Fig 2: Townes mouse HSPCs edited at HBG1 engraft efficiently but fail to recapitulate human hereditary persistence of fetal hemoglobin (HPFH). (A) Experimental strategy. Lin- HSPCs from Townes mice were transfected with Cas9+gRNA RNP targeting the human HBG1 promoter or Cas9 only, then transplanted into irradiated C57Bl/6 hosts. (B) Kaplan–Meier curves showing survival of recipients after transplantation of edited or control HSPCs. (C) Engraftment of donor HSPCs, as determined by the fraction of circulating CD45.2+ mononuclear cells in recipient mice. (D) Percentage of HBG1 promoter indels in blood mononuclear cells after transplantation. (E) HbF immunostaining cells (F-cells) determined by flow cytometry. (F) Percentage of HbF protein in red blood cell lysates, determined by ion-exchange high-performance liquid chromatography. Graphs in C-F show data as mean±s.e.m. of three independent transplantation experiments, n=13 HBG-edited and n=9 Cas9 control mice. **P<0.01, ***P<0.001, ****P<0.0001, by unpaired, two-tailed Student's t-test. (G) %RBC HbF protein versus %peripheral blood mononuclear cell indels in recipient mice transplanted with HBG1-edited Townes HSPCs (teal circles) or HBG-edited human CD34+ cells from SCD patients (orange circles). Orange data points are adapted from Métais et al. (2019) with permission.

Fig 3: Editing the ?-globin transgenes eliminates bone marrow engraftment of Berkeley mouse HSCs. (A) Experimental strategy. Lin- HSPCs from Berkeley mice (CD45.2) were transfected with RNP targeting HBG1/HBG2 or Rosa26, transplanted into irradiated C57Bl/6 (CD45.1) hosts and analyzed serially. (B) Kaplan–Meier curves showing survival of irradiated recipients after transplantation of edited HSPCs. (C) Engraftment of HBG1/HBG2-edited or Rosa26-edited Berkeley mouse donor HSPCs over time, as determined by the fraction of circulating CD45.2+ mononuclear cells in recipient mice. (D) Frequency of on-target indels at the HBG or Rosa26 loci in blood mononuclear cells at the indicated times following transplantation. Graphs show mean±s.e.m. of three biological replicate experiments with n=10 HBG-edited, n=6 Rosa26-edited and n=10 control (Cas9 only-transfected) mice. ns, not significant; **P<0.01, ***P<0.001, ****P<0.0001, by unpaired, two-tailed Student's t-test, survival calculations Log-rank (Mantel–Cox) test.

Fig 4: Schematic representation of known and identified point mutations in HBG promoter region that elevates fetal hemoglobin (HbF): The proximal promoter region of HBG2 and HBG1 is represented from transcription start site (TSS) till –205 bases.Novel clusters identified from this study are highlighted in Sage (five clusters), and known clusters are highlighted in Melon (two clusters). Among these clusters, known base conversions are represented in black and identified hereditary persistence of fetal hemoglobin (HPFH)-like mutations are represented in red text. The novel base conversions from our study are represented in bold font. Transcriptional activators (lavender) and repressors (orange) that bind to the known clusters are also depicted in the figure.

Fig 5: Editing the human ?-globin transgenes impairs the viability of Berkeley but not Townes mouse hematopoietic stem and progenitor cells (HSPCs). (A) Experimental strategy. Three million lineage-negative (Lin-) bone marrow cells from Berkeley (B-F) or Townes (G-K) mice were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9+guide RNA (gRNA) targeting the -118 to -114 BCL11A binding site (TGACCA) in the HBG1/2 promoter, or a control gRNA targeting Rosa26 intron 1, and grown in culture for 5 days with mouse stem cell factor (mSCF), human FLT3 (hFLT3) ligand, mouse interleukin 3 (mIL-3) and mouse interleukin 11 (mIL-11). (B,G) On-target insertion-deletion (indel) mutations in genomic DNA, determined by next-generation sequencing (NGS) of PCR products generated across the targeted region after editing. Controls include electroporation with Cas9 alone or non-electroporated cells (Ctrl). (C,H) Live cell numbers measured by Trypan Blue exclusion. (D,I) Percentages of apoptotic (Annexin V+) cells measured by flow cytometry. (E,J) Percentages of S-phase cells measured by incorporation of bromodeoxyuridine (BrdU). (F,K) Fold change in the TP53-induced Cdkn1 (p21) mRNA measured by real-time quantitative PCR (RT-qPCR), normalized to mouse Gapdh mRNA. All graphs show data as mean±s.e.m. from two biological replicate experiments with cells from two mice for each replicate (n=4 mice total). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by two-way ANOVA for HBG- versus Rosa26-edited cells.