Functional Characterisation of Novel Variants in SCD Modifier Genes Using CRISPR-Cas9 Cellular Models: Implications for Therapeutic Targeting

Sickle cell disease clinical heterogeneity is shaped by modifier genes that influence HbF levels, inflammatory pathways, and vascular biology beyond the primary HBB mutation. While several modifier loci have been identified in GWAS, the functional consequences of novel African-specific variants in these genes remain unvalidated experimentally. This dissertation conducted functional characterisation of eight novel variants in SCD modifier genes (BCL11A, HBS1L-MYB intergenic region, KLF1, and NRF2) using CRISPR-Cas9 base editing and prime editing in erythroid cellular models. Variants were identified through prior targeted resequencing of 500 Nigerian SCD patients from Lagos and Kano with extreme HbF phenotypes. Guide RNAs and base editor constructs were designed and validated in HEK293T cells before application in HUDEP-2 erythroid progenitor cells differentiated to orthochromatic erythroblasts. Allele-specific effects on BCL11A enhancer activity were assessed by ATAC-seq and CUT&RUN chromatin profiling. KLF1 variant effects on downstream target gene expression were quantified by RNA-seq. Three variants demonstrated significant allele-specific regulatory activity, with two novel BCL11A enhancer variants associated with reduced BCL11A expression and elevated HbF. These functionally validated variants represent candidate therapeutic targets for HbF reactivation strategies in Nigerian SCD patients. Keywords: sickle cell disease, CRISPR-Cas9, modifier genes, BCL11A, HbF reactivation