SRP125811 PRJNA387385 Characterization of Chromosomal Alterations Using a Zinc-Finger Nuclease Targeting the Beta-globin Gene Locus in Hematopoietic Stem/Progenitor Cells The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease (SCD) mutation in hematopoietic stem and progenitor cells (HSPCs). However, due to the high sequence homology existing between the adjacent human beta- and delta-globin genes, off-target cleavage events have been observed at delta-globin when using some endonucleases targeted to the sickle mutation in beta-globin. The introduction of multiple double stranded breaks by endonucleases has the potential to induce chromosomal alterations such as translocations, deletions, and inversions. Using a novel Droplet Digital PCR (ddPCR) assay and high throughput sequencing we have characterized the frequency of deletions, inversions and translocations between the beta- and delta-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony forming units (CFUs) from SCD bone marrow (BM) donors were treated with nuclease only or with nuclease and a DNA donor template (as an integrase defective lentiviral vector or as a single stranded oligonucleotide); and then analyzed for each of the potential chromosomal rearrangements. It was observed that in both pooled CD34+ cells and CFU samples, the intergenic beta-delta-globin deletion was the most frequent rearrangement event, followed by the inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. Additionally, it was observed that an endonuclease possessing only on-target beta-globin cleavage events did not produce these rearrangements. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted chromosomal alterations. Homo sapiens