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Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

eLife (2017) 6
DOI: 10.7554/eLife.27873
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Abstract

Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.

Figures

  • 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 postelectroporation highlight the CCR5 tNGFRhigh population (red gate) generated by the addition of Cas9 RNP compared to cells with low reporter expression (green gate) and reporternegative 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 reporterhigh (red), reporterlow (green), and reporterneg (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, reporterhigh 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, reporterhigh 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 2. Identification and enrichment of biallelic genome-edited CD34+ human hematopoietic stem and progenitor cells (HSPCs). (a) Left, Schematic showing biallelic targeting strategy for ASXL1 using GFP and BFP-encoding rAAV6 donors for integration into each allele of ASXL1. The SFFV promoter drives reporter expression. Middle, FACS plot from an ‘AAV only’ sample day four post electroporation, showing low episomal reporter expression (BFP and GFP) in cells without the CRISPR system. Right, FACS plot of CD34+ HSPCs treated with both Cas9 RNP and the two rAAV6 donors highlighting the generation of BFPhigh/GFPhigh double positive cells that have undergone ASXL1 dual-allelic targeting. (b) HSPCs were targeted at both alleles of HBB (Cas9 RNP with GFP and tdTomato rAAV6 donors) and at day four post electroporation, dual positive cells were sorted and cultured for 16 days while analyzing reporter expression. Error bars representing S.E.M. are present, but too small to be visible (N = 3 different HSPC donors). (c) Gel images showing PCR genotyping of six methylcellulose-derived clones from (e) confirming integration into each of the HBB alleles. (d) Human primary T cells were CD3/CD28 stimulated for three days and then electroporated with CCR5-targeting Cas9 RNP and transduced with two CCR5-specific rAAV6 donors encoding GFP and mCherry, respectively. FACS plots show GFPhigh/mCherryhigh biallelic targeting frequencies at day four post-electroporation.
  • Figure 3. Identification, enrichment, and long-term engraftment in NSG mice of di-genic genome-edited CD34+ human hematopoietic stem and progenitor cells (HSPCs). (a) Left, Schematic depicting HBB and IL2RG di-genic targeting. Middle, FACS plot of an ‘AAV only’ sample at day four post electroporation, showing low episomal reporter expression (HBB-tdTomato and IL2RG-GFP) in cells without the CRISPR system. Right, FACS plot at day four post-electroporation of HSPCs electroporated with Cas9 RNP targeting both HBB and IL2RG followed by transduction with HBB-tdTomato and IL2RG-GFP rAAV6 donors showing the generation of tdTomatohigh/GFPhigh cells with di-genic targeting at HBB and IL2RG. (b) Double-positive HSPCs targeted at HBB (GFP) and CCR5 (mCherry) were sorted at day four post-electroporation and cultured for 15 days while analyzing reporter expression. Error bars represent S.E.M. (N = 3 different HSPC donors). (c) Representative gel images showing PCR genotyping of six (out of 57 total) HBB-GFPhigh (gene reporter 1)/CCR5-mCherryhigh (gene reporter 2) methylcellulose-derived clones confirming integration at each locus (d) Representative fluorescence microscopy images of methylcellulose-derived clones with di-genic targeting at HBB and CCR5 show myeloid and erythroid progenitors with both GFP and mCherry expression. (e) HSPCs were targeted at the HBB and AAVS1 loci with a GFP and BFP expression cassette, respectively. Representative FACS plot (left panel) shows analysis seven days after targeting. All four gated populations were sorted and genomic DNA was subject to TIDE analysis for determining INDEL frequencies at the two loci (middle panel), and subject to ddPCR quantification of one of the two possible monocentric translocations between HBB and AAVS1 (right panel) (see also Figure 3—figure supplement 2). (f) Representative FACS plots from cells targeted at the HBB and AAVS1 loci with a GFP and BFP expression cassette, respectively. Representative FACS plot shows analysis four days after targeting at which point the four populations were sorted and transplanted intrafemorally into NSG mice that were irradiated 24 hr before transplantation. (g) Bone marrow from the injected femurs from the mice transplanted as described in (f) was analyzed 12 weeks after transplantation. Representative FACS plots are from a mouse from each of the four groups depicted in (f) as well as a mouse transplanted with mock-electroporated cells. The middle row depicts human engraftment gated as positive for the human leukocyte antigen complex (HLA-ABC). The upper and lower rows
  • Figure 4. Multiplexing homologous recombination in CD34+ human hematopoietic stem and progenitor cells (HSPCs). (a) HSPCs were electroporated with Cas9 RNP targeting ASXL1 and RUNX1 followed by rAAV6 transduction with two donors for ASXL1 (mCherry and GFP) and two donors for RUNX1 (E2Crimson and BFP). Tetra-allelically targeted HSPCs were identified as mCherryhigh/GFPhigh/BFPhigh/E2Crimsonhigh (N = 3 see Supplementary file 1e) (b) Cells modified at both alleles for RUNX1 and ASXL1 (as in (a)) were subjected to a methylcellulose assay (triplicates) and scored as BFU-E, CFU-M, CFU-GM or CFU-GEMM based on morphology 14 days after sorting. (c) PCR was performed on colony-derived gDNA to detect targeted integrations at both genes. 73 individual colonies were analyzed. Color coding for colonies with triple-allelic integration are as follows: grey: RUNX1 biallelic/ASXL monoallelic; white: RUNX1 monoallelic/ASXL1 biallelic. (d) For tri-genic targeting of HSPCs, cells were electroporated with Cas9 RNP targeting IL2RG, HBB, and CCR5 followed by transduction of three rAAV6 donors homologous to each of the three genes (IL2RG-GFP, HBB-tdTomato, and CCR5tNGFR). Tri-genic-targeted cells were identified as reporterhigh for all three reporters (N = 5 see Supplementary file 1e). (e) Methylcellulose clones from the triple-positive cells in (d) were subjected to genotyping PCR and gel images show colonies with targeted integration at all three genes in 9/11 colonies (note that GFP shows a faint band in colony 6). (f) Left, Schematic showing strategy for targeting four different genes (HBB, RUNX1, ASXL1, and CCR5) simultaneously (tetra-genic). Four different genes are targeted by electroporation of four different Cas9 RNPs followed by transduction with four different rAAV6 donors that each targets a gene with a different reporter. Right, Tetra-genic targeting at the above-mentioned four genes was identified as reporterhigh for all four reporters (N = 3 see Supplementary file 1e).
  • Table 1. Overview of targeting experiments in hematopoietic stem and progenitor cells (HSPCs). Overview of all HSPC targeting experiments performed in this study with the number of independent experiments (N) for each experiment type, and the mean targeting efficiency (±SD). See also Supplementary file 1a, c, and e.

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Bak, R. O., Dever, D. P., Reinisch, A., Hernandez, D. C., Majeti, R., & Porteus, M. H. (2017). Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. ELife, 6. https://doi.org/10.7554/eLife.27873

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