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

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 proge...

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Bibliographic Details
Published in:International journal of molecular sciences 2022-04, Vol.23 (9), p.5002
Main Authors: Zhang, Haokun, Zhou, Qinlinglan, Chen, Hongyan, Lu, Daru
Format: Article
Language:English
Subjects:
alpha-Globins - genetics
Apoptosis
beta-Globins - genetics
beta-Thalassemia - genetics
beta-Thalassemia - therapy
beta-thalassemia mutation
Blood diseases
Cell culture
Cell viability
Cells (biology)
Cloning
Design optimization
Erythroid Precursor Cells - metabolism
Erythropoiesis
Flow cytometry
Gene therapy
Genome editing
Genomes
HBB gene
Hematopoietic stem cells
HUDEP-2 cells
Humans
Mutation
Phenotypes
Plasmids
prime editor 3
Progenitor cells
Reporter gene
RNA editing
Thalassemia
the HBB gene
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Summary: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 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.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms23095002