560. Therapeutic Editing of the HBB Locus Using the Endogenous HBD Locus as a Donor Template

Sickle Cell Anemia is a recessive disorder caused by a single point mutation in the human beta globin (HBB) gene. Affecting nearly 1 million people worldwide, this disease is severely lacking in long-term treatment options and is a prime candidate for a gene editing therapeutic approach. Here we report the use of the CRISPR/Cas system to target the human HBB gene in the region of the sickle cell anemia-causing mutation.Utilizing two different Cas9 nickases as well as the wild type nuclease, we are able to introduce blunt double-strand breaks, single strand nicks, and dual-nicks in which the nicks are placed on opposite strands and leave either 3’ or 5’ overhangs of varying lengths. Using either single strand oligonucleotide (ssODN) or plasmid DNA donors, we characterize several different DNA repair outcomes including indel mutations resulting from non-homologous end-joining, homology-dependent repair (HDR) using the donor as a template, and finally HDR using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene. The region of gene conversion includes the sequence most commonly mutated in sickle cell anemia. The frequency of this event depends on the nature of the break. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.