Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells

TL;DR

Researchers used CRISPR to edit a specific mutation in blood cells from a patient with a corneal disease, showing it's technically possible but not yet clinically applicable.

Contribution

Demonstrates precise CRISPR editing of the TGFBI R555W mutation in patient-derived PBMCs using optimized sgRNA and ssODN combinations.

Findings

sgRNA3–ssODN3 achieved 98.2% homology-directed repair efficiency in edited cells.

GFP-positive cells represented 2–4% of electroporated PBMCs after enrichment.

HRM and NGS confirmed the precision of the CRISPR editing process.

Abstract

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.

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