| Abstract |
CRISPR-Cas9 gene editing offers the potential to transform the treatment of sickle cell disease by correcting the sickle mutation in β-globin gene (HBB). However, in addition to alleles with homology-directed repair (HDR), Cas9 editing at HBB generates a diverse spectrum of outcomes, including small insertions and deletions (indels), large deletions (LDs), and loss of allele (LOA) events, that can compromise genomic integrity and raise significant safety concerns. While new pharmacological modulators have been developed to increase the HDR rates, they may also elevate the risk of large gene modifications. To better understand the complex HBB gene editing outcomes, we engineered a live-cell, dual-fluorescent reporter cell model enabling allele-specific monitoring of HBB expression via GFP and blue fluorescent protein (BFP) tagging. Based on fluorescence intensities, this model can discriminate in-frame alleles, frameshift indels, LDs, and LOA, supporting high-throughput genotype-phenotype mapping. By applying HDR-enhancing agents, we further show that the reporter sensitively captures shifts in LOA outcomes that are missed by other bulk assays. This cell model provides a valuable tool for dissecting gene editing outcomes due to different DNA repair pathways and quantitatively linking editing genotypes to erythroid phenotypes and thus can be used to evaluate the safety of CRISPR/Cas9-based therapies.
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