLURP1 gene. The study was started by selecting suitable plasmids and designing epegRNA suitable for the c.286C>T mutation in the SLURP1 gene. The designed epegRNA oligonucleotides were annealed and cloned into the pU6-tevopreq1-GG-acceptor plasmid with a truncated mRFP1 region using the Golden Gate method. The cloned plasmid was transferred to DH5α bacteria via transformation and the isolated plasmid DNA was verified after colony selection. The Prime Editor system (nCas9-RT + epegRNA) prepared for lymphocyte cell culture obtained from the patient's peripheral blood was transfected. In order to evaluate the transfection effıciency and editing effıciency, target region-specific primers were designed and a limited-scope Next Generation Sequencing (NGS) analysis was applied only for the target variant after PCR amplification. Each stage was strengthened with optimization and the next stage was not passed until the desired result was obtained. As a result of NGS, sequence data in FASTQ format were analyzed using Notepad++ software and editing rates were calculated. It was determined that corrections were achieved at 0.40% and 0.23% levels in SH1 and SH2 samples, respectively. Despite the relatively low correction rates, the analyses show that a specific correction was successfully achieved in the targeted variant region.