Hflt3l

Lentivirus was produced and concentrated as previously described^{4 (link)}. A high-titre virus of IK6-GFP and Ctrl-GFP was prepared by ultracentrifugation. Lin⁻CD34⁺CD38⁻ HSPCs were infected with lentivirus at a multiplicity of infection of 100 for 24 h in serum-free Stem/Span (Stem Cell Technologies, Canada) medium supplemented with growth factors (hSCF 50 ng/ml, hFlt3-L 50 ng/ml, hIL-6 10 ng/ml and hTPO 10 ng/ml; Peprotech, USA).

Mobilized peripheral-blood-derived human CD34⁺ HSPCs were cultured and electroporated as previously described^{55 (link)}. In brief, cryopreserved cells were thawed and cultured in StemSpan SFEMII medium (StemCell, 09655) supplemented with 1% penicillin–streptomycin (10,000 U ml⁻¹), 1% l-glutamine (200 mM), 125 ng ml⁻¹ hSCF (Peprotech, 300-07), 125 ng ml⁻¹ hFLT3L (Peprotech, 300-19), 62.5 ng ml⁻¹ hTPO (Peprotech, 300-18), 0.75 μM StemRegenin-1 (SR1, StemCell, 72344) and 35 nM UM171 (Sellekchem, S7608) unless stated otherwise. Then, 48 h after thawing (unless stated otherwise), cells were collected and electroporated using the Lonza 4D-Nucleofector system by resuspending the cells in P3 solution (Lonza, V4XP-3024) supplemented with 100–250 nM base editor mRNA, 15–20 μM sgRNA (IDT) and 1.2 U μl⁻¹ RNase inhibitor (Promega RNAsin Plus, N2611) or 1.5–3% (v/v) glycerol. After electroporation, cells were then counted and transplanted after 24 h for in vivo experiments, or cultured for an additional 5–7 days in the same medium described above at 0.5 M ml⁻¹ for in vitro experiments.

Day-seven cultures were processed to FACS-sort CD34⁺CD115⁺ DCPs (or mock-sorted) using a BD FACSAria II SORP apparatus. Sorted DCPs were cultured in StemSpam SFEMII medium (STEMCELL Technologies) supplemented with 50 units per ml of penicillin (Gibco), 50 μg ml^–1 streptomycin (Gibco), 20 ng ml^–1 hGM-CSF, 100 ng ml^–1 hFLT3L, 20 ng ml^–1 hSCF (all from PeproTech) and 1,000 IU ml^–1 hIFNa2b (InvivoGen) for 7 days. Day-14 cultures contained differentiated cell populations identified as indicated in the Reporting Summary.

Lin⁻ mouse BM cells were isolated by flushing femurs, tibias, and iliac crests of 6- to 8-week-old CD45.2 C57BL/6 or CD45.2 Berkeley SCD mice (BERK-SCD, JAX stock #003342) followed by lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin− cells were pre-stimulated at 1 × 10⁶ cells/mL in Stem Cell Growth Medium (CellGenix) supplemented with mouse SCF (100 ng/mL), hTPO (100 ng/mL), mouse IL-3 (mIL-3) (20 ng/mL), and hFlt3-L (100 ng/mL), all from Peprotech. Following a 36- 40-h pre-stimulation, cells were transduced at a density of 1 × 10⁶ cells/mL in the presence of LentiBOOST enhancer, and transduced cells (without sorting) were transplanted by retro-orbital injection into lethally irradiated (7 + 4 Gy, split dose) CD45.1 recipients (B6.SJL-Ptprca Pepcb/BoyJ, Jax Strain #002014) 24 h after transduction. PB samples were collected at weeks 4, 8, 12, and 16 to measure engraftment by flow cytometry (CD45.2/CD45.1), determine RBC indices, and quantitate sickled cells. At week 16, mice were euthanized, and BM cells were used to measure engraftment by flow cytometry (CD45.2/CD45.1), VCN, and mRNA expression, spleens were collected to weigh. All animal experiments were approved by the Boston Children's Hospital's Institutional Animal Care and Use Committee.

Human leukemia cell lines (K562, isogenic K562 with a CRISPR-HDR corrected TP53 allele^{38 (link)}), kindly provided by Benjamin Ebert (Dana Farber Cancer Institute) were maintained in RPMI media (Gibco, 11875-119) supplemented with 10% FBS, penicillin and streptomycin (Gibco). Primary cultures of bone marrow fibroblasts were established and maintained in Chang D media (Irvine Scientific, T105). Mobilized peripheral blood CD34+ and bone marrow mononuclear cells were maintained in GMP SCGM Serum-free Media (Cellgenix, 20802–0500) supplemented with 100 ng/mL hSCF (Peprotech, 300-07), hTPO (Peprotech, 300-18), hFLT3-L (Peprotech, 300-19), and for bone marrow mononuclear cell culture, 20 ng/mL of IL-3 (R&D systems 203-IL-010/CF) was added. All cells lines were cultured at 37 °C under 5% CO₂ and routinely screened for mycoplasma^{39 (link)}.