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 mCherry^high/GFP^high/BFP^high/E2Crimson^high (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 CCR5-tNGFR). Tri-genic-targeted cells were identified as reporter^high 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 reporter^high for all four reporters (N = 3 see Supplementary file 1e).

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 in Figure 4a. (c) FACS plot showing very low frequency of tetra-reporter^high cells without Cas9. (d) FACS plots of cells from single methylcellulose colonies derived from tetra-reporter^high cells from Figure 4a. (e) Schematic showing targeting both alleles of RUNX1 and HBB for HR with four distinct reporters. (f) Top, FACS plots of HSPCs transduced with four rAAV6s (no Cas9 RNPs) showing the gating scheme and low episomal reporter expression without a nuclease. Bottom, HSPCs were electroporated with RNPs targeting HBB and RUNX1 and then transduced with four rAAV6s. FACS plots from day four post electroporation show MFI shift for each reporter alone. HBB-tNGFR rAAV6 has reproducibly shown lower episomal expression than all other rAAV6 we have used. (g) Images from fluorescence microscopy showing an mCherry/BFP/GFP positive CFU-GM clone that has undergone tetra-allelic HR. The colony was not stained for HBB-tNGFR. (h) Left, FACS plots show very low frequency of tetra-reporter^high cells without Cas9. Right, Nuclease addition increases the frequency of bi and tetra-reporter^high HSPCs.

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 Figure 4d. (c) Schematic outlining another tri-genic targeting experiment for RUNX1, ASXL1, and HBB. (d) Top, FACS plots of HSPCs transduced with three rAAV6 donors (no RNPs). Bottom, HSPCs were electroporated with gene-specific RNPs and then transduced with three rAAV6 donors. FACS plots at Day 4-post electroporation show MFI shift for each reporter alone. (e) FACS plots from same sample as in (d), but showing different combinations of di-genic reporter^high populations that contain the same frequency of tri-genic reporter^high cells.

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 Live/Dead and Annexin V stains. Viable cells are defined as live, non-apoptotic (Annexin V⁻) and plotted as percentage of a single AAV6 donor alone. Error bars represent SD, ns = not statistically significant, Mann-Whitney test, N = 2 different HSPC donors.

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 targeting in HSPCs was determined by electroporating cells with an HBB-targeting Cas9 RNP followed by transduction with HBB-GFP (homologous) and CCR5-mCherry (non-homologous) rAAV6 donors. FACS plots show a false-positive rate of 0.24% dual reporter^high cells. Note that 4% dual reporter^high cells was reported in Figure 3—figure supplement 1 when performing di-genic targeting at CCR5 and HBB, giving a false positive rate of 6% of targeting. (b) Left, To determine false-positive frequencies of tri-genic targeting in HSPCs, we electroporated IL2RG-RNP and HBB-RNP into HSPCs followed by transduction with the rAAV6 donors IL2RG-GFP (homologous), HBB-tdTomato (homologous), and CCR5-tNGFR (non-homologous). FACS plots show a false-positive frequency of 0.47%. Note that Figure 4d shows a tri-genic targeting frequency of 4.1% (a false-positive rate of 11% of targeting). Right, We employed a similar strategy to determine false-positive frequencies of tri-genic targeting, but this time used different combinations of on-target nucleases. The false-positive rate detected here was 0.1% (2.4% of targeting). (c) To determine tetra-genic false-positive frequencies, we electroporated HSPCs with three on-target nucleases (IL2RG, HBB, and CCR5) and then transduced with three homologous rAAV6 donors (IL2RG, HBB, and CCR5) and one non-homologous donor (CXCL12). FACS plots show a frequency of 0.09% that are reporter^high for all four reporters with Figure 4f showing a tetra-genic targeting frequency of 1.0% (a false-positive rate of 9% of targeting).

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 followed by a cassette encoding a reporter gene (here GFP). WT cDNA is expressed from the endogenous promoter as reported by Voit et al. (2014), Hubbard et al. (2016), and Dever et al. (2016), which maintains endogenous regulatory control over gene expression. The other donor encodes another reporter (here BFP), which disrupts the targeted gene. Double positive cells (GFP⁺/BFP⁺) are heterozygous for the knockout allele. (b) A population heterozygous for a particular SNP can be generated using two donors that knock in cDNA expression cassettes followed by different reporter genes. One cDNA is WT while the other carries the SNP of interest. Double positive cells (GFP⁺/BFP⁺) are heterozygous for the SNP allele. Endogenous 3’ UTRs may be incorporated to preserve posttranscriptional regulation. Heterozygous SNP cDNA knock-in may be expanded to two or more genes, which may be of particular interest in studies of leukemia-mutated genes such as DNMT3A, IDH1/2, JAK2, and KRAS, which often occur in various combinations as heterozygous gain-of-function or dominant negative mutations. In addition, reporter knock-in combined with WT cDNA knock-in (as depicted in a) as well as SNP cDNA knock-in (SNP that disrupts gene or gene function) combined with WT cDNA knock-in (as depicted in b) could be used to study haploinsufficiencies. Though not depicted, all genes in the schematic are followed by polyadenylation signals.