Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6
- Stanford University, United States
Figures
Figure 1 with 2 supplements
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 tNGFRhigh population (red …
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 …
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 …
Figure 2 with 2 supplements
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. Mid…
Figure 2—figure supplement 1
Cas9 and rAAV6-mediated biallelic homologous recombination (HR) in human CD34+HSPCs.
Top, Representative FACS plots from HSPCs transduced with two rAAV6 (two fluorescent reporters for each gene), that have homology for the genes listed on the bottom panel, show low episomal …
Figure 2—figure supplement 2
Toxicity assessment of biallelic integration at the CCR5 locus in primary human T cells.
Human primary T cells were isolated from buffy coats and stimulated for three days using anti-CD3 and anti-CD28 antibodies. Cells were then electroporated with CCR5-targeting Cas9 RNP and transduced …
Figure 3 with 3 supplements
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 …
Figure 3—figure supplement 1
Cas9 and rAAV6-mediated di-genic homologous recombination (HR) in human CD34+ HSPCs.
Top, Representative FACS plots of HSPCs transduced with two rAAV6 donors targeting two genes with two distinct fluorescent reporters (listed in FACS plots in lower panel) show low episomal …
Figure 3—figure supplement 2
Measuring translocations after HBB and AAVS1 di-genic targeting.
(a) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. (b) The …
Figure 3—figure supplement 3
Analysis of mice transplanted with different sorted populations of cells targeted at the HBB and AAVS1 locus.
The table shows an overview of the 11 NSG mice that were transplanted intrafemorally with either mock-electroporated cells or sorted cells from the four populations displayed in Figure 3f. 12 weeks …
Figure 4 with 5 supplements
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). …
Figure 4—figure supplement 1
Targeting two genes for biallelic homologous recombination (HR) in primary CD34+ HSPCs.
(a) Schematic showing experimental strategy for Figure 4a for targeting both alleles of RUNX1 and ASXL1. (b) FACS plots, gating scheme, and frequencies of HR at each allele for the experiment shown …
Figure 4—figure supplement 2
Multiplexing homologous recombination at three genes simultaneously in HSPCs.
(a) Schematic showing experimental strategy for Figure 4d targeting three genes, IL2RG, CCR5, and HBB. (b) FACS plots show gating scheme and HR frequencies at each locus for the experiment shown in F…
Figure 4—figure supplement 3
Toxicity assessment of multiplexed HR.
CD34+ cells from mobilized peripheral blood were targeted at one, two, or three genes with Cas9 RNP and rAAV6 donors. Viabilities were measured by flow cytometry 72 hr post-electroporation using …
Figure 4—figure supplement 4
Assessment of false-positive frequencies of FACS-based identification of multiplexed HR in HSPCs.
Since capture of rAAV6 donors at the site of a DSB via NHEJ has been reported, we measured the false-positive rate of multiplexing HR via flow cytometry. (a) False-positive frequencies of di-genic …
Figure 4—figure supplement 5
Controlling genotype with cDNA knock-in.
(a) A heterozygous knockout population can be generated with two HR donors. The first donor is designed to knock-in a wild-type (WT) cDNA cassette into the start codon (ATG) of the gene of interest …
Author response image 1
Author response image 2
Tables
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 Supplemen…
| Experiment | N | % efficiency ± SD |
|---|---|---|
| Monogenic | 47 | 21.7 ± 13.4 |
| Biallelic | 16 | 5.5 ± 4.2 |
| Di-genic | 17 | 8.1 ± 8.1 |
| Tetra-allelic | 3 | 0.9 ± 0.3 |
| Tri-genic | 6 | 4.5 ± 4.8 |
| Tetra-genic | 3 | 0.7 ± 0.3 |
Additional files
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Supplementary file 1
(a) Overview of Cas9 and rAAV6 mono-genic targeting experiments performed in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB)-derived human CD34+HSPCs.
This table summarizes all independent experiments targeting HBB, CCR5, IL2RG, RUNX1, ASXL1, STAG2, and AAVS1 in HSPCs and the reporter genes used. GFP: green fluorescent protein, tNGFR: truncated Nerve Growth Factor Receptor, BFP: blue fluorescent protein. Efficiencies were averaged across 47 independent experiments, N = 47. (b) Overview of genotypes for the non-integrated alleles in mono-genic integration experiments. The three tables show the different INDELs that were identified by Sanger Sequencing of the non-edited allele in mono-genic targeting experiments (CCR5, IL2RG, and RUNX1) used to analyze genotype frequencies shown in Figure 1—figure supplement 2b and d. Alleles are grouped into WT (blue), INDELs that preserve the reading frame (red) and INDELs that disrupt the reading frame (green). Note that INDELs that preserve the reading frame can potentially be disruptive depending on the size and location. For example, the 147 bp deletion in RUNX1 is considered disruptive because of its large size and because it deletes the splice donor site in the intron between exon 2 and 3. For IL2RG, one clone was found to have an allele with integration of 230 bp from the donor (at the end of the RHA and 72 bp into the ITR). (c) Overview of di-genic and biallelic targeting experiments in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB)-derived human CD34+HSPCs. This table summarizes the experiments targeting HSPCs for biallelic and di-genic HR and the reporter genes used. GFP: green fluorescent protein, tNGFR: truncated Nerve Growth Factor Receptor, BFP: blue fluorescent protein. Efficiencies were averaged across 16 and 17 independent experiments, respectively, N = 16 and N = 17. (d) Overview of genotypes for the non-integrated alleles in clones with tri-genic integrations. Each row of the table represents the genotype of a colony established from a tri-genic targeting experiment (IL2RG, HBB, and CCR5). Alleles are grouped into WT (blue), INDELs that preserve the reading frame (red) and INDELs that disrupt the reading frame (green). Note that INDELs that preserve the reading frame can potentially be disruptive depending on the size and location. For HBB we identified one clone where HBD had been used as repair template and three clones with mono-allelic integration of part of the SFFV promoter indicative of HR events that ended prematurely. (e) Overview of tetra-allelic, tri-genic, and tetra-genic targeting experiments performed in human CD34+HSPCs derived from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB). This table summarizes the independent multiplexing HR experiments performed for tetra-allelic, tri-genic, and tetra-genic targeting and the reporter genes used. GFP: green fluorescent protein, tNGFR: truncated Nerve Growth Factor Receptor, BFP: blue fluorescent protein. Efficiencies were averaged across independent experiments, N = 3 (tetra-allelic and tetra-genic) and N = 6 (tri-genic).
- https://doi.org/10.7554/eLife.27873.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.27873.021
