Epitope editing enables targeted immunotherapy of acute myeloid leukaemia – Nature

Sleeping Beauty transgene overexpression

Bidirectional Sleeping Beauty plasmids encoding the codon-optimized cDNA of the gene of interest (FLT3, KIT, CD123) or their mutated variants were cloned downstream of an EEF1A1 promoter. The same construct included a reporter cassette comprising a fluorescent protein (mCherry or mTagBFP2 for FLT3 and KIT, respectively) and puromycin-acetyl-transferase (PAC) separated by a P2A peptide downstream a RPBSA chimeric promoter. CD123 constructs included human CSF2RB (cytokine receptor common subunit β) cDNA in place of the reporter cassette. dsDNA inserts used for cloning were synthesized by Genewiz or IDT (IDT gBlocks). Human (K562, HEK293T) or mouse (NIH-3T3 or BaF3) cell lines were electroporated using the Lonza 4D-Nucleofector system according to the manufacturer instructions with 500 ng pSB100x transposase plasmid (Addgene, 34879) and 100 ng of the transfer plasmid, unless stated otherwise. Cells underwent puromycin selection (1–5 μg ml⁻¹) starting 3 days after electroporation and were analysed by flow cytometry 7–14 days after the start of the selection.

FLT3 combinatorial library

We designed a combinatorial plasmid library in which the human or the mouse codons were randomly selected at each of 16 positions (Asn354, Ser356, Asp358, Gln363, Glu366, Gln378, Thr384, Arg387, Lys388, Lys395, Asp398, Asn399, Asn408, His411, Gln412, His419) within FLT3 ECD4 (GenScript). The library was cloned to allow a total theoretical complexity of 65,536 (covered at least 10 times). Single-colony sequencing showed a rate of n = 4 mutations in 68.75% of the colonies, and n = 5 mutations in 18.75% of colonies. NGS sequencing of K562 cells transduced with the library, among 9,166 filtered reads, detected 4,375 unique amino acid sequences with a frequency of >0.0001. K562 cells were electroporated with 5–15 ng of library plasmid as described above and selected with 1 μg ml⁻¹ puromycin. Cells were then stained with anti-FLT3 control antibodies (BV10A4, BioLegend, 313314) and FLT3 therapeutic antibodies (4G8, BD, 563908). The single-positive (BV10A4⁺4G8⁻) and double-positive (BV10A4⁺4G8⁺) populations were sorted using the BD FACS Aria sorter and expanded in culture. After gDNA extraction, the library region was PCR-amplified with primers bearing Illumina partial adapters for NGS sequencing (250 bp paired-end, Genewiz, Azenta Life Sciences). Amplification of the integrated transgene was ensured using exon-spanning primers specific for the codon-optimized sequence. Read alignment and trimming was performed by Genewiz (Azenta), after which the sequences were translated to protein sequence and indels were discarded. After aggregation of reads encoding the same protein variant, the sequences were filtered by minimum abundance >0.001 within single-positive and double-positive samples, and the relative representations of each amino acid at each position within the sorted fractions were plotted as sequence logos. Analysis was performed using R Studio 2022.07.0 (R v.4.1.2).

KIT degenerated codon library

We cloned a bidirectional Sleeping Beauty plasmid encoding a codon-optimized human KIT cDNA bearing unique restriction sites flanking each ECD and a reporter cassette composed of mTagBFP2-P2A-puromycin N-acetyl-transferase. To introduce random amino-acids, we generated a library insert by PCR amplification of 350-bp-long pooled ssDNA oligos (IDT oPools), each encoding for the KIT ECD4 bearing degenerated bases (NNN) at each amino-acid position, flanked by homology arms for the backbone plasmid. The gel-purified insert was then cloned into the Sleeping Beauty transfer by HiFi cloning (NEB, E2621L) and plated onto ten 15 cm agar dishes to estimate the library complexity. Electroporation of HEK293T cells with low plasmid doses (50–250 ng) enabled us to select cells transduced with (~5–10%) efficiency to obtain approximately 1 variant integration per cell. Sorting of cells positive for KIT control antibodies (104D2 or Ab55 clones) and negative for the therapeutic Fab-79D clone, and cells positive for both antibodies was performed using the BD FACS Melody sorter. gDNA of the sorted fractions was extracted and the library region was amplified by PCR using primers bearing Illumina partial adapters for NGS sequencing (Genewiz, Azenta Life Sciences). Read alignment and trimming was performed by automated analysis (Genewiz), after which the read sequences were translated into protein and indels were discarded. After aggregation of reads encoding the same protein variant, the relative abundance of each protein sequence within each sample and the enrichment in single-positive versus double-positive samples was calculated as the log[fold change]. Sequences were filtered by log[fold change] > 0.5, and the minimum-abundance fraction within single-positive samples >0.0015 of each amino acid substitution was plotted as a sequence logo. Analysis was performed using R Studio 2022.07.0 (R v.4.1.2).

Gene overexpression through promoter integration

Reporter K562 cells overexpressing FLT3, KIT and CD123 from the endogenous genomic locus were generated using CRISPR–Cas nuclease and HDR integration of a full EEF1A1 promoter upstream of the gene reading frame. A dsDNA donor template encoding the EEF1A1 promoter flanked by 50 bp homology arms (HA) was prepared by PCR amplification (Promega GoTaq G2, M7845) of plasmid DNA templates using primers with 5′-tails encoding the sHA. K562 cells were electroporated with 100 pmol SpCas9 gRNAs (for FLT3 and KIT) or AsCas12a sgRNAs (CD123) complexed with 12 μg of corresponding Cas protein (IDT; SpCas9 V2, 1081058; or AsCas12a, 10001272) to form RNPs, along with 0.5 to 10 μg donor template using the Lonza 4D-Nucleofector system. Edited cells were evaluated by flow cytometry 3–5 days after electroporation and FACS-sorted (BD FACS Melody) to select cells expressing the gene of interest. Single-cell cloning was performed by limiting dilution for FLT3 and CD123 to isolate clones with the highest expression for subsequent editing experiments.

Fluorescent ligand-binding assay

Human FLT3L (Peprotech, 300-19), hSCF (Peprotech, 300-07) or hIL-3 (200-03) were resuspended at 1 mg ml⁻¹ and conjugated with AF488 or AF647 (Invitrogen AF-488 or AF-647 Antibody Labeling Kit, A20181 and A20186) for 1 h at room temperature. The reaction was quenched with 1% 1 M Tris-HCl pH 8. Cell lines transduced with the respective cytokine receptor by Sleeping Beauty transposase were incubated in the presence of AF488-conjugated FLT3L, SCF or IL-3 for 15 min at room temperature, washed with PBS + 2% FBS and analysed by flow cytometry.

Modelling of antibody–ligand affinity curve

The data obtained from the antibody and ligand affinity assays at different concentrations were analysed using the drc R package (v.3.0). The LL.4 model was used to fit the affinity curves and estimate the parameters of interest, including the maximum response (ED₅₀), the slope of the curve at the inflection point (Hill slope), the lower asymptote (lower limit) and the upper asymptote (upper limit). The difference among affinity curves was tested using a likelihood ratio test in which the parameters were fixed (model0) or antibody-dependent (model1).

Western blotting

K562 (for FLT3) or NIH-3T3 (for KIT) cells overexpressing the receptor of interest by Sleeping Beauty transposase-mediated integration were starved overnight in IMDM without FBS. The next day, cells were collected, washed, counted and stimulated with the respective ligand at different concentrations (0, 0.1, 1, 10, 100 ng ml⁻¹) for 15 min at 37 °C. Cells were then collected, washed twice in ice-cold PBS and lysed in cell extraction buffer (Thermo Fisher Scientific, FNN0011) + protease inhibitor (1 mM PMSF) for 30 min on ice. Protein-containing supernatants were then collected, and the concentration was measured using the BCA assay. A total of 35 µg of protein lysate was then mixed with Laemmli loading buffer (Bio-Rad, 161-0747) supplemented with 0.1× volumes of 2-mercaptoethanol and incubated for 5 min at 95 °C. Denatured protein samples and a prestained protein ladder (Bio-Rad Precision Plus Kaleidoscope, 1610375) were loaded onto 4–12% Tris-Glycine gel (Invitrogen Novex WedgeWell, XP04125BOX), run at 150 V for 90 min and transferred onto PDVF membranes by blotting at 100 mA for 60 min. Washed membranes were then blocked with TBST + 5% BSA, washed three times with TBST and cut at the 75 kDa mark to separate FLT3 and KIT (110–140 kDa) and actin (42 kDa) bands and incubated with primary antibodies at 1:1,000 concentration (pKIT Tyr568/570, Cell Signalling, 48347S; pFLT3 Y589-591, Cell Signalling, 3464S) for 1 h at room temperature. The membrane was then washed three times and incubated with the appropriate secondary antibodies (anti-rabbit IgG, HRP-linked Antibody, Thermo Fisher Scientific, 31460), washed and developed with Thermo Scientific SuperSignal West Femto substrate (34095). The membranes were imaged using the ImageQuant LAS 4000 system. To reprobe the same samples with anti-FLT3 or anti-KIT antibodies, the membranes were incubated for 30 min with Thermo Scientific Restore Stripping Buffer (Thermo Fisher Scientific, 21059), blocked, washed and incubated with primary antibodies (anti-FLT3, Origene, TA808157; anti-KIT, Invitrogenc MA5-15894). The membrane was then washed and incubated with secondary antibodies (anti-mouse IgG, HRP-linked antibodies, Thermo Fisher Scientific, 62-6520; or anti-rat IgG, HRP-linked antibodies, Thermo Fisher Scientific, 31470) and reimaged as described above.

Base editing of cell lines

Human K562 cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin–streptomycin (10,000 U ml⁻¹), 2% l-glutamine (200 mM). For base-editing experiments, K562 cells were collected and resuspended in electroporation solution⁵¹ supplemented with 500 ng base editor plasmid and 150–360 pmol of sgRNA (IDT) in 20 μl. Cells were electroporated using the Lonza 4D-Nucleofector system and cultured for 72 h before evaluation by flow cytometry or gDNA collection.

Base-editing evaluation by Sanger sequencing

Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69506) or QuickExtract solution (Lucigen, QE0905T). PCR amplification of the 450–600 bp region surrounding the target residue was performed using GoTaq G2 polymerase (Promega, M7848) and purified using the SV-Wizard PCR Clean-Up kit (Promega, A9282) or sparQ PureMag magnetic beads (MagBio, 95196). Sanger sequencing was performed by Genewiz (Azenta Life Sciences) and base-editing outcomes were estimated using the editR v.1.08 R package⁵².

CAR T cell generation

Peripheral blood mononuclear cells were isolated by ficoll density-gradient centrifugation from healthy blood donor samples (non-HLA-matched with CD34⁺ HSPCs). Total PBMCs or purified T cells (Pan T Cell Isolation Kit, Miltenyi, 130-096-535) were cultured in IMDM supplemented with 10% FBS, 1% penicillin–streptomycin (10,000  U ml⁻¹), 2% l-glutamine (200 mM), 5 ng ml⁻¹ IL-7 (Peprotech, 200-07) and 5 ng ml⁻¹ IL-15 (Peprotech, 200-15) in the presence of CD3-CD28 Dynabeads (Gibco, 11141D) at a 3:1 bead:cell ratio. On day 2–3 of culture, cells were counted and transduced with a third-generation bidirectional lentiviral vector encoding the CAR and an optimized version³⁶ of the truncated EGFR⁵³ marker at a multiplicity of infection (MOI) of 5 to 10. Dynabeads were removed at day 7 and the culture was continued for a total of 14 days before either vital freezing or in vitro/in vivo experiments. The CAR T cell phenotype and transduction efficiency was evaluated by flow cytometry.

CAR T cell co-culture assays

Target cells (K562 cells, AML PDXs or CD34⁺ HSPCs) were plated in flat-bottom 96-well plates, 10,000 cells per well in IMDM supplemented with 10% FBS. Freshly cultured or thawed CAR T or unstransduced T cells were marked with CellTrace Yellow or Violet reagent (Invitrogen, C34567 and C34557) for 15 min at 37 °C and then washed with complete medium. T cells then were added to the target cells at several effector:target ratios (10, 5, 2.5, 1.25, 0.625, 0) in a total volume of 200 μl in triplicate or quadruplicate. The plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for 6 h (early timepoint) or 48 h (late timepoint). For analysis, cells were transferred to V-bottom plates supplemented with 10–15 μl CountBeads (BioLegend, 424902), centrifuged and resuspended in staining mix containing human Fc-blocking reagent (Miltenyi, 130-059-901), and CD3 (or CD4/CD8 cocktail), CD69 and CD107a antibodies unless stated otherwise. In K562 cell experiments, FLT3 BV10A4 and/or CD123 9F5 or KIT 104D2 antibodies were included in the staining mix to evaluate the residual target expression at the end of co-culture. In CD34⁺ HSPC experiments, cells were stained for CD34, CD90 and CD45RA. After incubation at room temperature for 15 min, the cells were washed and resuspended in annexin V binding buffer (BioLegend, 422201) containing annexin V FITC or PacificBlue (BioLegend, 640945 or 640918) and 7-AAD. The plates were analysed using the BD Fortessa high-throughput system (HTS) and acquired using BD FACS Diva (v.6). Absolute counts of live (Annexin V⁻7-AAD⁻) target cells were calculated using CountBeads and normalized to the E:T = 0 (no T cells) condition (absolute count of target cells divided by the median of the target cells in E:T = 0 replicates). The MFI of target genes (FLT3, CD123, KIT) was normalized in the same manner to account for slight differences in transgene expression.

Base editor mRNA in vitro transcription

Base editor mRNA was prepared by T7 run-off in vitro transcription using a custom plasmid template encoding for a T7 promoter, a minimal 5′-UTR, the base editor reading frame, 2× HBB 3′-UTR and a 110–120 bp poly(A) sequence⁵⁴. The plasmid template was linearized by BbsI-HF restriction digestion (NEB, R3539) and purified by phenol–chloroform extraction (Sigma-Aldrich, P2069). mRNA IVT was performed using the NEB HiScribe T7 kit (E2040S) and co-transcriptionally capped with ARCA (3′-O-Me-m7G(5′)ppp(5′)G RNA cap analogue, NEB, S1411L) or CleanCap AG (Trilink, N-7113). Partial (75–85%) or total UTP substitution with N1-methyl-pseudo-UTP (Trilink, N-1103) or 5-methoxy-UTP (Trilink, N-1093) was performed as indicated. Dephosphorylation with QuickCIP (NEB, M0525L) or DNase treatment (NEB, M0303L) was added after IVT reaction (30 min at 37 °C). IVT mRNA was purified using the NEB Monarch RNA Clean up kit (T2050L) or sparQ PureMag magnetic beads (MagBio, 95196) and resuspended in RNase-free water. mRNA was quantified using the Nanodrop-8000 spectrophotometer and quality control was performed using the Agilent Fragment Analyzer with RNA Kit-15NT (Agilent, DNF-471).

CD34⁺ base editing

Mobilized peripheral-blood-derived human CD34⁺ HSPCs were cultured and electroporated as previously described⁵⁵. 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.

Transduction and expansion of human AML xenografts

Human patient-derived AML xenografts (PDX) 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), 50 ng ml⁻¹ hSCF (Peprotech, 300-07), 50 ng ml⁻¹ hFLT3L (Peprotech, 300-19), 25 ng ml⁻¹ hTPO (Peprotech, 300-18), 10 ng ml⁻¹ IL-3, 10 ng ml⁻¹ G-CSF, 0.75 μM StemRegenin-1 (SR1, StemCell, 72344), 35 nM UM171 (Sellekchem, S7608) and 10 μM PGE2. Cells were transduced with a third-generation LV vector expressing mNeonGreen under a hPGK promoter (titre, ~2×10¹⁰ TU ml⁻¹) at an MOI of 100 and cultured overnight at 37 °C in a humidified incubator under 5% CO₂. Cells were collected the next day and transplanted into 4–8-week-old NBSGW female mice by tail-vein injection. Engraftment was monitored by peripheral blood collection and FACS analysis. Mice were euthanized when PDX AML cells exceeded around 20% of total white blood cells. BM and spleen were collected and either vitally frozen or FACS-sorted (BD FACS Melody sorter) to isolate mNeonGreen⁺ cells and retransplanted into new NBSGW recipients to obtain fully transduced PDXs.

In vivo xenotransplantation experiments

All of the animal experiments were performed in accordance with regulations set by the American Association for Laboratory Animal Science and Institutional Animal Care and Use Committee (IACUC) approved protocol (DFCI, 21-002). Mice were housed in sterile individually ventilated cages and fed autoclaved food and water, under a standard 12 h–12 h day–night light cycle. Female NOD.Cg-Kit^W-41JTyr⁺Prkdc^scidIl2rg^tm1Wjl/ThomJ mice (aged 4–8 weeks; termed NBSGW, Jackson, 026622) were xenotransplanted with 1 million human CD34⁺ HSPCs per mouse by tail-vein injection. Human engraftment was monitored by peripheral blood FACS analysis at 7–9 weeks. In AML co-engraftment experiments, mice then received 0.75 M human patient-derived AML xenograft cells (PDX), previously transduced with a mNeonGreen, by tail-vein injection. Then, 10 days after AML transplantation, mice received 2.5 M CAR T cells by tail-vein injection unless stated otherwise. The mice were monitored by peripheral blood analysis for a period of 2  weeks after CAR T cell administration and then euthanized. At euthanasia, the bone marrow, spleen and peripheral blood were collected for FACS and genomic analysis.

Colony-forming assays

CFU assays were performed by plating 1,000 CD34⁺ cells per well for in vitro CD34⁺ HSPCs experiments, or 25,000 total bone marrow cells per well for xenotransplanted BM-derived assays, unless stated otherwise. Cells were resuspended in Methocult H4034 medium (StemCell, 04034) and plated into SmartDish meniscus-free six-well plates. Wells were imaged and analysed after 2 weeks using the StemCell STEMvision system and STEMvision software using the 14 day bone marrow setting. For flow cytometry analysis, methylcellulose medium was softened with prewarmed PBS, collected and washed twice before analysis. The percentage of leukaemia cells within the BM-derived CFU was determined by mNeonGreen fluorescence.

Lineage differentiation cultures from CD34⁺ HSPCs

CD34⁺ HSPCs were thawed and edited as previously described. At day 7 after electroporation, cells were placed into differentiation media as follows: myeloid culture (SFEMII, penicillin–streptomycin 1%, Q 1%, FLT3L 100 ng ml⁻¹, SCF 100 ng ml⁻¹, TPO 50 ng ml⁻¹, GM-CSF 100 ng ml⁻¹, IL-6 50 ng ml⁻¹ and IL-3 10 ng ml⁻¹), macrophage (RPMI, FBS 10%, M-CSF 100 ng ml⁻¹ and GM-CSF 50 ng ml⁻¹) on non-tissue-treated plates, DC (RPMI, FBS 10%, GM-CSF 50 ng ml⁻¹ and IL-4 20 ng ml⁻¹), megakaryocyte (SFEMII, penicillin–streptomycin 1%, Q 1%, SCF 25 ng ml⁻¹, TPO 100 ng ml⁻¹, IL-6 10 ng ml⁻¹, IL-11 5 ng ml⁻¹), granulocyte (SFEMII, penicillin–streptomycin 1%, Q 1%, GM-CSF 100 ng ml⁻¹, then transitioned to RPMI + FBS 20% + G-CSF 50 ng ml⁻¹ after 7 days). The immunophenotype of differentiated cells was evaluated by flow cytometry.

Flow cytometry

Cell lines were collected, washed in PBS + 2% FBS, resuspended in 100 μl, incubated with human or mouse Fc-blocking reagent (Miltenyi 130-059-901 and 130-092-575) and then stained with the indicated antibodies for 20–30 min at 4 °C and washed. Viability was assessed by LiveDead yellow (Invitrogen, L34959) 7-AAD (BioLegend, 420404) or propidium iodide (PI, BioLegend, 421301) staining. Immunophenotyping of in vitro base-edited CD34⁺ HSPCs was evaluated by staining for 30 min at 4 °C with CD34 BV421, CD45RA APC-Cy7, CD90 APC, CD133 PE and, in some experiments, CD49f PE-Cy7 antibodies after incubation with Fc-blocking reagent (Miltenyi, 130-059-901). Viability was assessed by 7-AAD or PI staining.

For assessment of reactive oxygen species production, cells from the myeloid differentiation culture were collected and incubated with PMA (5 ng μl⁻¹) for 15 min at 37 °C, then CellROX Green reagent (Invitrogen, C10444) was added at a final concentration of 500 nM and the sample was kept for an additional 30 min at 37 °C (ref. ¹⁷). Cells were then stained with hFC block and HLA-DR PacificBlue, CD11b APC-Fire750, CD14 BV510, CD33 PE-Cy7 and CD15 AF700 antibodies, and washed and analysed. PI was included for viability. To test phagocytosis, in vitro differentiated macrophages were incubated with pHrodo green E. coli bioparticles (Thermo Fisher Scientific, P343666) for 60 min at 37 °C, washed and analysed by flow cytometry¹⁷. To assess signal transduction of in vitro differentiated myeloid culture, cells were collected, cytokine-starved overnight in SFEMII (not supplemented with cytokines) and then stimulated with GM-CSF 50 ng ml⁻¹, IL-4 50 ng ml⁻¹, IL-6 100 ng ml⁻¹, type I IFNα A/D 5,000 U ml⁻¹, PMA 100 ng ml⁻¹ plus ionomycin 1 μg ml⁻¹ or LPS 100 ng ml⁻¹ for 30 min at 37 °C. Cells were then fixed with BioLegend fixation buffer (420801) and permeabilized with True-Phos Perm Buffer (425401) according to the manufacturer’s instructions. Cells were then aliquoted into two fractions, stained with the following antibody panels and analysed using flow cytometry: (1) pSTAT1 PE-Cy7, pSTAT3 BV421, pSTAT5 PE and pSTAT6 AF647; (2) pERK1/2 BV421, pCREB AF647, p38/MAPK PE and pRPS6 PE-Cy7. In vitro differentiated dendritic cells were stained with hFC block, CD1c APC, CD33 PE-Cy7, CD303 APC-Cy7, CD141 KiraviaBlue520 and FLT3 PE antibodies and 7-AAD for viability. To assess upregulation of co-stimulatory molecules, dendritic cells were incubated with 100  ng ml⁻¹ LPS for 30 min at 37 °C and stained with hFC block, CD1c APC, CD303 APC-Cy7, CD11c BUV661, FLT3 PC7, HLA-DR Pacific Blue, CD80 BV605 and CD86 FITC antibodies, plus PI for viability. To evaluate macrophage differentiation towards an M1-like (CD80⁺CD86⁺HLA-DR⁺) or M2-like (CD86⁻CD200R⁺CD206⁺HLA-DR⁻) phenotype, in vitro differentiated macrophages were plated in a 96-well plate and stimulated overnight with LPS 100 ng ml⁻¹ or IL-4 20 ng ml⁻¹, respectively. Macrophages were then detached and stained with hFC block, CD11b APC-F750, CD33 BB515, CD200R PE-Cy7, CD206 PE, CD209 AF647, HLA-DR PacificBlue, CD80 BV605 and CD86 AF700 antibodies, and PI for viability and analysed by flow⁵⁶. Neutrophil extracellular trap (NETosis) induction in differentiated granulocytes was performed as previously described⁵⁶. In brief, cells were seeded in a 96-well plate, stimulated with PMA 50 ng ml⁻¹ for 4 h at 37 °C and then stained with hFC block and CD15 FITC, CD66b PE-Cy7 and CD33 APC antibodies for 15 min at room temperature. Granulocytes were then washed and fixed with PFA, supplemented with DAPI and PI staining solution to stain exocytosed nucleic acid, and analysed by flow cytometry. In vitro differentiated megakaryocytes were analysed by staining for 30 min at 4 °C with hFC block and CD41 PE, CD42b AF647, CD61 FITC, CD34 BV421 and CD45 BV786 antibodies. For megakaryocyte ploidy, cells were stained with stained with hFC block and CD42b AF647 and CD61 FITC antibodies, and then washed and resuspended in 500 μl FxCycle PI/RNase staining solution (Molecular Probes, F10797) and analysed by flow cytometry⁵⁷.

Peripheral blood collected from in vivo experiments was lysed twice for 10 min at room temperature using ACK reagent (StemCell, 07850), washed, incubated with human and mouse Fc-blocking reagent (Miltenyi, 130-059-901 and 130-092-575), and stained at room temperature for 15 min with hCD45 BV421, mCD45 PE, CD3 AF647, CD19 BV605 and CD33 PE-Cy7 antibodies, unless stated otherwise. 7-AAD was included as viability stain. To comprehensively identify haematopoietic populations in BM samples⁵⁸, collected cells were lysed using ACK reagent, washed twice and supplemented with CountBeads for absolute quantification. The samples were then incubated with human and mouse Fc-blocking reagent and stained for 30 min on ice with hCD45 BV786, mCD45 PerCP-Cy5.5, CD3 PE-Cy5, CD7 AF700, CD10 BUV737, CD11c BUV661, CD14 BV510, CD19 BV605, CD33 PE-Cy7, CD38 BUV396, CD45RA APC-Cy7, CD56 BUV496, CD90 APC, FLT3 PE or BV711, and either KIT BV711 or CD123 PE antibodies (depending on the experimental design) (Supplementary Table 2), with the addition of 50 μl per sample Brilliant Stain buffer (BD, 659611). A T-cell-focused panel was applied to the spleen and BM samples by staining for 30 min on ice with hCD45 BV786, mCD45 PerCP-Cy5.5, EGFR AF488, CD3 BUV396, CD4 APC, CD8 PacificBlue, CD62L BV605, CD69 PE-Cy7, CD45RA APC-Cy7, PD1 BUV737 and LAG3 PE antibodies. Propidium iodide (3.5 μl per 100 μl) was included as a viability stain shortly before analysis. Cells were acquired on the 5-laser BD Fortessa flow cytometer using BD FACS Diva (v.6) and analysed using FCS Express 6 (DeNovo Software).

Gene expression analysis of edited CD34⁺ HSPCs

CD34⁺ cells from healthy donors were either base edited for FLT3, CD123, KIT or AAVS1 or untreated (electroporation only). Then, 4 days after editing, to enable complete turnover of the edited receptors, cells were starved overnight in SFEMII without cytokines. On day 5 after editing, CD34⁺ HSPCs were stimulated with either FLT3L 500 ng ml⁻¹, SCF 500 ng ml⁻¹ or IL-3 100 ng ml⁻¹ according to the editing condition. AAVS1 and the electroporation-only conditions were divided into three separate wells and incubated with the three cytokines, to provide appropriate controls for each donor and stimulation. After 24 h of stimulation, cells were collected and RNA was extracted using the Qiagen RNAeasy Mini kit (74104) with on-column DNase I treatment (Qiagen, 79254). RNA was quantified using the Nanodrop and sent for sequencing (DNBSEQ stranded mRNA library, BGI). Variations in the gene expression profile in response to specific ligand stimulation were compared among FLT3-, KIT– and CD123-edited samples, control AAVS1-edited samples and electroporation-only samples. Raw sequencing files were filtered for quality control and aligned to the reference human genome hg38 using STAR workflow⁵⁹, obtaining, as a result, the gene-based count matrices. On the basis of a preliminary principal component analysis, we identified one sample (HD-2, KIT-edited, unstimulated) as an outlier and removed it from the subsequent analysis. We also filtered poorly detected genes with cumulative read counts smaller than 10 or measured (read count > 0) in less than two samples. Statistical analysis was performed in R software, using the DEseq2 package⁶⁰. Gene counts were modelled using a negative binomial model, the significance of specific coefficients was tested using the Wald test, and P values were adjusted for multiplicity using the Holm method⁶¹. We analysed the expression pattern for each gene by using the full model configuration: FullModel: GeneExpr_i = b_0 + b_Don. Donor + b_Edit. Editing+ b_Stim Stimulation +  b_(Edit.Stim) Editing x Stimulation. We then tested the significance of the following parameters: b_Edit(UT VS AAVS1): genes differentially expressed in UT with respect to the reference group AAVS1; b_Edit(FLT3 | KIT | CD123 VS AAVS1): genes differentially expressed in sample edited using FLT3, KIT or CD123 sgRNA with respect to the reference group AAVS1; b_Stim: genes differentially expressed in stimulated samples (FLT3L, SCF or IL-3) with respect to the unstimulated group; b (Edit.Stim): genes differentially expressed in an editing group-specific manner after stimulation.

The expression values for the significant genes identified in each comparison are displayed as heat maps in Extended Data Fig. 5c,e. The figures were generated using the R package ggplot2⁶².

Phosphoproteome analysis by mass spectrometry

CD34⁺ cells were base edited for FLT3, CD123, KIT or AAVS1. Four days after editing, cells were starved overnight in SFEMII without cytokines. On day 5 after editing, CD34⁺ HSPCs were incubated at 37 °C with FLT3L (500 ng ml⁻¹), SCF (500 ng ml⁻¹) or IL-3 (100 ng ml⁻¹) according to the editing condition (AAVS1 was stimulated independently with each of the three cytokines). After 6 h, cells were collected and dry pellets were flash-frozen in liquid nitrogen. Cell pellets were lysed in 5% SDS, 50 mM TEAB pH 8.5 with protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich, phosphatase inhibitor cocktails 2 and 3) for 30 min with shaking. The protein concentration was determined by BCA (Thermo Fisher Scientific). Aliquots of protein (30 µg) were treated with 250 U of benzonase and incubated for 5 min at room temperature. Proteins were then reduced with DTT (final concentration, 10 mM; 56 °C for 30 min), alkylated with iodoacetamide (25 mM) for 30 min at room temperature, acidified 1:10 with 12% phosphoric acid, further diluted 6× with binding buffer (90% methanol and 100 mM TEAB) and then applied to S-trap micro columns (Protifi). The columns were washed four times with 150 µl binding buffer, and proteins digested with trypsin overnight at 37 °C. Peptides were extracted from the column by centrifugation after applying elution buffers (1–50 mM TEAB, 2–0.2% formic acid and 3–50% acetonitrile), and dried by vacuum centrifugation. Peptides were resuspended in 100 mM HEPES, pH 8.0, and treated with TMT-Pro isobaric labelling reagents for 1 h at room temperature using 8 out of the 16 possible reagents (126, 127N, 128N, 129N, 130N, 131N, 132N and 133N, or 127C, 128C, 129C, 130C, 131C, 132C, 133C and 134N). Each experiment included proteins from target and control edits ± ligand stimulation for 2 separate donors. After labelling, the reactions were quenched with 5% hydroxylamine in 50 mM TEAB, combined, dried, reconstituted in 0.1% TFA and desalted by C18. Phosphopeptides were then enriched by Fe³⁺-NTA-IMAC as described previously⁶³ and analysed by nanoflow liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS)⁶⁴. Peptides were loaded onto an analytical column with an integrated ESI emitter tip (75 µm internal diameter, fused, packed with 25 cm of 1.9 µm Reprosil C18, ESI Source Solutions), desalted and resolved with an LC gradient (1–5% B in 5 min, 5–18% B in 115 min, 18–27% B in 50 min, 27–39% B in 30 min, 39–80% B in 15 min, where A is 0.1% formic acid in water and B is 0.1% formic acid in acetonitrile). Peptides were analysed on the TimsTOF Pro II mass spectrometer with 6 PASEF ramps (mobility range 0.8–1.4 V s cm⁻², ramp time 100 ms), MS/MS stepping enabled, a 1.5 Da isolation width and a target intensity of 17,500. Active exclusion was enabled with a release time of 30 s. Data files were searched against a forward reverse human protein database (UCSG) using MSFragger with precursor and product ion tolerances of 25 ppm and 0.05 Da, respectively. Search parameters specified variable oxidation of methionine, variable phosphorylation (STY), fixed carbamidomethylation of cysteine and fixed TMT-Pro modification of peptide N termini and lysine residues. Custom Python scripts⁶⁵ were used to filter search results to 1% FDR, as well as extract TMT reporter ion intensities and correct for isotopic impurities. After filtering out PSMs with missing TMT values, reporter ion evidence was summed for peptides of the same modification and charge state, with values of less than 20 counts imputed to the estimated noise level (20 counts). Data were then quantile normalized after removing peptides with a median reporter ion intensity of less than 100 counts. We analysed the phosphorylation pattern for each peptide using the full model configuration: FullModel: PhosphoPeptide_i = b_0 + b_Don. Donor + b_Edit. Editing+ b_Stim Stimulation; and tested the significance of b_Edit and b_Stim using a likelihood ratio test and the following reduced models: ReducedModel for testing b_Edit: PhosphoPeptide_i = b_0 + b_Don. Donor + b_Stim Stimulation; ReducedModel for testing b_Stim: PhosphoPeptide_i = b_0 + b_Don. Donor + b_Edit. Editing.

Given the high signal-to-noise ratio of this methodology and the limited sample size, the results were analysed descriptively. We calculated the average (across donor) fold change between the phosphorylation levels in the unstimulated and stimulation conditions for both editing settings.

Off-target evaluation

GUIDE-seq was conducted in HEK293T cells⁶⁶. The GUIDE-seq dsODN was prepared by annealing PAGE-purified oligos GUIDE-seq dsODN sense and antisense (a list of the sequences is provided in Supplementary Table 1) using the following conditions: 95 °C for 2 min, slow ramp down to 25 °C with an increment of −0.1 °C per cycle for 700 cycles. HEK293T cells were electroporated with 4 µg SpRY Cas9 mRNA, 200 pmol of dsODN and 600 pmol of guide RNA using the SF Cell Line 4D Nucleofector X Kit S (Lonza, V4XC-2024). The dsODN concentration was selected after a titration to identify the maximal dose permitting at least 60% cell viability 24 h after delivery by electroporation. Genomic DNA was collected 4–6 days after electroporation. We performed GUIDE-seq library construction with some modifications as previously described⁶⁷. Adapter oligos (from IDT) were annealed using the following conditions: 95 °C for 2 min, slow ramp down to 25 °C with an increment of −0.1 °C per cycle for 700 cycles. We added Tn5-transposase (86% reaction volume) to pre-annealed oligo (14% reaction volume) for transposome assembly at room temperature for 1 h. We prepared tagmentation reactions by titrating the assembled transposome into 200 ng of genomic DNA (gDNA) in the presence of 1× TAPS-DMF, incubating at 55 °C for 7 min. 1× TAPS-DMF buffer contains 50 mM TAPS-NaOH at pH 8.5 (at room temperature; Thermo Fisher Scientific, J63268AE), 25 mM magnesium chloride (MgCl₂; Thermo Fisher Scientific, AM9530G), 50% dimethylformamide (Thermo Fisher Scientific, 20673). The transposome was inactivated with 0.2% SDS at room temperature for 5 min. Tagmentation conditions were selected that yielded DNA fragment size distribution concentrated at 500–1,000 bp. After the tagmented DNA was isolated, we performed a two-step PCR amplification with the first step (PCR 1) for GUIDE-seq oligo-specific amplification and the second step (PCR 2) for indexing. The PCR 1 reaction mix contained 50 mM magnesium chloride (MgCl₂; Thermo Fisher Scientific, AM9530G), 2× Platinum SuperFi PCR Master mix (Thermo Fisher Scientific, 12358050), 0.5 M tetramethylammonium chloride solution (TMAC; Sigma-Aldrich, T3411), 10 µM GUIDE-Seq oligo-specific primer (GSP, IDT) (Supplementary Table 1), 10 µM i5 amplification primer (IDT, 5′-AATGATACGGCGACCACCGAGATC-3′) and tagmented DNA. The PCR 2 reaction mix was the same as for PCR 1, but without 50 mM MgCl₂, and we added 10 µM i7 barcode primer (IDT) instead of GSP. The PCR 1 product was size-selected with 45 µl (0.9× original reaction) of AMPure XP Beads (Agencourt, A63882) and eluted in nuclease-free water before proceeding to the next step. The PCR 2 amplified product was double-size selected with 25 µl (0.5× original reaction) of AMPure XP Beads and then with 17.5 µl (0.35× original reaction) of the beads to isolate PCR products in the range of 200–500 bp, quantified using the Qubit dsDNA High-Sensitivity Assay (Thermo Fisher Scientific, Q32851), checked for size and purity using the TapeStation (Agilent 4200 System) and KAPA qPCR (KAPA Library Quantification Kit Illumina Platform), before samples were equimolar pooled and sequenced. The libraries were deep-sequenced as a pool using a paired-end 150 bp run on the Illumina MiniSeq system (~1.5 million reads per sample) with the following parameters: 25–35% PhiX and 151|8|17|141 (Read1|Index1|Index2|Read2). Deep sequencing data from the GUIDE-seq experiment were analysed using GS-Preprocess (https://github.com/umasstr/GS-Preprocess) and Bioconductor Package GUIDEseq (v.1.4.1)⁶⁸. The GS-Preprocessing script demultiplexed the raw Illumina FASTQ files based on the index information and mapped the sequence files to the reference genome. It generated the input files (BAM files of plus and minus strands, gRNA fasta files and a unique molecular index (UMI) for each read) for Bioconductor Package GUIDEseq. The window size for peak aggregation was set to 50 bp. Off-target site identification parameters were set for SpRY-Cas9 as follows: PAM.pattern = “NNN”, max.mismatch = 10, includeBulge = TRUE, max.n.bulge = 2L, upstream = 50, downstream = 50, max.mismatch = 6 and allowed.mismatch.PAM = 3. To filter false-positive signals, we called the peaks and calculated their frequencies in the control group. These background peaks were used to filter out potential hot spots or sequencing noise in Cas9–sgRNA treatment groups. This analysis provided a list of potential off-target sequences that were ranked with regard to unique reads on the basis of the incorporated UMI and the adapter ligation position, which has been correlated with the activity of SpRY-Cas9 at each sequence (Supplementary Table 16). The results were filtered by the number of mismatch + bulge < 6 to identify high-risk off-target sites.

Candidate in silico predicted off-target sites were selected on the basis of the CRISPOR online tool (top six intronic and top six exonic sites based on sequence homology for FLT3, CD123 and KIT sgRNAs when using a SpRY-Cas9 PAM (NRN). We amplified the off-target sequences by PCR on genomic DNA extracted from edited or control CD34⁺ cells, using three different healthy donors for each condition. PCR products were designed to be <400 bp in size. PCR amplicons for each condition were pooled together and sent for library preparation and NGS sequencing (300 bp paired-end reads, Azenta). One exonic and two intronic sites for CD123 sgRNA-R were eliminated from the final analysis owing to an insufficient number of reads. Reads were processed using the CRISPResso2 tool (CRISPRessoPooled), setting as the quantification window the amplicon interval overlapping with the sgRNA. We modelled the proportion of EditedReads over TotalReads (unedited + edited) using a binomial response generalized linear model (logistic regression) including the following covariates: DonorID, factor distinguishing cell donor; treatment, a binary variable having value treatment = 1 for edited and treatment = 0 for mock samples. Standard logistic regression estimation frameworks have convergence issues when the input data table is sparse (multiple samples with EditedReads = 0). To address this issue, we adopted a bias-reduction approach⁶⁹, a second-order unbiased estimator that returns finite standard errors for the coefficients under all conditions. We performed the regression and analysed the results for each locus separately and calculated: (1) EditedReads proportion estimation and related 95% confidence interval for all samples; (2) the significance of the treatment coefficient (Wald test for the null hypothesis β = 0 and α = 0.05). Suppose the coefficient estimate is positive and significant. In that case, the expected EditedReads proportion in the edited samples (treatment = 1) is greater than in the mock samples, and therefore bona fide, off-target, base editor activity is detected. For the quantification of on-target indels proportion, a similar procedure was used by selecting from the edited reads (Unedited=“FALSE”) only those having either n_deleted > 0 or n_inserted > 0. The analysis was performed using R software. For the estimation of the logistic model, we used the R package brglm2 (ref. ⁷⁰).

RNA-editing analysis

The presence and extent of guide-independent RNA editing activity caused by ABE have been verified using the data generated in the RNA-seq experiment described in the ‘Gene expression analysis of edited CD34⁺ HSPCs’ section. Each sample’s R1 and R2 reads files were processed using REDItools2⁷¹ using a base quality filtering threshold (-bq 30) and a hg38 transcript annotation database. On the basis of the output generated by REDItools2, for each sample, we selected the adenine nucleotides in the reference genome with the highest coverage (top 5%) by transcriptomics reads. We then calculated the percentage of detected A>G transition events (number of A>G transition)/A coverage). The distribution of A>G transition rates across all A nucleotides considered for each sample is graphically represented using box plots in Extended Data Fig. 8c. The sample-specific coverage threshold and the total number of A nucleotides considered is reported in Supplementary Table 21.

Statistical analyses

The n values indicate the number of biologically independent samples, animals or experiments. Data are summarized as mean ± s.d. Inferential techniques were applied in presence of adequate sample sizes (n ≥ 5), otherwise only descriptive statistics are reported. Comparisons between two groups were performed using unpaired t-tests. FDR-adjusted P values are reported when appropriate. When one variable was compared among more than two groups, one-way ANOVA was used. When multiple variables were compared among more than two groups, two-way ANOVA was used. The P values of the row or column effect is reported as appropriate to describe the significance of the selected parameter on the measured variable. Multiple comparisons between groups are performed with two-stage step-up procedure of Benjamini, Krieger and Yekutieli to control the FDR (Q = 0.05) when appropriate. In all of the analyses, the significance threshold was set at 0.05; NS, not significant. Analyses were performed using GraphPad Prism v.9.4 (GraphPad). The methods for the statistical analyses of genomic off-target sites, RNA-seq, phosphoproteomic profiling by MS, RNA editing and antibody/ligand affinity curves are reported in each Methods section.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.