Scopus
🔓 Açık Erişim
Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells
International Journal of Molecular Sciences · Mart 2026
Makale Bilgileri
DergiInternational Journal of Molecular Sciences
Yayın TarihiMart 2026
Cilt / Sayfa27
Scopus ID2-s2.0-105032669792
Erişim🔓 Açık Erişim
Özet
Over 70 mutations in the transforming growth factor beta-induced (TGFBI) gene are associated with corneal dystrophies that impair vision. The R555W hotspot mutation is a major cause of granular corneal dystrophy type 1 (GCD1). Here, we evaluated the technical feasibility of CRISPR/Cas9-mediated editing of the R555W mutation in peripheral blood mononuclear cells (PBMCs) obtained from a patient with GCD1. Three single guide RNAs (sgRNA1–3) and matched single-stranded oligodeoxynucleotide donors (ssODN1–3) were designed and co-transfected into PBMCs. Transfected cells were enriched by flow cytometric sorting, with GFP-positive cells representing approximately 2–4% of the total electroporated population. Editing outcomes were initially screened using high-resolution melting (HRM) analysis, and the sgRNA3–ssODN3 combination identified as the most promising candidate was subsequently validated by next-generation sequencing (NGS). Sequencing revealed a homology-directed repair efficiency of 98.2% among GFP-positive sorted cells, demonstrating efficient and precise genome editing within the enriched population. Because PBMCs are not disease-relevant corneal epithelial cells and only genomic endpoints were assessed, the clinical applicability of this study is limited and the work should be considered a technical proof-of-concept. This framework supports optimization of CRISPR-based strategies prior to studies in biologically relevant corneal models.
Yazarlar (4)
1
Burak Dağdelen
ORCID: 0000-0002-4362-3852
2
Hilal Arikoglu
ORCID: 0000-0002-6600-6603
3
Dudu Erkoc-Kaya
ORCID: 0000-0003-0114-6602
4
Banu Bozkurt
ORCID: 0000-0002-9847-3521
Anahtar Kelimeler
CRISPR/Cas9
genome editing
granular corneal dystrophy
homology-directed repair
peripheral blood mononuclear cells
R555W mutation
TGFBI
Kurumlar
Selçuk Tip Fakültesi
Konya Turkey