Figure 2.
Schematic representation of the CYBB gene correction protocol. (A) Genomic organization of the first three exons of CYBB with consensus splice donor (GT), splice acceptor (AG) and polypyrimidine tract (10-12Y) shown for each intron. An enhanced view showing the relevant segment of the intron 1/exon 2 boundary is also shown to highlight the location of the CGD2 patient's T > G mutation within the polypyrimidine tract and the location of the two CRISPR-gRNA target sites (gp912 and gp913, underlined), along with their respective PAMs (bold). (B) Induced pluripotent stem cells generated from patient CGD2 were grown under feeder free conditions, electroporated with CRISPR-gRNA constructs and repair template, and selected for transfected cells using puromycin. The residual heterogeneous population of cells was assayed for gene-editing frequency by sequencing and was subsequently passaged onto MEF feeder cells. Single-cell clones were picked, grown on MEFs, and assayed individually for their sequence at the CYBB gene. Successfully modified clones were expanded, converted to feeder-free culture, assayed for pluripotency and intact karyotype, and differentiated into monocytes/macrophages using an embryoid-body-based protocol [15]. Monocytes harvested from the factory supernatant, and subsequently M-CSF-differentiated macrophages, were then assayed for phenotypic correction of the CGD mutation.