The work presented in this thesis combines two hugely promising nascent technologies by establishing a CRISPR-Cas9 based genome editing toolkit for use in the immortalized erythroblast line BEL-A (Bristol Erythroid Line - Adult). Various approaches to CRISPR-Cas9 editing were tested to achieve knockouts, knock-ins, and transcriptional activation. An efficient workflow for generation of lentiviral CRISPR-mediated knockouts was established and multiple enucleation competent cell lines deficient in individual blood groups were produced as tools for diagnostics and as proof of principal for transfusion therapy. Multiple blood group knockouts were combined to generate a cell line which could be differentiated to form reticulocytes deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (H0), Rh (Rhnull), Kell (K0), Duffy (Fynull) and GPB (S- s- U-). This represents a significant step towards the generation of engineered red blood cells for transfusion of patients with specific blood group matching requirements, such as those with very rare blood types or those with diseases requiring regular transfusion therapy. The genetic toolkit was expanded to demonstrate gene regulation in the BEL-A cell line and efficient activation of erythroid genes ICAM4 and SLC14A1 (Kidd) and non-erythroid genes CD4 and CD8A was achieved using the CRISPR activator SunTag system. The genome editing techniques established here provide valuable tools for the generation of engineered red blood cells which holds great promise for basic research purposes, for the future provision of a sustainable blood source for transfusion, and as a platform for erythrocyte-based therapeutic applications.
|Date of Award||1 Oct 2019|
- The University of Bristol
|Supervisor||Abigail Spear (Supervisor), Ash M Toye (Supervisor) & Jan Frayne (Supervisor)|
- Gene editing
- Universal donor