Development of HEK293T cell reference materials for β-thalassemia genetic testing using prime editing

Objectives β-Thalassemias, caused by mutations in HBB , are hereditary blood disorders that impose a significant global health burden. Detecting these mutations through accurate genetic analysis is essential. This study aimed to create a panel of cell type reference materials for β-thalassemia genetic testing using prime editing (PE), a flexible and precise genomic editing method. Methods PE3 systems were designed to target specific HBB mutations, including single nucleotide variants (SNVs) (−32 (C>A), CD 17 (A>T)), insertions (CD 14/15 (+G), CD 71–72 (+A)), and deletions (CD 31 (−C), CD 41/42 (−TTCT)). HEK293T cells were edited to carry these mutations. Sanger sequencing was performed to confirm the accurate introduction of homozygous and heterozygous mutations. The cell lines were further verified using commercial reverse dot-blot hybridization and melting curve assays. Results We successfully constructed 12 stable HEK293T cell lines carrying the intended homozygous or heterozygous HBB mutations using PE3 systems. No off-target mutations in the HBB were detected by Sanger sequencing in these stable cell lines, even after more than 10 weeks of culturing. Additionally, all target mutations were accurately and consistently detected using two reverse dot-blot hybridization kits and one melting curve assay kit. Conclusions The 12 stable cell lines exhibited accurate and stable HBB mutations, making them valuable reference materials for β-thalassemia genetic testing. PE3 systems show potential for generating a comprehensive panel of HBB mutations, particularly those that are clinically rare.