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

Abstract

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 p47 phox subunit of the oxidase complex carry the deletion c.75_76delGT (DGT). 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 p47 phox) but carry the DGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-DGT 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.

Differential Transgene Silencing of Myeloid-Specific Promoters in theAAVS1Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells

“…Two different p47 phox -deficient iPSC lines, p47-ΔGT (generated by CRISPR-Cas9 modification of healthy wild type (WT) iPSC derived from CD34 + cells) and p47-CGD (generated via reprogramming of peripheral blood from a CGD patient) were used in this study 27 . For genetic correction, 5 x 10 4 p47 phox -deficient iPSC were seeded in a 24-well plate on Geltrex (Thermo Fisher) in the presence of 10 µM ROCK inhibitor Y-27632.…”

“…We measured an on-target activity of 43-47% on average for the AAVS1-specific sgRNA at different multiplicities of infection ( Figure S1B-S1D). After validation of the sgRNA, two different p47 phox -deficient iPSC lines (p47-ΔGT generated by CRISPR-Cas9 and p47-CGD generated via reprogramming 27 The screening results of all analyzed iPSC clones were summarized in Figure 1D. In most cases, we achieved more monoallelic corrected clones than biallelic corrected clones most likely due to the low frequency of HDR.…”

Mechanistic and Translational Advances Using iPSC-Derived Blood Cells

“…For example, patients with chronic granulomatous disease (CGD) have recurrent bacterial and fungal infections caused by defective phagocytic functions. By correcting one of the NADPH oxidase mutations responsible for 90% of the cases of CGD in a patient-derived iPSC line, NADPH oxidase function and cellular bacterial killing activity were restored in vitro [47].…”

Mechanistic and Translational Advances Using iPSC-Derived Blood Cells

“…For example, patients with chronic granulomatous disease (CGD) have recurrent bacterial and fungal infections caused by defective phagocytic functions. By correcting one of the NADPH oxidase mutations responsible for 90% of the cases of CGD in a patient-derived iPSC line, NADPH oxidase function and cellular bacterial killing activity were restored in vitro [50]. iPSC models can also provide cellular screening platforms for novel immunodeficiency syndrome treatments.…”