Efficient and in situ correction of hemoglobin Constant Spring mutation by prime editing in human hematopoietic cells

Hemoglobin Constant Spring (Hb CS) is the most common non-deletional and clinically significant α-thalassemic mutation, and it is caused by an anti-termination mutation at the α2-globin gene stop codon. We developed a prime editing strategy for the creation and correction of Hb CS. We showed that prime editing could efficiently introduce Hb CS mutations in both human erythroblast cell lines (an average frequency of 32%) and primary hematopoietic stem and progenitor cells (HSPCs) from healthy donors (an average frequency of 27%). By targeting the established Hb CS homozygous erythroblasts, we achieved an average frequency of 32% in situ correction without selection. Notably, prime editing corrected the Hb CS mutation to wild type at an average frequency of 21% in HSPCs from three patients with hemoglobin H Constant Spring (HCS). Erythrocytes that differentiated from prime-edited erythroblasts or HSPCs exhibited a significant reduction in the amount of α^CS-globin chains. Insertions and deletions on HBA2 locus and Cas9-dependent DNA off-target editing were detected with relatively low frequency after prime editing. Our findings showed that prime editing can successfully correct Hb CS in erythroblasts and patient HSPCs, which provides proof of principle for its therapeutic potential in HCS.