Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes

Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47phox subunit of the oxidase complex carry the deletion c.75_76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47phox) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach. [Display omitted] •CRISPR/Cas9-mediated disease modeling of p47-CGD in iPSCs mimics pathogenesis•Targeted repair allows correction of p47-CGD without cleavage in the pseudogenes•Corrected iPSC-derived granulocytes have a restored NADPH oxidase function•Corrected iPSC-derived macrophages phagocytose and kill bacteria Klatt, Schambach and colleagues generated patient-like p47-ΔGT iPSCs for disease modeling of chronic granulomatous disease and subsequent gene correction of NCF1 (encodes p47phox) using CRISPR/Cas9. Their strategy avoided cleavage of the highly homologous NCF1 pseudogenes, which might have a functional role. Corrected granulocytes and macrophages demonstrated restored NADPH oxidase activity, formation of neutrophil extracellular traps, and killing of phagocytosed bacteria.