Prime Editor 3 Mediated Beta-Thalassemia Mutations of the HBB Gene in Human Erythroid Progenitor Cells

International Journal of Molecular Sciences

Daru Lu¹  Qinlinglan Zhou¹  Haokun Zhang¹  Hongyan Chen¹ 

[1] State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China;

关键词: prime editor 3;    beta-thalassemia mutation;    the HBB gene;    HUDEP-2 cells;    genome editing;   

DOI  :  10.3390/ijms23095002

来源: DOAJ

【 摘 要 】

Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in the erythroid progenitor cell line HUDEP-2. We inserted the mCherry reporter gene cassette into editing plasmids, each including the prime editing guide RNA (pegRNA) and nick sgRNA. The plasmids were electroporated into HUDEP-2 cells, and the PE3 modified cells were identified by mCherry expression and collected using fluorescence-activated cell sorting (FACS). Sanger sequencing of the positive cells confirmed that PE3 induced precise beta-thal mutations with editing ratios from 4.55 to 100%. Furthermore, an off-target analysis showed no unintentional edits occurred in the cells. The editing ratios and parameters of pegRNA and nick sgRNA were also analyzed and summarized and will contribute to enhanced PE3 design in future studies. The characterization of the HUDEP-2 beta-thal cells showed typical thalassemia phenotypes, involving ineffective erythropoiesis, abnormal erythroid differentiation, high apoptosis rate, defective alpha-globin colocalization, cell viability deterioration, and ROS resisting deficiency. These HUDEP-2 beta-thal cells could provide ideal models for future beta-thal gene therapy studies.

【 授权许可】

Unknown