Fig. 4

ZFN-mediated gene editing leads to enriched biallelic edited cells and allele-additive increases in HbF. (a) Total editing levels in erythroid colonies derived from single-cell CD34 + HSPC from healthy and SCD donors. (b) Frequency of the most common indel patterns in single-cell derived colonies. The unedited (wild type) sequence is shown in bold, with a rectangle highlighting the core GATAA site and lines representing ZFN contacts. (c and d) Frequency of wild-type, monoallelic, and biallelic-edited genotypes derived from single-cell clonal analysis of plerixafor-mobilized HSC from (c) 5 healthy and (d) 3 SCD donors. The distribution of genotype was significantly skewed towards biallelic clones. The expected distribution of clones was calculated using an equation analogous to the Hardy–Weinberg equilibrium (Online Methods). P-values from χ² tests comparing the observed and expected number of wild-type, monoallelic, and biallelic clones for each sample are reported on the figure. Significant P-values reflect a skew of the distributions towards wild-type and biallelic clones. (e and f) Association of γ-globin levels measured by reversed-phase ultra-performance liquid chromatography with genotypes in (e) healthy donors (linear regression P = 8 × 10⁻⁸⁴; beta = 14.9; standard error = 0.7) and (f) SCD donors (linear regression P = 7 × 10⁻⁸; beta = 14.1; standard error = 2.5). Mean differences between biallelic-edited and unedited clones were 32% (HD 1), 38% (HD 2), 28% (HD 3), 30% (HD 4), 21% (HD 5), 28% (SCD 1), 32% (SCD 2), and 20% (SCD 3). (g) Effect of indels on gamma-globin expression in healthy donor cells, stratified by whether they disrupt the core GATAA site (GATAA-, left) or not (GATAA + , right). For (e, f, and g), beta and P-values were calculated by fitting linear regression models of HbF (gamma-globin) against genotype in all clones, adjusted for the different donors (HbF ~ genotype + donor). The beta coefficient for the genotype effect is reported. HbF, fetal hemoglobin; HD, healthy donor; HSC, hematopoietic stem cell; SCD, sickle cell disease; WT, wild type.