(F1300) ENHANCEMENT OF ERYTHROCYTE DIFFERENTIATION VIA THE PRECISE INTRODUCTION OF A NATURALLY OCCURRING VARIANT IN EPOR USING CRISPR/CAS9 GENOME EDITING
Program Specific Researcher Center for iPS Cell Research Amd Application, Kyoto University Kyoto, Kyoto, Japan
Abstract: Biomanufacturing with induced pluripotent stem (iPS) cells for industrial or therapeutic applications faces significant limitations, including scalability, cost-efficiency, and consistency of the differentiated cell product. Naturally occurring variants in humans are often studied for their impact on disease, yet some variants confer traits seen as beneficial in vitro. For example, C-terminal truncating variants in the erythropoietin receptor (EpoR) increase sensitivity to erythropoietin (Epo), resulting in uncontrolled expansion of red blood cells, a condition known as congenital erythropoiesis (CE). Genetic engineering of the EPOR gene in iPS cells represents an approach to overcome biomanufacturing challenges and meet global demands for blood and blood cell products. In this work, we engineered human iPS cells with a 7-bp deletion (NM_000121.4: c.1299_1305del) resulting in truncated EpoR reported in CE. Our strategy employs CRISPR/Cas9 genome editing coupled with DNA repair via endogenous microhomology-mediated end-joining to achieve precise editing without the need for an exogenous DNA template. In two iPS cell lines commonly used for research, the 7-bp deletion variant accounted for 90.0% of all indels where the target site was modified at the frequency of 86.5%. Our approach was reproducible in an additional eight iPS cell lines established from healthy donors, with an average of 90.8% of edited alleles carrying the 7-bp deletion variant. During differentiation of EPOR-edited iPS cells into erythrocytes, iPS cells homozygous for the truncated EPOR allele demonstrated a selective advantage in proliferation, with a remarkable 93-fold increase in the production of erythroid cells compared to the wildtype allele. Moreover, cell surface markers of the erythrocyte lineage CD71 and CD253a were presented earlier in iPS cells with truncated EPOR, indicating accelerated differentiation. Finally, a survey of single nucleotide polymorphisms (SNPs) in population genomic data suggests that our guide RNA (gRNA) targets a highly conserved region of EPOR across various ethnicities. Taken together, these data suggest that our editing approach is broadly applicable to the general human population and may be integrated into biomanufacturing processes to treat hematological disorders.
