Therapeutic editing of the BCR-ABL fusion gene in chronic myeloid leukemia cell line using prime editing technology

Objective: Prime Editing (PE) is one of the new approaches in gene editing, among CRISPR-Cas technologies, showing high efficiency and high specificity to the selected sequence. This study aimed to apply PE in the K562 cell line and to offer a new strategy in the field of gene therapy for the treatment of the disease through genetic editing by trying a different approach than current approaches in the BCR-ABL1 fusion oncogene of Chronic Myeloid Leukemia (CML). Materials and Methods: In this study, a novel method targeting BCR-ABL1 fusion, the fundamental mechanism of CML pathogenesis, was developed. Using the K562 cell line, a specific termination sequence was added to the BCR-ABL1 junction to terminate translation. First, the target region sequence was determined. The most specific part of the study, the epegRNA molecule, was designed. EpegRNA synthesis was performed using the Golden Gate Cloning method. The epegRNA was ligated to the vector. Transformation and transfection procedures were applied. The PE efficiency rate was analyzed using the Next Generation Sequencing (NGS) method using two different and complementary bioinformatics approaches. First, the total wild-type (WT) allele population was determined with the P2 primer set. In the next step, the specifically designed P3 primer set was used to enrich and amplify only the edited alleles. The raw data obtained from NGS were analyzed using Integrative Genomics Viewer (IGV) by both direct counting with custom scripts and alignment to custom-generated reference sequences. has been confirmed. Results: Editing efficiency was calculated as 10.64%. It has been shown that the standard NND analysis is insufficient to detect low-frequency edits due to negative Polymerase Chain Reaction (PCR) bias. To overcome this limitation, a selective PCR approach was applied that enriches only edited alleles. This allowed for the precise calculation of the edit rate. Integration of data from both stages confirmed that editing occurred with high accuracy and provided a reliable determination of the edit rate in the population. Conclusion: This thesis demonstrates a novel and unique approach that offers the potential for a durable treatment for CML by targeting the oncogenic BCR-ABL1 fusion gene for the first time using PE technology. Successful editing was demonstrated with an efficiency of 10.64% in the K562 cell line, and a unique two-stage NGS analysis method was developed to overcome the inadequacy of standard methods for the detection of these low-frequency editing. This rate was interpreted as a meaningful starting point in the K562 cell line, which is considered difficult to edit. These promising results, combined with the high specificity and safety of PE technology, provide a solid foundation for future studies to increase editing efficiency and translate it to in vivo models, reinforcing the promise of this approach as a durable solution for the treatment of CML.