Abstract

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2,3,4,5,6. CRISPR–Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

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Data availability

The data that support the findings of this study are available within the paper and its supplementary information files. The deep sequencing data that support the findings of this study are publicly accessible from the National Center for Biotechnology Information Bioproject database with the accession number PRJNA517275, including the editing efficiency, pre- or post-mice-transplant data in Figs. 14 and the off-target assessment in Extended Data Fig. 6. The analytical results and statistics used to generate Figs. 14 and Extended Data Fig. 6 are provided in Supplementary Table 9. There are no restrictions on availability of the data from this study.

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Acknowledgements

We thank D. Chui for genetic analyses, N. Barteneva for imaging flow cytometry analysis, R. Mathieu for flow cytometry assistance, Z. Herbert for deep sequencing, and G. Menard and R. Rosales for help with hemoglobin HPLC. We appreciate useful discussions with J. Hsu, B. Croker, C. Lindsley, K. Holden, M. Hoban, M. Canver, and S. Orkin. This project was funded in part by the Translational Research Program at BCH. D.A.W. and C. Brendel were supported by NHLBI (grant no. U01HL11772) and D.A.W. and E.B.E. by NHLBI (grant no. R01HL137848). The trial for SCD HSPC procurement was supported by research funding from bluebird bio to A.B. L.P. was supported by NHGRI (grant no. R00HG008399). S.A.W. was supported by NIAID (grant no. R01AI117839) and NIGMS (grant no. R01GM115911). D.E.B. was supported by NIDDK (grant nos. K08DK093705 and R03DK109232), NHLBI (grant nos. DP2OD022716, P01HL053749 and P01HL032262), Harvard Stem Cell Institute Seed Grant, St. Jude Children’s Research Hospital Collaborative Research Consortium, Burroughs Wellcome Fund, American Society of Hematology, and the Doris Duke Charitable, Charles H. Hood, and Cooley’s Anemia Foundations.

Author information

Author notes

  1. These authors contributed equally: Yuxuan Wu, Jing Zeng.

Affiliations

  1. Division of Hematology/Oncology, Boston Children’s Hospital , Boston, MA, USA

    • Yuxuan Wu
    • , Jing Zeng
    • , Qiuming Yao
    • , Mitchel A. Cole
    • , Cristina Baricordi
    • , Anne H. Shen
    • , Chunyan Ren
    • , Erica B. Esrick
    • , John P. Manis
    • , David A. Williams
    • , Alessandra Biffi
    • , Carlo Brugnara
    • , Luca Biasco
    • , Christian Brendel
    •  & Daniel E. Bauer
  2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Yuxuan Wu
    • , Jing Zeng
    • , Qiuming Yao
    • , Mitchel A. Cole
    • , Cristina Baricordi
    • , Anne H. Shen
    • , Chunyan Ren
    • , Erica B. Esrick
    • , John P. Manis
    • , David A. Williams
    • , Alessandra Biffi
    • , Carlo Brugnara
    • , Luca Biasco
    • , Christian Brendel
    •  & Daniel E. Bauer
  3. Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA

    • Yuxuan Wu
    • , Jing Zeng
    • , Qiuming Yao
    • , Mitchel A. Cole
    • , Cristina Baricordi
    • , Anne H. Shen
    • , Chunyan Ren
    • , Erica B. Esrick
    • , John P. Manis
    • , David A. Williams
    • , Alessandra Biffi
    • , Carlo Brugnara
    • , Luca Biasco
    • , Christian Brendel
    •  & Daniel E. Bauer
  4. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    • Yuxuan Wu
  5. Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA

    • Benjamin P. Roscoe
    • , Pengpeng Liu
    • , Kevin Luk
    •  & Scot A. Wolfe
  6. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA

    • Qiuming Yao
    • , Kendell Clement
    •  & Luca Pinello
  7. Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA

    • Qiuming Yao
    • , Kendell Clement
    •  & Luca Pinello
  8. Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    • Cicera R. Lazzarotto
    •  & Shengdar Q. Tsai
  9. Gene Therapy Program, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, USA

    • Cristina Baricordi
    • , Alessandra Biffi
    • , Luca Biasco
    •  & Christian Brendel
  10. Department of Pathology, Brigham and Women’s Hospital , Boston, MA, USA

    • David M. Dorfman
  11. Department of Pathology, Harvard Medical School, Boston, MA, USA

    • David M. Dorfman
  12. University College of London, Great Ormond Street Institute of Child Health, Faculty of Population Health Sciences, London, UK

    • Luca Biasco

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Contributions

D.E.B. conceived and supervised this study. D.E.B. and Y.W. designed the experiments. Y.W. and J.Z. performed all experiments in human CD34+ HSPC, RNP editing, human CD34+ HSPC transplant, and engraftment analysis. D.E.B., Y.W., and J.Z. analyzed data. B.P.R., P.L., K.L., C.R., and S.A.W designed and purified 3xNLS-SpCas9 protein. D.M.D. assisted with hemoglobin HPLC analysis. E.B.E., J.P.M., D.A.W., and A.B. helped obtain plerixafor-mobilized SCD CD34+ HSPCs. C. Brugnara helped obtain β-thalassemia CD34+ HSPCs. C. Baricordi and L.B. assisted with flow cytometry of HSPCs. C. Brendel contributed to xenotransplant experiments and flow cytometry. C.R.L. and S.Q.T. performed CIRCLE-seq experiments and analyzed data. Q.Y., K.C., M.A.C., A.H.S., and L.P. performed computational data analyses. D.E.B. and Y.W. wrote the manuscript. All of the authors contributed to editing the manuscript.

Competing interests

Y.W., J.Z., S.A.W., and D.E.B. have applied for patents related to therapeutic gene editing, including US Patent applications nos. 13/72236, 15/572,523, 18/34618, and 18/43073.

Corresponding author

Correspondence to Daniel E. Bauer.

Extended data

  1. Extended Data Fig. 1 Cas9 RNP dose-dependent editing of BCL11A enhancer for HbF induction in CD34+ HSPCs.

    a, Comparison of indel frequencies with in vitro transcribed (IVT), synthetic (syn) and modified synthetic (MS) sgRNAs in CD34+ HSPCs by TIDE analysis. b, Comparison of viability of CD34+ HSPCs edited with in vitro transcribed (IVT), synthetic (syn) and modified synthetic (MS) sgRNAs. c, Dose-dependent editing rates with Cas9 coupled with MS-sgRNA-1617 and -1639 targeting BCL11A enhancer and -e2 targeting BCL11A exon2 in CD34+ HSPCs by TIDE analysis. d, Comparison of indel frequencies with different molar ratios of Cas9 to MS-sgRNA in CD34+ HSPCs by TIDE analysis. e, Comparison of viability of CD34+ HSPCs edited with different molar ratios of Cas9 to MS-sgRNA. f, Percentage HbF+ cells by flow cytometry analysis in erythroid cells in vitro differentiated from CD34+ HSPCs edited by RNP coupled with various sgRNAs (each targeting BCL11A enhancer). Error bars indicate standard deviation (n = 3 replicates). g, Summary of deep sequencing data derived from the Cas9 RNP (coupled with MS-sgRNA-1617) edited CD34+ HSPCs. Asterisk indicates unedited allele. h, HbF induction by HPLC analysis in erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs. i, Genotyping and β-like globin expression analysis of clonal erythroid cells derived from single CD34+ HSPCs. Error bars indicate standard deviation (n = 3 technical replicates per colony). j, BCL11A expression in CD34+ HSPCs edited with Cas9 coupled with various MS-sgRNAs targeting BCL11A enhancer. Expression normalized to CAT, measured by RT–qPCR on day 11 of in vitro differentiation. Error bars indicate standard deviation (n = 3 replicates). k, Correlation of γ-globin mRNA expression determined by RT–qPCR versus HbF by HPLC. Black dots represent samples edited with 2xNLS-Cas9 coupled with various MS-sgRNAs. l, Correlation of BCL11A mRNA versus γ-globin mRNA determined by RT–qPCR. Black dots represent samples edited with 2xNLS-Cas9 coupled with various sgRNAs. m,n, Genotyping and HbF level by HPLC of clonal erythroid cells derived from single CD34+ cells from two independent healthy donors (βAβA#1 in m and βAβA#3 in n edited with MS-sgRNA-1617. o, Correlation of percentage γ-globin mRNA determined by RT–qPCR versus HbF by HPLC. Black dots represent single colonies edited with 2xNLS-Cas9 coupled with MS-sgRNA-1617. The Pearson correlation coefficient (r) is shown. In all panels, data are plotted as mean ± s.d. Data are representative of three biologically independent replicates.

  2. Extended Data Fig. 2 Indel frequencies from deep sequencing.

    a. Frequency distribution of alleles with and without indels (shown in blue and red, respectively) from deep sequencing of CD34+ HSPCs edited with 2xNLS-Cas9 RNP with indicated MS-sgRNAs targeting BCL11A enhancer. b, Correlation of indel frequencies by deep sequencing versus indel frequencies by TIDE analysis. The Pearson correlation coefficient (r) is shown.

  3. Extended Data Fig. 3 Long-term multilineage engraftment of BCL11A enhancer edited HSPCs in immunodeficient mice.

    CD34+ HSPCs from two healthy donors were electroporated with 2xNLS-SpCas9 RNP (coupled with MS-sgRNA-1617) and transplanted into NBSGW mice. Non-electroporated cells were transplanted as controls. A total of 0.4 million cells per mouse were infused for donor βAβA#1, and 0.8 million cells per mouse for donor βAβA#2. a, Mouse bone marrow (BM) was analyzed for human cell chimerism by flow cytometry 16 weeks after transplantation, defined as %hCD45+/(%hCD45+ + %mCD45+) cells. Each symbol represents a mouse, and mean for each group is shown. b, Indels at the human BCL11A enhancer were determined by TIDE analysis in the input HSPCs before transplant and in the mouse bone marrow 16 weeks after transplant. Each engrafted dot represents one mouse, and mean for each group is shown. c, BM collected 16 weeks after transplantation was analyzed by flow cytometry for multilineage reconstitution (calculated as percentage of hCD45+ cells). d, BM collected 16 weeks after transplantation was analyzed by flow cytometry for CD235a+ erythroid cells (calculated as percentage of mCD45hCD45 cells). eg, Gene expression analysis by RT–qPCR in human cells (from donor βAβA#2) from BM of engrafted mice. BCL11A expression normalized by CAT in human B cells (e) or human erythroid cells (f) sorted from BM of engrafted mice, and β-like globin expression (g) by RT–qPCR in human erythroid cells sorted from BM. h, BM from one engrafted mouse with unedited control or edited cells (from donor βAβA#1) were transplanted to three secondary NBSGW mice each (control mouse shown with black circle and edited mouse with green diamond symbol in a,b,d. After 16 weeks, BM was analyzed for human cell chimerism by flow cytometry. i, Indel frequencies within human BCL11A enhancer in BM 16 weeks after secondary transplantation. Each symbol represents an individual recipient mouse. Data are plotted as mean ± s.d. for c. Median of each group with 2–4 mice is shown as line for the other panels.

  4. Extended Data Fig. 4 Highly efficient editing of BCL11A enhancer in CD34+ HSPCs.

    a, Dose-dependent viability enhancement with glycerol or glycine after electroporation. 0.27 M = 2% glycerol, 0.2 M = 1.5% glycine. b, Quantification of editing frequency from deep sequencing of CD34+ HSPCs edited with 3xNLS-Cas9 RNP with MS-sgRNA-1617. c, Length distribution of alleles with and without indels (shown in blue and red, respectively) from deep sequencing of the 2xNLS-Cas9 RNP with MS-sgRNA-1617. d,e, Reduction of BCL11A mRNA by RT–qPCR or protein by western blot after editing of human BCL11A enhancer with 2xNLS-Cas9 or 3xNLS-Cas9 RNP with MS-sgRNA-AAVS1 or -1617 on various days of in vitro differentiation. Relative areas under curve (AUCs) are indicated. f,g, β-like globin expression by RT–qPCR and HbF level by HPLC in erythroid cells in vitro differentiated from 3xNLS-Cas9 RNP coupled with MS-sgRNA-1617 edited CD34+ HSPCs. All data represent the mean ± s.d. Statistically significant differences are indicated as follows: *P < 0.05 as determined by unpaired t-test. P = 0.0152 for f, 0.0443 for g. In all panels, data are plotted as mean ± s.d. and analyzed using unpaired two-tailed Student’s t-tests. Data are representative of three biologically independent replicates.

  5. Extended Data Fig. 5 Long-term multilineage reconstituting HSCs edited with 3xNLS-Cas9.

    ad, NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited healthy donor CD34+ HSPCs 2 h (day 0), 24 h (day 1) or 48 h (day 2) after electroporation. BM was collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (a), multilineage reconstitution (b), or human erythroid cells (c) in BM, as well as indel frequencies determined by TIDE analysis (d). eh, NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited healthy donor CD34+ HSPCs supplemented with 2%, 4%, or 6% glycerol for electroporation. BM was collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (e), multilineage reconstitution (f), or human erythroid cells (g) in BM, as well as the indel frequencies determined by TIDE analysis (h). i, Multilineage reconstitution analysis of BM collected from mice engrafted with control or edited CD34+ HSPCs (from donor βAβA#4). Error bars indicate standard deviation. Data are plotted as mean ± s.d. for b,f,i. Median of each group with 1–3 mice is shown as line for the other panels.

  6. Extended Data Fig. 6 Off-target analysis of human CD34+ HSPCs edited by SpCas9 RNP targeting BCL11A enhancer.

    a, Off-target sites detected by CIRCLE-seq for MS-sgRNA-1617 targeting human BCL11A enhancer. b, Deep sequencing analysis of potential off-target sites detected by CIRCLE-seq or in silico computational prediction within human CD34+ HSPCs edited by 2xNLS-Cas9 or 3xNLS-Cas9 RNP (coupled with MS-sgRNA-1617) targeting BCL11A enhancer. On-target sequence is at the BCL11A enhancer. Dotted line at 0.1% denotes sensitivity of deep sequencing to detect indels. c, RT–qPCR analysis of p21 expression after gene editing. Relative expression to GAPDH is shown. Data are plotted as mean ± s.d. and representative of three biologically independent replicates.

  7. Extended Data Fig. 7 Editing of BCL11A enhancer in SCD patient (βSβS) HSPCs.

    ad, NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited βSβS#1 CD34+ HSPCs 24 h (day 1) or 48 h (day 2) after electroporation. BM was collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (a), multilineage reconstitution (b), or human erythroid cells (c) in BM, as well as the indel frequencies determined by TIDE analysis (d). Error bars indicate standard deviation. e, Editing efficiency of 3xNLS-Cas9 coupled with MS-sgRNA-AAVS1 for control and -1617 for BCL11A enhancer editing in βSβS#2 CD34+ HSPCs as measured by TIDE analysis. f, β-like globin expression by RT–qPCR analysis in erythroid cells in vitro differentiated from RNP edited βSβS#2 CD34+ HSPCs. Error bars indicate standard deviation (n = 3 replicates). g, Multilineage reconstitution analysis of BM collected from mice engrafted with control or edited CD34+ HSPCs (from donor βSβS#2). h, Analysis of in vitro sickling of unedited control or edited enucleated βSβS#2 erythroid cells. Images were taken every 1 min after MBS treatment. Result shown as percentage sickled cells at each time point. Data are plotted as mean ± s.d. for b,e,f,g. Median of each group with 1–3 mice is shown as line for the other panels.

  8. Extended Data Fig. 8 Summary of engraftment analysis.

    a, Indel frequencies of indicated input HSPCs and engrafted human cells in 16 week BM. b. Correlation between input cell number and human engraftment rates in 16 week BM. c, Correlation of BCL11A mRNA versus γ-globin mRNA determined by RT–qPCR. Black dots represent erythroid cells from CD34+ HSPCs edited with SpCas9 coupled with various sgRNAs differentiated in vitro without engraftment; red dots represent erythroid cells sorted from mice BM engrafted from human CD34+ HSPCs edited with SpCas9 coupled with MS-sgRNA-1617. The Pearson correlation coefficient (r) is shown. d, BM cells (engrafted from donor βAβA#1 and βSβS#1) collected from engrafted mice were in vitro differentiated to human erythroid cells for HbF level analysis by HPLC. Each dot represents erythroid cells differentiated from BM of one mouse, and mean ± s.d. for each group is shown. e, Relative loss of indels in HSC-enriched CD34+ CD38 CD90+ CD45RA sorted population compared with bulk pre-sorted HSPCs after editing by 2 µM or 5 µM RNP. All data represent the mean ± s.d. Statistically significant differences are indicated as follows: ****P < 0.0001, **P < 0.01 (P = 0.0046) as determined by unpaired t-test. f, Comparison of β-like globin expression by RT–qPCR between erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs (pre-engraftment) and engrafted bone marrow (post-engraftment). Statistically significant differences are indicated as follows: ****P < 0.0001, ***P < 0.001 (P = 0.0006), **P < 0.01 (P = 0.0092) as determined by unpaired t-test. In all panels, data are plotted as mean ± s.d. and analyzed using unpaired two-tailed Student’s t-tests. Data are from indicated number of mice for a,b,d or representative of three biologically independent replicates for c,e,f.

  9. Extended Data Fig. 9 Indel spectra of engrafted bone marrow and corresponding input cells.

    a,b, Indel spectra of engrafted bone marrow (BM) and corresponding input cells from four donors electroporated with 2xNLS-Cas9 or 3xNLS-Cas9 coupled with MS-sgRNA-1617 (a) or -AAVS1 (b) supplemented with different concentration of glycerol (0%G to 6%G). c, Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ in mice BM 16 weeks after transplant. The indel spectrum was determined by TIDE analysis. Indel length from −8 to +6 bp was calculated as NHEJ, and from −9 to −20 bp as MMEJ. These data comprise 28 mice transplanted with eight BCL11A enhancer edited inputs and five mice transplanted with two AAVS1 edited inputs. Median of each group is shown as line, **P < 0.005, ****P < 0.0001 as determined by Kolmogorov–Smirnov test. d,e, Summary of most frequent indels by deep sequencing of bone marrow cells from primary recipient (d) and secondary recipient (e) engrafted with BCL11A enhancer edited CD34+ HSPCs. Asterisk indicates unedited allele. f,g, Indel spectra of HSPCs stained and sorted 2 h after RNP electroporation with 3xNLS-Cas9 with sgRNA-AAVS1. HSPCs prestimulated for 24 h before electroporation. HSPCs stained with CD34, CD38, CD90, CD45RA in f and with Pyronin Y, Hoechst 33342 in g. Indels determined by Sanger sequencing with TIDE analysis after culturing cells for 4 days after sort. Data are representative of three biologically independent replicates.

  10. Extended Data Fig. 10 Flow cytometry of CD34+ HSPCs with 24 h of culture.

    ad, Cryopreserved G-CSF mobilized CD34+ HSPCs were stained with CD34, CD38, CD90, and CD45RA antibodies (in a), or stained with Hoechst 33342 and Pyronin Y (in b) at 0 h following thaw or after 24 h in culture with SCF, TPO and FLT3-L. HSPCs were electroporated with RNP with 3 × -NLS-SpCas9 with BCL11A enhancer or AAVS1 targeting sgRNA. After 2 h recovery, cells were stained with CD34, CD38, CD90, and CD45RA or with Hoechst 33342 and Pyronin Y, and sorted according to gates as shown in c and d.

Supplementary information

  1. Supplementary Information

    Supplementary Tables 2 and 3

  2. Reporting Summary

  3. Supplementary Tables

    Supplementary Tables 1, 4, 5,6, 7, 8 and 9

  4. Supplementary Video 1

    Video of MBS-induced in vitro sickling of unedited enucleated βSβS erythroid cells.

  5. Supplementary Video 2

    Video of MBS-induced in vitro sickling of BCL11A enhancer edited enucleated βSβS erythroid cells.

Source data

  1. Source Data Fig. 2

    Unprocessed Western Blots for Fig 2g

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DOI

https://doi.org/10.1038/s41591-019-0401-y

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