A novel molecular approach for the generation of the t315i mutation in the K562 cell line by prime editing

Chronic Myeloid Leukemia (CML) is a hematopoietic malignancy characterized by constitutive tyrosine kinase activity of the BCR-ABL1 fusion protein and is commonly treated with tyrosine kinase inhibitors (TKIs). However, resistance to TKI therapy can develop in some patients, often due to point mutations in the BCR-ABL1 gene. These mutations hinder TKI binding, reducing treatment efficacy and accelerating disease progression. In this thesis, the T315I mutation, which is the most frequently observed mutation conferring TKI resistance in CML, was modeled in the K562 cell line using prime editing technology. Two different methodologies were applied, one of which was developed by the researchers and incorporates a dual-fluorescent marker–based enrichment strategy. This novel approach allows visual monitoring of target cells and selection of cells carrying the resistance gene, thereby enhancing editing efficiency. The results indicate that the developed strategy significantly increases the editing rate compared to the previous methodology. This approach enables reliable in vitro modeling of CML-specific mutations and provides a platform to improve prime editing efficiency in systems with limited transfection efficiency or high cellular heterogeneity. Consequently, the developed methodology represents a valuable tool for both basic research and future functional and therapeutic studies.