Figure 1.
Generation of Rh null and D-- iPSCs. (A) Schematic for CRISPR-Cas9–mediated disruption of RHCE in RhD-negative iPSC to generate Rh null iPSCs. (B) PCR products amplified with primers targeting intron 1 and intron 2 identifies iPSC clones with Cas9-mediated large deletion of ∼1 kb. (C) Schematic for ZFN-mediated insertion of RHD cDNA into the safe harbor AAVS1 locus of Rh null iPSCs to generate D-- iPSCs. (D) PCR products amplified using primers indicated in panel C to identify clones with successful integration of the RHD cDNA cassette. (E) Cell surface Rh protein visualized by flow cytometry using a pan-Rh antibody in control donor RBCs (red), and untargeted parent, Rh null, and D-- iRBCs (blue). CAG, CAG promoter; F, forward primer; gRNA, guide RNA; HA, homology arm; KO, knockout; Neo, neomycin resistance cassette; R, reverse primer.

Generation of Rh null and D-- iPSCs. (A) Schematic for CRISPR-Cas9–mediated disruption of RHCE in RhD-negative iPSC to generate Rh null iPSCs. (B) PCR products amplified with primers targeting intron 1 and intron 2 identifies iPSC clones with Cas9-mediated large deletion of ∼1 kb. (C) Schematic for ZFN-mediated insertion of RHD cDNA into the safe harbor AAVS1 locus of Rh null iPSCs to generate D-- iPSCs. (D) PCR products amplified using primers indicated in panel C to identify clones with successful integration of the RHD cDNA cassette. (E) Cell surface Rh protein visualized by flow cytometry using a pan-Rh antibody in control donor RBCs (red), and untargeted parent, Rh null, and D-- iRBCs (blue). CAG, CAG promoter; F, forward primer; gRNA, guide RNA; HA, homology arm; KO, knockout; Neo, neomycin resistance cassette; R, reverse primer.

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