CRISPR-Cas9 Functional Validation of Sickle Cell Disease Modifier Genes Identified in Nigerian Population Genome-Wide Studies: An Original Gene Editing Platform for Therapeutic Target Confirmation

This dissertation establishes an original CRISPR-Cas9 functional validation platform for confirming candidate sickle cell disease (SCD) modifier genes identified in Nigerian population genome-wide association studies, bridging the gap between statistical genomic associations and mechanistic therapeutic target verification. Genome-wide studies have identified multiple SCD modifier loci, but functional validation using disease-relevant human erythroid models has lagged significantly. Using an integrative approach, this dissertation selected seven top modifier candidate genes from large-scale SCD GWAS datasets with relevance to Nigerian patients at Lagos University Teaching Hospital. Patient-derived CD34+ haematopoietic stem and progenitor cells from HbSS donors were obtained and differentiated to the erythroid lineage. CRISPR-Cas9 perturbation of each candidate gene was performed using optimised electroporation delivery to CD34+ cells. Functional readouts included HbF percentage (by HPLC), erythroid differentiation efficiency, enucleation rate, sickling index under deoxygenation, and oxidative stress marker profiles. Four of seven candidates showed significant HbF modulation upon CRISPR perturbation, with KLF1 and ZBTB7A knockouts producing the most reproducible HbF increases (mean 9.4 and 7.1 percentage points respectively). An original Erythroid CRISPR Functional Priority Score is developed, ranking modifier candidates by their therapeutic actionability combining HbF effect size, specificity, and absence of cytotoxicity. This platform provides a translatable framework for accelerating gene therapy target validation for SCD in African genetic contexts. Keywords: CRISPR-Cas9, sickle cell disease modifier genes, gene editing, erythroid model, therapeutic target.