Figure 4.
Consequences of on-target editing of HBG1 and HBG2. (A) Scale diagram of the HBG1 and HBG2 genes with exons shown as rectangles and on-target Cas9:sgRNA-1 DNA cleavage sites indicated by vertical black arrows. The genes differ by a single nucleotide in exon 3 (open arrows). Primer pair If+Ir amplifies a region surrounding the RNP cleavage site of both genes; NGS of this PCR product identified small on-target indels. Larger deletions presumed to arise from simultaneous dsDNA breaks in HBG2 and HBG1, with the loss of the intervening 4.9 kb (dotted line), were indicated by 2 proxy methods. “ΔHBG2” denotes dropout of HBG2 exon 3 based on the single-nucleotide difference between HBG2 and HBG1, determined by high-throughput sequencing of PCR products generated by a single primer pair (not shown). “Δ366+” denotes a quantitative TaqMan PCR assay that detects deletions ≥366 nt upstream of the Cas9:sgRNA-1 cleavage site in HBG1. (B) Large deletions detected by the ΔHBG2 and Δ366+ assays in CD34+ cells 4 days after editing (Pre-BMT) and 17 weeks after xenotransplantation (Post-BMT). Data reflect studies from 2 different CD34+ cell donors (blue or red dots), with each dot after bone marrow transplantation representing a single mouse. (C) Mutational analysis and %HbF in single BFU-E colonies. Each dot represents a BFU-E colony grouped by number of 4.9-kb deletions (Del) using the ΔHBG2 assay as proxy and the number of indels in the remaining BCL11A binding motifs of HBG2, HBG1, or the modified HBG1 fusion gene formed by deletion repair. Thus, heterozygosity for the 4.9-kb deletion leaves 3 potential remaining BCL11A-binding motifs and associated HBG coding regions, whereas homozygosity leaves only 2 such motifs. The bars represent the percentages of HbF as the means ± SDs. **P < .01, ***P < .001, ****P < .0001 (by unpaired Student t test). (D) Inversions resulting from simultaneous editing of HBG2 and HBG1 target sites were characterized by PCR analysis using the indicated primer pairs. The agarose gel image shows PCR products from the bulk-edited HUDEP-2 (H2) erythroid cell line and CD34+ cells. ns, not significant.

Consequences of on-target editing of HBG1 and HBG2. (A) Scale diagram of the HBG1 and HBG2 genes with exons shown as rectangles and on-target Cas9:sgRNA-1 DNA cleavage sites indicated by vertical black arrows. The genes differ by a single nucleotide in exon 3 (open arrows). Primer pair If+Ir amplifies a region surrounding the RNP cleavage site of both genes; NGS of this PCR product identified small on-target indels. Larger deletions presumed to arise from simultaneous dsDNA breaks in HBG2 and HBG1, with the loss of the intervening 4.9 kb (dotted line), were indicated by 2 proxy methods. “ΔHBG2” denotes dropout of HBG2 exon 3 based on the single-nucleotide difference between HBG2 and HBG1, determined by high-throughput sequencing of PCR products generated by a single primer pair (not shown). “Δ366+” denotes a quantitative TaqMan PCR assay that detects deletions ≥366 nt upstream of the Cas9:sgRNA-1 cleavage site in HBG1. (B) Large deletions detected by the ΔHBG2 and Δ366+ assays in CD34+ cells 4 days after editing (Pre-BMT) and 17 weeks after xenotransplantation (Post-BMT). Data reflect studies from 2 different CD34+ cell donors (blue or red dots), with each dot after bone marrow transplantation representing a single mouse. (C) Mutational analysis and %HbF in single BFU-E colonies. Each dot represents a BFU-E colony grouped by number of 4.9-kb deletions (Del) using the ΔHBG2 assay as proxy and the number of indels in the remaining BCL11A binding motifs of HBG2, HBG1, or the modified HBG1 fusion gene formed by deletion repair. Thus, heterozygosity for the 4.9-kb deletion leaves 3 potential remaining BCL11A-binding motifs and associated HBG coding regions, whereas homozygosity leaves only 2 such motifs. The bars represent the percentages of HbF as the means ± SDs. **P < .01, ***P < .001, ****P < .0001 (by unpaired Student t test). (D) Inversions resulting from simultaneous editing of HBG2 and HBG1 target sites were characterized by PCR analysis using the indicated primer pairs. The agarose gel image shows PCR products from the bulk-edited HUDEP-2 (H2) erythroid cell line and CD34+ cells. ns, not significant.

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