CRISPR-directed therapeutic correction at the NCF1 locus is challenged by frequent incidence of chromosomal deletions

Abstract Resurrection of non-processed pseudogenes may increase the efficacy of therapeutic gene editing, upon simultaneous targeting of a mutated gene and its highly homologous pseudogenes. To investigate the potency of this approach for clinical gene therapy of human diseases, we corrected a pseudogene-associated disorder, the immunodeficiency p47phox-deficient chronic granulomatous disease (p47phox CGD), using clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR-Cas9) to target mutated neutrophil cytosolic factor 1 (NCF1). Being separated by less than two million base pairs, NCF1 and two pseudogenes are closely co-localized on chromosome 7. In healthy people, a two-nucleotide GT deletion (ΔGT) is present in the NCF1B and NCF1C pseudogenes only. In the majority of patients with p47phox CGD, the NCF1 gene is inactivated due to a ΔGT transfer from one of the two non-processed pseudogenes. Here we demonstrate that concurrent targeting and correction of mutated NCF1 and its pseudogenes results in therapeutic CGD phenotype correction, but also causes potentially harmful chromosomal deletions between the targeted loci in a p47phox-deficient CGD cell line model. Therefore, development of genome editing-based treatment of pseudogene-related disorders mandates thorough safety examination as well as technological advances, limiting concurrent induction of multiple double-strand breaks on a single chromosome.