Extended Data Fig. 1: ZNF410 targeted HUDEP-2 cells display normal cell growth and elevated γ-globin expression.

HUDEP-2 cells were edited at ZNF410 or AAVS1 (control) using sgRNA:Cas9 RNP electroporation. a, Efficient editing was achieved with all five ZNF410 targeting gRNAs (n = 1 for each gRNA). Cells were cultured in EDM2 for 8 days and (b) cell count and (c) viability data were recorded on alternate days for five individual gRNAs targeting ZNF410 (sg 1-5, n = 3 for sg 1, n = 1 for sg 2-5) in comparison to mock (n = 3) and AAVS1 (control, n = 3) targeted cells. Data are presented as mean values and error bars are standard deviation. d, Robust γ-globin induction was obtained with all five ZNF410 targeting gRNAs (n = 1 for each gRNA) in comparison to mock (n = 1) and AAVS1 (control, n = 1) targeted cells. e, HUDEP-2/Cas9 cells nontransduced (mock, n = 3) or transduced with ZNF410 targeting sgRNA (n = 4) assayed on day 9 of erythroid differentiation with RT-qPCR for HBE1 (p = 0.1426, ns), HBB (p = 0.0353) and HBA (p = 0.0122) expression. All values are relative to Catalase expression (endogenous control) and expressed as fold change relative to mock for each biological replicate. Data are presented as mean values and error bars are standard deviation. Statistically significant differences were determined using paired Student’s t-test comparing ZNF410 targeted cells to mock. f, Three HUDEP-2 ZNF410 biallelic KO clones were generated using paired genomic cleavages that delete the entire coding sequence. Clones were generated in two successive steps. In the first step a HUDEP-2 clone heterozygous for ZNF410 deletion was isolated. The ZNF410 null allele in this clone is designated Null cl. allele 1 and its sequence is shown on the left. In the second step this heterozygous ZNF410 deletion clone was retargeted and three biallelic ZNF410 null clones were isolated with the sequences of the second ZNF410 null allele in each clone shown on the right of the figure.