Figure 1. FACS-based identification and enrichment of monogenic genome-edited CD34⁺ human hematopoietic stem and progenitor cells (HSPCs).

(a) HSPCs were electroporated with CCR5-RNP and transduced with CCR5-tNGFR rAAV6 HR donor. Representative FACS plots from day four post-electroporation highlight the CCR5 tNGFR^high population (red gate) generated by the addition of Cas9 RNP compared to cells with low reporter expression (green gate) and reporter^negative cells (black gate). Numbers reflect percentage of cells within gates. (b) Day four post-electroporation, CCR5 (tNGFR or GFP) and IL2RG (GFP)-targeted HSPCs from reporter^high (red), reporter^low (green), and reporter^neg (blue) fractions were sorted and cultured for 20-22 days while monitoring the percentage of cells that remained GFP⁺. Error bars represent S.E.M. N = 6 for CCR5, N = 3 for IL2RG, all from different CD34⁺ donors. (c) HSPCs were targeted at CCR5 (with GFP or tNGFR donor) or at IL2RG (GFP donor; only female cells for IL2RG). At day four post-electroporation, reporter^high cells were single-cell sorted into methylcellulose for colony formation. PCR was performed on colony-derived gDNA to detect targeted integrations. 338 CCR5 and 177 IL2RG myeloid and erythroid methylcellulose colonies were screened from at least two different CD34⁺ HSPC donors. (d) HSPCs were targeted at the STAG2 gene or the AAVS1 locus with a GFP reporter cassette. Cells that only received the STAG2-GFP AAV6 donor and not Cas9 RNP were included as an additional control. At day four post-electroporation and transduction, reporter^high cells from the STAG2 and AAVS1 targeting experiments and bulk cells from the STAG2 AAV6 only population were plated in methylcellulose for colony formation. After 14 days, colonies were scored as either erythroid or myeloid based on morphology. Error bars represent S.E.M, N = 3, ***p<0.001, n.s. = p≥0.05, unpaired t-test.

Figure 1—figure supplement 1. Analysis of cell fractions with different fluorescence intensity.

(a) Schematic showing the general layout of the AAV6 donors employed. ITR: inverted terminal repeat; SFFV promoter: spleen focus forming virus promoter; GFP: green fluorescent protein; polyA: bovine growth hormone polyadenylation signal; RHA: right homology arm. Approximate sizes are shown below each component. (b) Cells were targeted at the HBB locus by electroporation of Cas9 RNP followed by transduction of a homologous rAAV6 donor carrying a GFP expression cassette. At 4 days post electroporation and transduction, cells with different GFP intensities (GFP^high, GFPLow^High, GFPLow^Med, GFPLow^Low) were FACS-sorted and cultured for an additional 16 days. At day 20 post targeting, cells were analyzed for GFP expression by flow cytometry and the red gates show the GFP^high population at this time point. (c) The cells from b) were analyzed at different time points after sorting, and data points show the percentage of cells within the GFP^high gate for the different populations as well as a population receiving only the rAAV6 donor and not Cas9 RNP.

Figure 1—figure supplement 2. Genotypes of clones with mono-genic targeting.

(a) Left, schematic representation of the three-primer PCR used to genotype CCR5 alleles for integrated (green PCR product) and non-integrated (red PCR product) alleles. One forward primer is located in the left homology arm (LHA), one forward primer is located in the poly A, and a common reverse primer is located outside the region of the right homology arm (RHA). Right, gel image of representative genotyped clones from Figure 1c (CCR5) showing colonies with biallelic and monoallelic integrations. (b) A subset of the CCR5 and IL2RG clones (only female cells for IL2RG) from Figure 1c with monoallelic integration had the genotype on the non-integrated allele analyzed by Sanger sequencing of purified PCR products. Note that in-frame INDELs can be gene-disrupting depending on the location and size of the INDEL. (c) As in Figure 1c, HSPCs were targeted at RUNX1 and at day four post-electroporation, reporter^high cells were single cell-sorted into methylcellulose-containing 96-well plates to establish colonies. After 14 days, PCR was performed on colony-derived gDNA to detect targeted integrations. A total of 36 myeloid and erythroid methylcellulose colonies were screened. (d) The monoallelically targeted clones from c) had the genotype assessed on the non-integrated allele by Sanger sequencing of purified PCR products. See Supplementary file 1b for complete list of genotypes.