# Complex HBB gene editing outcomes revealed by a fluorescent reporter cell model

## TL;DR

A fluorescent reporter cell model helps study gene editing outcomes at the β-globin gene, revealing complex and unintended changes that affect safety and efficacy.

## Contribution

A novel fluorescent reporter cell model enables allele-specific monitoring of HBB gene editing outcomes with high resolution.

## Key findings

- The model discriminates in-frame alleles, frameshift indels, large deletions, and loss of allele events.
- HDR-enhancing agents increase LOA outcomes, which are undetected by traditional bulk assays.
- The model links editing genotypes to erythroid phenotypes, aiding safety evaluation of gene therapies.

## 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.

In this work, a fluorescent cell model was established to study gene editing outcomes at the β-globin locus near the sickle mutation. This model enables high-resolution analysis of editing efficacy and safety, uncovering complex and unintended genotypes that highlight both the therapeutic benefits and potential risks of gene editing therapies.