Off-target genomic DNA loci with partial homology to the BCL11A enhancer-directed sgRNA

Off-target genomic DNA loci with partial homology to the BCL11A enhancer-directed sgRNA refer to unintended sites in the human genome where the CRISPR-Cas9 complex may bind and induce double-strand breaks during the manufacturing of exagamglogene autotemcel (Casgevy). The intended target is the erythroid-specific enhancer of the BCL11A gene, which, when disrupted, prevents the repression of fetal hemoglobin (HbF) production, providing a therapeutic benefit for patients with sickle cell disease and transfusion-dependent beta-thalassemia (Frangoul et al., 2021, NEJM). Because the 20-nucleotide guide RNA (sgRNA) sequence may share high similarity with other genomic regions, there is a risk of off-target editing at these homologous sites. Such unintended modifications could lead to genotoxicity, including the disruption of tumor suppressor genes or the activation of oncogenes, potentially causing secondary malignancies (FDA Briefing Document, 2023). Extensive preclinical assessments using methods like GUIDE-seq and CIRCLE-seq are employed to identify these loci and confirm that no frequent or deleterious editing occurs at these sites in patient-derived cells. Despite these risks, clinical data for Casgevy have shown no evidence of harmful off-target editing in treated patients to date. These loci represent a critical safety parameter rather than a therapeutic target, requiring rigorous monitoring throughout the lifecycle of gene-editing products.

Other names

CRISPR off-target sitesNon-specific Cas9 cleavage sitesBCL11A-enhancer off-targetsOff-target genomic loci

Unintended Cas9-mediated double-strand DNA cleavage at sites with sequence similarity to the target sgRNA, followed by non-homologous end joining (NHEJ) repair (FDA Briefing Document, 2023).