Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia

ARTICLE Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia Sachith Mettananda 1,2, Chris A. Fisher1, Deborah Hay1, Mohsin Badat1, Lynn Quek1, Kevin Clark3, Philip Hublitz3, Damien Downes1, Jon Kerry1, Matthew Gosden1, Jelena Telenius1, Jackie A. Sloane-Stanley1, Paula Faustino4,5, Andreia Coelho4, Jessica Doondeea1, Batchimeg Usukhbayar1, Paul Sopp3, Jacqueline A. Sharpe1, Jim R. Hughes 1, Paresh Vyas 1,6, Richard J. Gibbons1& Douglas R. Higgs 1,6 β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between αand β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia. DOI: 10.1038/s41467-017-00479-7 OPEN 1Medical Research Council (MRC) Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. 2Department of Paediatrics, Faculty of Medicine, University of Kelaniya, Ragama 11010, Sri Lanka. 3MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. 4Human Genetics Department, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, Lisbon 1649-016, Portugal. 5Institute of Environmental Health, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal. 6Oxford National Institute for Health Research Biomedical Research Centre, Blood Theme, Oxford University Hospital, Oxford OX3 9DU, UK. Richard J. Gibbons and Douglas R. Higgs contributed equally to this work. Correspondence and requests for materials should be addressed to D.R.H. (email: doug.hig[email protected]) NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications 1 Thalassemia is a disorder of hemoglobin synthesis characterized by severe anemia, which requires intensive supportive treatment from early childhood1. The most common and severe form of this disease (β-thalassemia) results from an absent or reduced production of normal β-globin chains2. Most aspects of the pathophysiology of β-thalassemia can be explained by the presence of excess α-globin chains, which can no longer pair with the reduced numbers of β-globin chains, found in patients with β-thalassemia, to produce normal hemoglobin tetramers (α 2 β 2 ). Excess α-globin chains precipitate both in red cell precursors (causing ineffective erythropoiesis) and mature red cells (causing hemolysis). Clinical and genetic data have clearly shown that when α-thalassemia is co-inherited with β-thalassemia, there is reduced expression of the α-globin genes, less globin chain imbalance, and reduced numbers of free α-globin chains, significantly ameliorating the clinical severity of β-thalassemia3.By contrast, inheritance of a higher than normal number of α-globin genes (5 or 6 rather than 4) substantially worsens the disease phenotype, emphasizing that excess α-globin chains are the major determinant of the clinical severity of β-thalassemia4,5. The human α-globin gene locus is situated in the short arm of chromosome 16 with two copies of the α-globin gene on each chromosome (αα/αα). Expression of the α-globin genes is controlled by four enhancers (MCS-R1 to R4) located 10–50 kb upstream of the genes6,7. Previous studies, including transgenic experiments combined with observations of naturally occurring mutations, have shown that a multi-species conserved sequence, which lies 40 kb upstream of the α-globin locus (MCS-R2, also known as HS-40), is the most powerful enhancer of α-globin gene expression8–10. We have previously characterized MCS-R2 in detail and shown that its activity is contained within a ~260 bp core fragment, including several well-conserved erythroid transcription factor binding sites11. A 1.1 kb deletion removing MCS-R2 in a humanized mouse model has been shown to result in a significant reduction of human α-globin expression9. More recently, Coelho et al.12 reported a patient homozygous for a rare 3.3 kb deletion, which uniquely removes MCS-R2 and results in a significant downregulation of α-globin gene expression. Thus we hypothesized that if a targeted mutation of MCS-R2 was to be created it should result in a reduction of α-globin expression to levels beneficial to patients with β-thalassemia. Genome editing using the CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR-associated protein 9) system provides a realistic approach to the treatment of human genetic diseases including hemoglobinopathies13. These nucleases create double-strand breaks at specific, chosen locations in the genome and, when repaired, create mutations at the targeted sites14. Although CRISPR/Cas9 can be used to promote either homology-directed recombination (HDR) or non-homologous end joining, hematopoietic stem cells (HSC) are currently largely limited to the latter form of editing since, despite some recent developments15, HDR remains an inefficient process in HSCs16. This means that deleting and inactivating an enhancer is currently a much more tractable approach to ameliorating β-thalassemia than directly repairing the β-globin gene or removing the α-globin genes by HDR. Here we show the use of CRISPR/Cas9 genome editing technology to create a targeted mutation of the MCS-R2 core element in human HSCs to mimic the effects of natural mutations, which knockdown α-globin expression. We demonstrate successful knockdown of α-globin expression in vitro in erythroid cells generated by genome-edited HSCs to levels beneficial in β-thalassemia without perturbing erythroid differentiation or having detectable off-target events. Finally, we show that this form of engineering occurs in long-term repopulating HSC (LT-HSC), the cell population currently used in clinical practice for HSC therapy in blood diseases, demonstrating the potential of this approach for translation into a therapy for β-thalassemia. Results A natural deletion that uniquely removes MCS-R2 enhancer. A previously reported patient (MC) is homozygous for a very rare 3.3 kb deletion (called (αα)ALT), which uniquely removes MCS-R2 but leaves the α-globin genes and all other enhancer elements intact12. MC is a member of a pedigree (Fig. 1a) that originates from Portugal and was evaluated in greater detail (Supplementary Table 1). Hematological analysis confirmed that MC has hypochromic microcytic anemia (Figs. 1b, c); 51% of his peripheral blood red blood cells (RBC) were positive for hemoglobin H (HbH) inclusion bodies confirming a diagnosis of α-thalassemia leading to HbH disease (Fig. 1d). α-Globin messenger RNA (mRNA) levels of MC’s peripheral blood are reduced and multiplex ligation-dependent probe amplification analysis12 confirmed the absence of MCS-R2 in its natural genomic location, but did not exclude the possibility of its presence in an ectopic locus. To examine this we performed Southern blot analysis using a probe specific to MCS-R2, which confirmed the absence of the MCS-R2 region anywhere in the genome (Fig. 1e). Interestingly, in the (αα)ALT mutation, the 3.3 kb deletion is associated with the insertion of a 39 nucleotide orphan segment, whose sequence was not found anywhere in the human genome. Our search for canonical binding motifs for both general transcription factors, and erythroid-specific factors within this orphan sequence did not reveal motifs with any known regulatory potential. Next, we examined the presence of histone 4 acetylation (H4Ac) or binding of the stem cell leukemia (SCL) complex, which are signatures of active chromatin within this orphan insert sequence. Chromatin immunoprecipitation (ChIP-qPCR) across this sequence confirmed the absence of enrichment for these activating chromatin marks (Figs. 1f, g). To assess whether the function of MCS-R2 was assumed by any other region within or beyond the α-globin cluster, we undertook ChIP-Seq (using an anti-pan-H4 acetylation antibody) to give the greatest likelihood of detecting any newly activated chromatin (Fig. 1h). No new putative regulatory elements were detected, with the only additional finding of note being the loss of a single H4Ac peak at the HBM promoter. This corresponds to a short deletion in the region of the HBM promoter, confirmed by Southern blot. This region does not contribute to normal α-globin transcription17 and its loss is not associated with any expected reduction in α-globin levels. These data support experimental studies in the mouse18, which define MCS-R2 as the major (but not the only) regulatory element controlling expression of α-globin RNA. Its loss from the natural chromosomal environment results in a significant reduction of α-globin transcription in cis. The clinical phenotypes of thalassemia trait in the heterozygous patient RC and of HbH disease in patient MC, who carries this deletion in homozygosity, demonstrate that deletion of MCS-R2 causes a significant (>50%), but not total reduction in α-globin expression in both homozygous and heterozygous states. However, of importance for this study, the absence of MCS-R2 in the homozygous state did not result in any other phenotypic abnormalities in MC, demonstrating that its unique functional significance is confined to expression of the α-globin locus. Targeted in vitro deletion of MCS-R2 to knockdown α-globin. Next, we hypothesized that creating a targeted deletion of MCS-R2 would phenocopy the effects seen in MC and RC in homozygous and heterozygous states, respectively. Thus the removal of the ~260 bp core element of MCS-R2 in human ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 2NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications erythroid cells should result in a reduction in α-globin expression to levels previously shown to be beneficial in patients with β-thalassemia. To do this, we designed several short guide RNAs (sgRNA) complementary to target sequences both upand downstream of the MCS-R2 core element (Fig. 2a; Supplementary Table 2) to generate CRISPR plasmid pairs to use in combination to create two double-strand breaks at either end of the enhancer. When repaired this should introduce targeted deletions. These sgRNAs were cloned into a plasmid vector (Addgene plasmid 48138: pSpCas9(BB)-2A-GFP (pX458)) that contains a gene 0 10 20 30 40 50 60 RHBDF1 ALT insert MCS-R1 Alpha promoter Beta actin promoter Enrichmet relative to input SCL ab c Control Control RC MC e MC RC Normal reference range Haemoglobin (g/dl) 9.5 12.9 Male:13.5–17.5 Female: 12.0–16.0 Mean corpuscular volume (fl) 65 75 80–100 Mean corpuscular hemoglobin (pg) 19.9 24.5 26–34 d fg MC RC Ch 16 ΔMCS R2 (homozygous) WT ENCODE UW Digital Genomic Footprinting (Raw, K562) 0 1 2 3 4 5 6 7 8 9 10 RHBDF1 ALT insert MCS-R1 Alpha promoter Beta actin promoter Enrichment relative to input H4 acetylation 23Kb 9.4Kb 6.5Kb 2.3Kb 2.0Kb h NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 ARTICLE NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications 3 encoding green fluorescent protein (GFP) fused to a Cas9 gene. We then co-transfected primary human CD34+ cells, obtained from normal, non-thalassemia controls, with pairs of CRISPR plasmids (Supplementary Fig. 1). The live-transfected cell populations were sorted by GFP expression (Supplementary Fig. 2; Fig. 2b) and screened for the predicted mutations. Through this preliminary screening we identified four optimal CRISPR plasmid pairs, which generated targeted deletions at the highest frequencies (Supplementary Fig. 3). Deletion efficiency of these CRISPR pairs was verified by two independent methods and was consistently over 70% when using three out of four pairs (Figs. 2c, d). The accuracy of editing was then determined by Sanger sequencing (Fig. 2e). Next we differentiated these transfected CD34+ cells along the erythroid lineage to evaluate globin gene expression. As expected, and similar to the observations made in MC, with all four CRISPR plasmid pairs, deletions of MCS-R2 (ΔMCS-R2) resulted in selective reduction in α-globin expression without altering the expression of the β-globin gene (Fig. 2f). Single-cell assays confirm α-globin knockdown by ΔMCS-R2. Next, to precisely dissect the effects of CRISPR/Cas9-mediated ΔMCS-R2, we performed single-cell assays using Terasaki multiwell plates, which were superior to the traditional methylcellulose-based colony assays in minimizing crosscontamination between clones. We transfected CD34+ cells with a pair of CRISPR plasmids (Cr2 + Cr12) and then sorted these cells using flow cytometry to generate single cells in individual wells. Following erythroid differentiation, individual clones were assayed for genotype and globin gene expression, which enabled us to determine the correlations between the dosages of mutant alleles and globin gene expression, and confirm the editing efficiency. Genotype analysis of these clones by PCR and sequencing revealed a variety of editing outcomes not only limited to the expected mutations resulting in a mixture of different genotypes, which included MCS-R2 non-deletions (wild type), heterozygous and homozygous ΔMCS-R2 mutations, MCS-R2 inversions, and unpredicted mutations (Figs. 3a, b; Supplementary Fig. 4). Analysis of globin gene expression of individual erythroid clones demonstrated significant reductions in α/β-globin mRNA ratios in clones with heterozygous (median 36% compared to normal) and homozygous (median 3% compared to normal) ΔMCS-R2 mutations (Fig. 3c) thus confirming the selective downregulation of α-globin by deletion of MCS-R2. The α/β-globin mRNA ratios in clones with inversions of the MCS-R2 were similar to those without mutations (Fig. 3d). Similar results were observed in single-cell clone analysis of CD34+ cells transfected with another independent CRISPR pair (Cr1 + Cr8) thus corroborating the results (Supplementary Figs. 5and 6). Genome-edited cells have no detectable off-target effects.To explore any off-target effects of MCS-R2 deletion by CRISPR, we identified potential genomic off-target loci for the four sgRNA (Cr1, Cr2, Cr8, and Cr12) using the Sanger off-target prediction tool (Supplementary Table 3). Sequencing of the DNA from genome-edited clones did not identify any off-target activity at these sites (Fig. 3e; Supplementary Fig. 5; Supplementary Table 4) confirming the specific on-target activity of the CRISPR plasmids used here. To examine the effect of deleting MCS-R2 on the differentiation of CD34+ cells following genome editing, we cultured the edited cells in conditions that favor the erythroid lineage. Morphological analysis of stained cytospins and immunophenotypic characterization using erythroid-specific cell surface markers were performed. CRISPR-edited cells demonstrated the same patterns of differentiation as unedited control cells (Supplementary Fig. 7). Genome-edited HSCs retain long-term repopulating ability.If genome editing of MCS-R2 is to become a realistic therapeutic approach in β-thalassemia, targeted mutations must be produced in LT-HSC. Human CD34+ cells are used in all current stem cell transplantation protocols for blood diseases, but they are known to be a heterogeneous collection of hematopoietic progenitors and rare LT-HSCs (so called HSCs;<0.5% of total nucleated bone marrow cells). To exclude the possibility that genome editing occurred in progenitors but not LT-HSCs, CRISPR-transfected CD34+ cells were injected into the bone marrows of four sub-lethally irradiated female NSG mice (80,000 cells per mouse) and their bone marrow was harvested after 12 weeks (Figs. 4a, b). All four mice demonstrated long-term multi-lineage (lymphoid and myeloid) engraftment (range 0.8–34% human leukocytes), and three had particularly high levels (27–34% hCD45+) (Fig. 4c; Supplementary Fig. 8). Human CD45+ cells obtained from these bone marrows were sorted and DNA was screened for the predicted edits from these three mice: two had genome-edited cells of which one (xenograft #3) had an edited allele frequency of 71% (Fig. 4d). Although not found in the in vitro analyses, in this analysis we found a slightly larger deletion (271 bp instead of 241 bp) presumably due to exonuclease cut back from the CRISPR/Cas9 cut sites (Fig. 4e). Nevertheless, this deletion removes the key transcription factor binding sites as in the experimental plan. Flow cytometry analysis of harvested bone marrow of xenograft #3 mice showed that 15.5% of all human CD45+ cells were positive for human CD34+. To determine if we had successfully edited LT-HSC, we performed serial, secondary transplantation with hCD45+ cells from the primary engrafted mice (100,000 cells per mouse, n=3). We detected multi-lineage engraftment of hCD45+ cells in 2/3 mice (range 29.7–36.9% human leukocytes). We also performed Fig. 1 Characterization of a rare natural mutation ((αα)ALT) confined to MCS-R2. aKindred showing patient (MC) homozygous for the (αα)ALT mutation and his heterozygote daughters. bHemoglobin level and red cell parameters of MC and RC (who is heterozygous for the (αα)ALT mutation). cPeripheral blood smear, Giemsa stained, from patient MC, which shows anisocytosis and poikilocytosis with some irregularly contracted cells; scale bar represents 10 μm. dPeripheral blood Hemoglobin H (HbH) preparation, brilliant cresyl blue stained, from patient MC, which shows HbH inclusions; scale bar represents 10 μm. eSouthern blot analysis using a probe specific for the core of MCS-R2, which gives an 19 kb band on BglII digest genomic DNA from normal controls and RC but not from MC confirming the absence of this segment. f,gAnalysis of enrichment of histone 4 (H4) acetylation fand SCL gby ChIP-qPCR in in vitro differentiated primary erythroid cells (Fibach culture35) of MC harvested at intermediated erythroblasts stage (day 8–10 of Phase 2). The y-axis represents enrichment relative to ChIP input, normalized to 18S control region. Mean values are presented and error bars represent SD (n=3). Amplicons are labeled thus: RHBDF1, intronic amplicon within the Rhomboid gene RHBDF1, used as a negative control; ALT insert, amplicon specific for 39 bp insert; MCS-R1, the human MCS-R1 which is one of the other enhancers of α-globin; alpha-promoter, α-globin promoter; beta actin promoter, a control amplicon over the β-actin promoter. hAnalysis of the abundance of panH4 acetylation across the α-globin locus by ChIP-seq in in vitro differentiated primary erythroid cells harvested at intermediated erythroblasts stage from patient MC (lower track) and a normal control (upper track), showing the absence of the peak over MCS-R2 in patient MC (red dashed box) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 4NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications methylcellulose colony assays with CD34+ cells harvested from the secondary xenograft, which again demonstrated the presence of granulocyte/macrophage and erythroid progenitor activity (Fig. 4f). Sanger sequencing of 40 colonies (20 erythroid and 20 myeloid) confirmed deletion of MCS-R2. These data confirm that we have successfully edited LT-HSC, which are able to reconstitute myeloid, lymphoid, and erythroid hematopoiesis. 0% 20% 40% 60% 80% 100% Cr1+8 Cr2+12 Cr9+12 Cr10+12 PCR band intensity Droplet digital PCR 0 0.2 0.4 0.6 0.8 1 1.2 1.4 NTC C9 Cr1+8 Cr2+12 Cr9+12 Cr10+12 α-globin β-globin α/β-globin ratio a Human chromosome 16 10 Kb α1α2 ζ MCS-R1 MCS-R4 MCS-R3 MCS-R2 50 bp 258 bp MCS-R2 core element sgRNA target sites cr9 cr12 cr7 cr2 cr8 cr10 cr1 GATA1 binding motif NF-E2 binding motif Globin genes cis-acting regulatory elements b 100 500 400 300 200 600 100bp ladder C9 Cr2+12 Cr10+12 Cr9+12 Cr1+8 WT 100 bp ladder c CRISPR efficiency f ** * * * * * ** Relative expression d FS NTC 105 104 103 102 105 104 103 102 105 104 103 102 105 104 103 102 105 104 103 102 105 104 103 102 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 C9 Cr1+8 Cr2+12 Cr10+12Cr9+12 0±0% 49±6%54±10%51±4% 56±10%60±9% e 27±5%25±4%29±3% 22±8%24±3%100±0% cr1 cr8 5’ cr2 cr10 cr9 cr12 113496 113497 / 113808 113808 cr1 cr8 310+311 bp deletion 113511 113753 cr2 cr12 241 bp deletion 113444 113752 cr12 cr12 cr9 cr10 307 bp deletion 113499 113752 252 bp deletion GFP NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 ARTICLE NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications 5 ΔMCS-R2 restores globin balance in β-thalassemia cells. Finally, to examine the ability of the edited deletion of MCS-R2 to rectify globin chain imbalance in the erythroid cells of patients with β-thalassemia, we performed single colony analysis on CRISPR-edited CD34+ cells obtained from patients with HbE β-thalassemia (Supplementary Table 5). Genome editing of CD34+ cells obtained from patients with HbE β-thalassemia produced a mixture of different genotypes similar to those observed in normal controls with comparable mutation frequencies (Supplementary Fig. 9; Fig. 5a). As expected, the α/β globin mRNA ratios in unmodified erythroid cells from patients with HbE β-thalassemia were higher than α/βglobin mRNA ratios in unmodified, normal erythroid cells. However when a heterozygous ΔMCS-R2 mutation was edited into the HbE β-thalassemia cells, the α/βglobin mRNA ratio decreased and many individual clones had α/βglobin mRNA ratios similar to normal control clones (Fig. 5b). When both alleles at MCS-R2 were edited in cells derived from patients with HbE β-thalassemia, the α/βglobin ratios were rebalanced and many individual clones had α/βglobin mRNA ratios similar to those found in normal control cells (Fig. 5b). Analysis of α/all β-like globins (α/(α+γ)) ratios demonstrated similar trends (Fig. 5c). These observations confirm that editing of MCS-R2 can efficiently restore globin chain balance in erythroid cells in patients with β-thalassemia and we predict that this would result in phenotypic improvements and improved survival of genome-edited cells with reduced levels of erythroid cell destruction and anemia in vivo. Discussion The clinical management of β-thalassemia still largely depends on supportive treatment with RBC transfusions and iron chelation in the majority of patients1. Allogenic bone marrow transplantation remains the only curative treatment; however its usefulness is limited to a minority of patients who have HLA-matched sibling donors19. Several new therapies for β-thalassemia are currently being investigated20. Except for a few studies21 all of these new and experimental therapies aim to resolve globin chain imbalance by increasing the production of γ-globin and fetal hemoglobin by genome editing of transcription factors22,23 (eg: BCL11A24 and LRF/ZBTB7A25) or by pharmacological methods (eg: histone deacetylase inhibitors26 and lysine-specific demethylase 127). Alternatively, here we present a novel approach by directly reducing expression of α-globin. Considering the central role of excess α-globin chains in the pathophysiology of β-thalassemia, and the extensive clinical evidence showing how co-inheritance of α-thalassemia ameliorates β-thalassemia, this presents an extremely promising approach to curing this disease until efficient gene correction of the defective β-globin by HDR in stem cells becomes routinely possible. This approach can be used on its own or to complement other on-going efforts of increasing the production of fetal hemoglobin. We have previously summarized the findings from several clinical studies showing that a reduction of α-globin expression to 75–25% of normal is an effective, safe, and tolerable level to provide sustainable beneficial effects in patients with β-thalassemia3. In genome-edited normal control cells many of the clones with heterozygous or homozygous deletions of MCS-R2 demonstrated reduction of the α/βratio to this desirable range. In cells with HbE β-thalassemia mutations, targeted deletion of MCS-R2 in both heterozygous and homozygous states resulted in amelioration of the disturbed globin ratios. In this paper we have demonstrated the use of CRISPR/ Cas9 system for genome editing of MCS-R2, the major α-globin enhancer, in human primary LT-HSCs. CD34+ cells, including LT-HSCs, are readily obtainable at a clinical scale and are currently used in all stem cell transplantation protocols for blood diseases. Thus the hematopoietic system offers the ideal model for translation of genome editing for clinical benefit, and successful genome editing of lymphoid cells using a zinc-finger nucleasebased technique has already progressed to clinical trials28. Translation of our strategy into clinical application would require a harvest of CD34+ cells from patients with β-thalassemia, ex vivo genome editing, and an autograft. This would therefore avoid the potentially life-threatening complications of graft rejection and graft versus host disease associated with allografting for patients with hemoglobinopathies. Using our protocol, we currently achieve transfection efficiency as high as 75% and in transfected cells the editing efficiency was 70–80%, providing an overall editing rate of 50–60% of live CD34+ cells. Previously, it has been shown that chimerism levels of only 10–20% of normal HSCs are sufficient to result in nearly complete hematologic and pathologic correction of β-thalassemia29,30. Therefore, using our strategy, we should be able to provide sufficient numbers of genome-edited HSCs to produce a clinically significant beneficial effect in patients with β-thalassemia. One potential limitation of this strategy is that deletion of MCS-R2 could lead to profound reduction in α-globin expression thus causing a critical decrease in total globin synthesis in some edited cells. However, it is likely that erythroid cells with more balanced globin chain synthesis generated from genome-edited HSCs will have less ineffective erythropoiesis and hemolysis and remain in the circulation for the full life-span thus demonstrating selective advantage for survival in vivo. Furthermore, genome engineering of the MCS-R2 enhancer could be used to fine-tune the level of α-globin expression by limiting the edit to already identified transcription factor binding sites within the enhancer. Therefore, the issue of excessive downregulation of α-globin need not be a significant barrier during future clinical use. Xenograft assays performed here show that CRISPR/Cas9 genome editing is not limited to progenitor cells but also occurs in LT-HSCs. These are the cells, which must be edited if a sustainable improvement is to be achieved in patients with β-thalassemia. We have shown that edited HSCs give rise to multiple lineages of hematopoietic cells in vivo. In the xenograft Fig. 2 Deletion of MCS-R2 using CRISPR/Cas9 genome editing in human CD34+ cells. aSchematic of sgRNA target sites. Four sgRNAs (Cr1, Cr2, Cr9, and Cr10) were designed to target the 5′end of the MCS-R2 core element, whereas three sgRNAs (Cr7, Cr8, and Cr12) target the 3′end. bRepresentative flow cytometry plots showing GFP expression and forward scatter (FS) after gating for live cells in non-transfection control (NTC), Cas9 control (C9), and CRISPR/Cas9 plasmid pair-transfected cells. Orange: GFP negative, blue: GFP positive (low), and green: GFP positive (high). Mean and SD of the percentage of cells within each region is indicated (n=3). Gating strategy is shown in Supplementary Fig. 10a. cRepresentative gel electrophoresis image of genomic DNA extracted from cells targeted by four CRISPR/Cas9 plasmid pairs analyzed by PCR. dGene editing deletion induction efficiency as measured independently by percentages of mutated alleles determined by band size in end-point PCR and subsequent Sanger sequence analysis and by determining inverse of proportions of amplicons inside: outside deletion (amplicons of same length) by multiplexed droplet digital PCR; mean values are presented and error bars represent SD (n=3). eCharacterization of deletion break points by sequencing (co-ordinates from Hum Mar 2006 (NCBI36/hg18) assembly). fα-and β-globin gene expression normalized to the expression of RPL13A and α/β-globin mRNA ratios relative to Cas9 control (C9) analyzed by qPCR; error bars represent SD (n=3); *P<0.05 and **P<0.01 relative to C9 (Student’st-test). C9 Cas9-only control ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 6NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications R2 non-del Het R2 del (241 bp) Hom R2 del (241 bp) 0.0 0.1 0.2 0.3 1 2 3 4 a 100 500 200 1000 WT ΔR2 *** * *** * Key: R2 non-deleted Heterozygous ΔR2 (241 bp) Homozygous ΔR2 (241 bp) Unpredicted WT ΔR2 100 500 200 1000 51 605958575655545352 61 706968676665646362 71 747372 100 bp ladder 100 bp ladder 75 848382818079787776 85 949392919089888786 95 989796 b Mutation type R2 non-deleted 6 (13%) Heterozygous ΔR2 (241 bp) 13 (28%) Homozygous ΔR2 (241 bp) 21 (45%) Unpredicted Frequency (%) (N=47) 4 (8%) Inversions 3 (6%) α/β globin mRNA ratio c *Inversion ********* ***** *** ********* **** *** ***** ** * * * P=0.09 P<0.0001 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 R2 non-del Het R2 del (241 bp) Hom R2 del (241 bp) R2 inversion d α/β globin mRNA ratio 1 2 4 3 56 7 9 8 Condition (potential off-target sites in Cr2 and Cr12 pools) Control (same genomic locations, in Cr1 and Cr8 pools, where they are not potential off-targets) e10,000 8000 6000 4000 2000 0 –2000 –4000 0 50 100 150 200 Fig. 3 Single-cell clone analysis of targeted deletion of MCS-R2. aGel electrophoresis image of genomic DNA from 48 individual single-cell clones genomeedited using CRISPR pair Cr2 and Cr12 (from three biological independent donors) analyzed by PCR. The amplicon from the wild-type allele is 613 bp and the mutated amplicon is 372 bp. Clones are numbered 51–98 and clone 59 failed to amplify. Extended genotype analysis of these clones by sequencing is presented in Supplementary Fig. 4.bFrequency of different types of mutations generated. cα/β-globin mRNA ratios of individual clones of erythroid cells which are non-deleted (R2 non-del) (n=6) and heterozygous (Het R2 del.) (n=13) or homozygous (Hom R2 del.) (n=21) for a 241 bp deletion of MCSR2 region analyzed by qPCR; median (horizontal bar) and 95% confidence interval (error bar) are shown and P-values were calculated using Mann–Whitney U-test. dα/β-globin mRNA ratios of erythroid cells, which has no deletion (n=6), heterozygous deletion (n=13), homozygous deletion (n=21), and inversion (n=3) of MCS-R2 analyzed by qPCR. Means and SEM are shown. eMeta-plot of all off-target loci for CRISPR pair Cr2 and Cr12. All captured sites are plotted on the same x-axis, showing ±100 bases from each potential off-target site. Counts deviating from the reference sequence, which are normalized to 10,000 counts are plotted in the y-axis. The values are means of the libraries where each library is a pool of five independent clones. The shaded violet area denotes ±1000 counts and only data over this threshold were considered as off-target. Error bars represent SEM for each base at each locus. Potential off-target hits for Cr2 and Cr12 (condition) are plotted alongside those of a control group (control). The numbers are annotated in Supplementary Table 4. All of the variations from the reference sequence were shown to be known SNPs or indels or novel variations common to both control and condition and therefore unrelated to potential off-target activity NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 ARTICLE NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications 7 GFP a b 53% 0.1%0.7% 46% Test 1-live cells c Forward scatter hCD45 APC 27% 27% 34% 0.8% d Marker Un-edited Human control Non-xenograft mouse control 100 500 400 300 200 600 700 WT % Deletion 0 7810 In-vitro edited Human control Xenograft #1 Xenograft #4 Xenograft #3 Xenograft #2 Non-template control ΔR2 7100 Cord blood CD34 selection CRISPR transfection Sort CD34+/GFP+ cells Transplant to irradiated NSG mice Bone marrow harvest 12 weeks Xenograft 3 Xenograft 4 Xenograft 1 Xenograft 2 ef Colony morphology Erythroid 292 (30%) Granulocyte/ macrophage 605 (63%) Mixed 66 (7%) Frequency (%) (N=963) 5’ 3’ 271 bp deletion 113510 113782 Secondary transplant to irradiated NSG mice Bone marrow harvest CD34 APC 105 104 103 102 50 100 150 FSC-A (× 1000) 200 250 50 100 150 FSC-A (× 1000) 200 250 50 100 150 FSC-A (× 1000) 200 250 50 100 150 FSC-A (× 1000) 200 250 103 104 105 102 103 104 105 Specimen_001-1 Specimen_001-3 Specimen_001-4 Specimen_001-2 103104105 0 –758 –542 0 M 640–670/30-A CD45 APC 640–670/30-A 102 103 104 105 102 103 104 105 CD45 APC 640–670/30-A CD45 APC 640–670/30-A CD45 APC 640–670/30-A 488–530/30-A 12 weeks Fig. 4 Xenograft assay. aSchematic of the workflow of mouse xenograft experiment. bFlow cytometry analysis of cells following CRISPR transfection demonstrating transfected (positive for GFP) CD34+ HSPCs. Gating strategy is shown in Supplementary Fig. 10b. cFlow cytometry plots of harvested bone marrow from xenograft mice gated for live cells demonstrating hCD45 expression. Gating strategy is shown in Supplementary Fig. 10c. dGel electrophoresis image of genomic DNA extracted from human cells obtained from xenograft mouse analyzed by PCR demonstrating genome-edited bands. eCharacterization of deletion break points by sequencing (co-ordinates from Hum Mar 2006 (NCBI36/hg18) Assembly) fMorphological analysis of hematopoietic colonies grown in methyl cellulose which were generated by HSCs harvested from secondary transplant mice (two independent mice) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 8NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications assays we observed a single, slightly larger deletion in mice that had genome-edited cells. This suggests highly oligo or monoclonal reconstitution that may have resulted from a limited number or even a single LT-HSC. This is not entirely surprising as LT-HSCs are rare within the CD34+ population of cells and this does not detract from the conclusion that the editing procedure does result in the engineering of this population of cells. A xenografted mouse is not a suitable model in which to study erythropoiesis from transplanted cells and therefore we were not able to analyze the effect of ΔMCS-R2 on globin gene expression in vivo. However, there is no reason to think that expression in vivo will be any different from that seen in erythroid cells differentiated from CD34+ cells in culture. An important consideration is whether editing a major enhancer would have effects on any other features of erythropoiesis or in other tissues. We addressed this by detailed analysis of a family with a rare natural deletion of MCS-R2. The two affected individuals with heterozygous and homozygous mutations of MCS-R2 are healthy with no other obvious phenotypes except for anemia validating MCS-R2 as a safe region of the genome to be targeted by genome editing. Furthermore, detailed evaluation of predicted off-target effects of genome editing tools used in our study did not reveal any off-target activity and the effects were specific to the target loci. In conclusion, we have shown that it is feasible to edit LT-HSCs and to inactivate an enhancer, which specifically and uniquely regulates α-globin expression. Importantly, this reduces α-globin expression to the levels known to be required to achieve beneficial effects in patients with β-thalassemia. These findings open up a new approach to ameliorate this life-limiting disease and provide useful insights into plausible therapeutic approaches to human genetic diseases in general. Methods Cell culture. Human umbilical cord blood and adult peripheral blood buffy coat residues were purchased from the National Health Service (NHS) Blood and Transplant, UK. Samples from β-thalassemia patients were collected after obtaining informed written consent. Ethical approval for the study was granted by North West Research Ethics Committee of NHS National Research Ethics Services (reference no. 03/08/097). Mononuclear cells were separated using Histopaque1077 Hybri-Max (Sigma), the CD34+ cells were purified using a CD34 MicroBead Kit (Miltenyl Biotech Cat. 130-046-702) and purity confirmed by FACS. Primary CD34+ cells were not tested for mycoplasma contamination. Then the CD34+ cells were differentiated into erythroid cells over 21 days using a two-phase liquid culture system which used StemSpan SFEM II (Stemcell Technologies). Phase 1 medium was supplemented with stem cell factor, interleukin-3, human recombinant erythropoietin (EPO) (0.5 U/ml) and cholesterol-rich lipids. Phase 2 medium was similar to phase 1 medium except for addition of iron saturated holotransferrin and higher concentration of EPO (3 U/ml). In single-cell experiments, single cells were sorted into the wells in Terasaki multiwell plates and were cultured in 20 μl culture medium. CRISPR plasmids. Several sgRNA-targeting MCS-R2 were cloned into the Addgene plasmid 48138 (pSpCas9(BB)-2A-GFP (pX458)) backbone vector. Guides were cloned into the BbsI restriction site containing two Guanine residues at positions 0 and 1 of the protospacer, the sequence of the targeting guides are listed in Supplementary Table 2. Transfection of CD34+ cells. Transfection of CRISPR plasmid DNA into CD34+ cells was done using Amaxa human CD34+ cell nucleofector kit (Lonza) and Amaxa nucleofector I device (Lonza)31. Culture medium containing 3×10 5–1×10 6cells was spun at 1200 rpm for 5 min. The cell pellet was Control - R2 non-del. β-thal. - R2 non-del. β-thal. - Het R2 del. β-thal. - Hom R2 del. β-thal. - R2 non-del. β-thal. - Het R2 del. β-thal. - Hom R2 del. 0.0 0.5 1.0 2 4 6 8 10 a Mutation type (N=59) R2 non-deleted 14 (24%) Heterozygous ΔR2 (241 bp) 21 (36%) Homozygous ΔR2 (241 bp) 20 (34%) Unpredicted 4 (7%) Frequency (%) b α/β globin mRNA ratio P=0.0014 P=0.023 P=0.016 0.0 0.1 0.2 0.3 1 2 3 4 5 c α/(β+γ) globin mRNA ratio P<0.0001 P=0.19 P<0.0001 P=0.17 Fig. 5 Deletion of MCS-R2 in CD34+ cells from HbE β-thalassemia patients using Cr2 + Cr12. aFrequency of different types of mutations generated. bα/β-globin mRNA ratios of individual clones of erythroid cells which are non-deleted normal control (n=6), non-deleted HbE β-thalassemia (n=13), HbE β-thalassemia heterozygous for the deletion of MCS-R2 (n=20), and HbE β-thalassemia homozygous for the deletion of MCS-R2 (n=20) analyzed by qPCR; median (horizontal bar) and 95% confidence interval (error bar) are shown and P-values were calculated using Mann–Whitney U-test. cData for α/(β+γ) globin mRNA ratios for individual clones of HbE β-thalassemia erythroid cells grouped according to genotype and normalized to median of non-deleted HbE β-thalassemia clones; median (horizontal bar) and 95% confidence interval (error bar) are shown and P-values were calculated using Mann–Whitney U-test NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00479-7 ARTICLE NATURE COMMUNICATIONS |8: 424 |DOI: 10.1038/s41467-017-00479-7 |www.nature.com/naturecommunications 9