Abstract |
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.
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