Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair

Abstract

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1–13 or 2–13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1–13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2–13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2–13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schemata of correction strategies for targeted CYBB cDNA insertion in X-CGD patient CD34+ HSPCs.
Fig. 2: Targeted genome editing efficiencies in HSPCs for correction strategies 1 through 3 using initial un-optimized editing conditions.
Fig. 3: Comparison of phenotypic correction of gp91phox expression in differentiated phagocytes after targeted cDNA insertion in X-CGD HSPCs.
Fig. 4: Comparison of functional correction of ROS production measured by DHR assay in differentiated phagocytes after targeted cDNA insertion in X-CGD HSPCs.
Fig. 5: Enhancement of targeted insertion efficiency using i53 mRNA in HSPCs with correction strategy 3.
Fig. 6: Engraftment data of X-CGD patient HSPCs corrected using strategy 2 with optimized targeted insertion conditions and transplanted into NSG mice.

Similar content being viewed by others

References

  1. Roos D, Kuhns DB, Maddalena A, Roesler J, Lopez JA, Ariga T, et al. Hematologically important mutations: X-linked chronic granulomatous disease (third update). Blood Cells Mol Dis. 2010;45:246–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Marciano BE, Spalding C, Fitzgerald A, Mann D, Brown T, Osgood S, et al. Common severe infections in chronic granulomatous disease. Clin Infect Dis. 2015;60:1176–83.

    Article  CAS  PubMed  Google Scholar 

  3. Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B, Kinner A, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med. 2010;16:198–204.

    Article  CAS  PubMed  Google Scholar 

  4. Ulrich S, Anna P, Heidi H-G, Elena K, Ulrike K, Eleonore DR, et al. Successful combination of sequential gene therapy and rescue allo-HSCT in two children with X-CGD—importance of timing. Curr Gene Ther. 2015;15:416–27.

    Article  Google Scholar 

  5. Grez M, Reichenbach J, Schwäble J, Seger R, Dinauer MC, Thrasher AJ. Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther. 2011;19:28–35.

    Article  CAS  PubMed  Google Scholar 

  6. Yahata T, Takanashi T, Muguruma Y, Ibrahim AA, Matsuzawa H, Uno T, et al. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells. Blood. 2011;118:2941–50.

    Article  CAS  PubMed  Google Scholar 

  7. Santilli G, Almarza E, Brendel C, Choi U, Beilin C, Blundell MP, et al. Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells. Mol Ther. 2011;19:122–32.

    Article  CAS  PubMed  Google Scholar 

  8. Kohn DB, Booth C, Kang EM, Pai S-Y, Shaw KL, Santilli G, et al. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med. 2020;26:200–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Moehle EA, Rock JM, Lee Y-L, Jouvenot Y, DeKelver RC, Gregory PD, et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA. 2007;104:3055–60.

    Article  CAS  PubMed  Google Scholar 

  13. DeKelver RC, Choi VM, Moehle EA, Paschon DE, Hockemeyer D, Meijsing SH, et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 2010;20:1133–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29:143–8.

    Article  CAS  PubMed  Google Scholar 

  15. Levetzow GV, Spanholtz J, Beckmann J, Fischer J, Kögler G, Wernet P, et al. Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells. Stem Cells Dev. 2006;15:278–85.

    Article  Google Scholar 

  16. Lesueur LL, Mir LM, André FM. Overcoming the specific toxicity of large plasmids electrotransfer in primary cells in vitro. Mol Ther Nucleic Acids. 2016;5:e291.

    Article  CAS  PubMed  Google Scholar 

  17. Sather BD, Romano Ibarra GS, Sommer K, Curinga G, Hale M, Khan IF, et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med. 2015;7:307ra156.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Wang J, Exline CM, DeClercq JJ, Llewellyn GN, Hayward SB, Li PW-L, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol. 2015;33:1256–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539:384–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. De Ravin SS, Reik A, Liu P-Q, Li L, Wu X, Su L, et al. Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol. 2016;34:424–9.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Pavel-Dinu M, Wiebking V, Dejene BT, Srifa W, Mantri S, Nicolas CE, et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat Commun. 2019;10:1634.

    Article  PubMed  Google Scholar 

  22. Pattabhi S, Lotti SN, Berger MP, Singh S, Lux CT, Jacoby K, et al. In vivo outcome of homology-directed repair at the HBB gene in HSC using alternative donor template delivery methods. Mol Ther Nucleic Acids. 2019;17:277–88.

    Article  CAS  PubMed  Google Scholar 

  23. Romero Z, Lomova A, Said S, Miggelbrink A, Kuo CY, Campo-Fernandez B, et al. Editing the sickle cell disease mutation in human hematopoietic stem cells: comparison of endonucleases and homologous donor templates. Mol Ther. 2019;27:1389–406.

    Article  CAS  PubMed  Google Scholar 

  24. Schiroli G, Conti A, Ferrari S, della Volpe L, Jacob A, Albano L, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell. 2019;24:551–65.e8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gundry Michael C, Brunetti L, Lin A, Mayle Allison E, Kitano A, Wagner D, et al. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9. Cell Rep. 2016;17:1453–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. DeWitt MA, Magis W, Bray NL, Wang T, Berman JR, Urbinati F, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med. 2016;8:360ra134.

    Article  PubMed  PubMed Central  Google Scholar 

  27. De Ravin SS, Li L, Wu X, Choi U, Allen C, Koontz S, et al. CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med. 2017;9:eaah3480.

    Article  PubMed  Google Scholar 

  28. Antony JS, Latifi N, Haque AKMA, Lamsfus-Calle A, Daniel-Moreno A, Graeter S, et al. Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors. Mol Cell Pediatr. 2018;5:9.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Park SH, Lee CM, Dever DP, Davis TH, Camarena J, Srifa W, et al. Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease. Nucleic Acids Res. 2019;47:7955–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hoban MD, Cost GJ, Mendel MC, Romero Z, Kaufman ML, Joglekar AV, et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood. 2015;125:2597–604.

    Article  CAS  PubMed  Google Scholar 

  31. Pannunzio NR, Watanabe G, Lieber MR, Nonhomologous DNA. end-joining for repair of DNA double-strand breaks. J Biol Chem. 2018;293:10512–23.

    Article  CAS  PubMed  Google Scholar 

  32. Miyaoka Y, Berman JR, Cooper SB, Mayerl SJ, Chan AH, Zhang B, et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep. 2016;6:23549.

    Article  CAS  PubMed  Google Scholar 

  33. Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Biol. 2008;9:297–308.

    Article  CAS  PubMed  Google Scholar 

  34. Genovese P, Schiroli G, Escobar G, Tomaso TD, Firrito C, Calabria A, et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature. 2014;510:235–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hubbard N, Hagin D, Sommer K, Song Y, Khan I, Clough C, et al. Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome. Blood. 2016;127:2513–22.

    Article  CAS  PubMed  Google Scholar 

  36. Sweeney CL, Zou J, Choi U, Merling RK, Liu A, Bodansky A, et al. Targeted repair of CYBB in X-CGD iPSCs requires retention of intronic sequences for expression and functional correction. Mol Ther. 2017;25:321–30.

    Article  CAS  PubMed  Google Scholar 

  37. Shaul O. How introns enhance gene expression. Int J Biochem Cell Biol. 2017;91:145–55.

    Article  CAS  PubMed  Google Scholar 

  38. Canny MD, Moatti N, Wan LCK, Fradet-Turcotte A, Krasner D, Mateos-Gomez PA, et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR–Cas9 genome-editing efficiency. Nat Biotechnol. 2017;36:95.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Escribano-Díaz C, Orthwein A, Fradet-Turcotte A, Xing M, Young Jordan TF, Tkáč J, et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol Cell. 2013;49:872–83.

    Article  PubMed  Google Scholar 

  40. Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33:985.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Moreno-Carranza B, Gentsch M, Stein S, Schambach A, Santilli G, Rudolf E, et al. Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells. Gene Ther. 2009;16:111–8.

    Article  CAS  PubMed  Google Scholar 

  42. Goodwin EC, Rottman FM. The 3’-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J Biol Chem. 1992;267:16330–4.

    Article  CAS  PubMed  Google Scholar 

  43. Gil A, Proudfoot NJ. Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit β-globin mRNA 3′ end formation. Cell. 1987;49:399–406.

    Article  CAS  PubMed  Google Scholar 

  44. Lanoix J, Acheson NH. A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA. EMBO J. 1988;7:2515–22.

    Article  CAS  PubMed  Google Scholar 

  45. Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther. 1999;10:2295–305.

    Article  CAS  PubMed  Google Scholar 

  46. Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 1999;73:2886–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Song L, Li X, Jayandharan GR, Wang Y, Aslanidi GV, Ling C, et al. High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo. PLoS ONE. 2013;8:e58757.

    Article  CAS  PubMed  Google Scholar 

  48. Hsiau T, Conant D, Rossi N, Maures T, Waite K, Yang J, et al. Inference of CRISPR edits from Sanger trace data. bioRxiv. 2019. https://doi.org/10.1101/251082.

  49. Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G. COSMID: a Web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol Ther Nucleic Acids. 2014;3:e214.

    Article  CAS  PubMed  Google Scholar 

  50. Sweeney CL, Merling RK, De Ravin SS, Choi U, Malech HL. Gene editing in chronic granulomatous disease. In: Knaus UG, Leto TL, editors. NADPH oxidases: methods and protocols. New York, NY: Springer New York; 2019. pp. 623–65.

  51. Zou J, Sweeney CL, Chou BK, Choi U, Pan J, Wang H, et al. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood. 2011;117:5561–72.

    Article  CAS  PubMed  Google Scholar 

  52. Merling RK, Sweeney CL, Chu J, Bodansky A, Choi U, Priel DL, et al. An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease. Mol Ther. 2015;23:147–57.

    Article  CAS  PubMed  Google Scholar 

  53. Yamauchi A, Yu L, Pötgens Andy JG, Kuribayashi F, Nunoi H, Kanegasaki S, et al. Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox. Microbiol Immunol. 2013;45:249–57.

    Article  Google Scholar 

  54. Komiya E, Kondoh M, Mizuguchi H, Fujii M, Utoguchi N, Nakanishi T, et al. Characteristics of transcription-regulatory elements for gene expression from plasmid vectors in human trophoblast cell lines. Placenta. 2006;27:934–8.

    Article  CAS  PubMed  Google Scholar 

  55. Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, et al. Optimization of plasmid vectors for high-level expression in lung epithelial cells. Hum Gene Ther. 1997;8:575–84.

    Article  CAS  PubMed  Google Scholar 

  56. Jayavaradhan R, Pillis DM, Goodman M, Zhang F, Zhang Y, Andreassen PR, et al. CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. Nat Commun. 2019;10:2866.

    Article  PubMed  Google Scholar 

  57. Marciano BE, Zerbe CS, Falcone EL, Ding L, DeRavin SS, Daub J, et al. X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability. J Allergy Clin Immunol. 2018;141:365–71.

    Article  CAS  PubMed  Google Scholar 

  58. Rees HA, Yeh W-H, Liu DR. Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nat Commun. 2019;10:2212.

    Article  PubMed  Google Scholar 

  59. Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, et al. Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nat Commun. 2019;10:3395.

    Article  PubMed  Google Scholar 

  60. Wienert B, Nguyen DN, Guenther A, Feng SJ, Locke MN, Wyman SK, et al. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. Nat Commun. 2020;11:2109.

    Article  CAS  PubMed  Google Scholar 

  61. Frazão JB, Thain A, Zhu Z, Luengo M, Condino-Neto A, Newburger PE. Regulation of CYBB gene expression in human phagocytes by a distant upstream NF-κB binding site. J Cell Biochem. 2015;116:2008–17.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Roesler J, Brenner S, Bukovsky AA, Whiting-Theobald N, Dull T, Kelly M, et al. Third-generation, self-inactivating gp91phox lentivector corrects the oxidase defect in NOD/SCID mouse–repopulating peripheral blood–mobilized CD34+ cells from patients with X-linked chronic granulomatous disease. Blood. 2002;100:4381–90.

    Article  CAS  PubMed  Google Scholar 

  63. Pinder J, Salsman J, Dellaire G. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 2015;43:9379–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Charpentier M, Khedher AHY, Menoret S, Brion A, Lamribet K, Dardillac E, et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun. 2018;9:1133.

    Article  CAS  PubMed  Google Scholar 

  65. Ye L, Wang C, Hong L, Sun N, Chen D, Chen S, et al. Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9. Cell Discov. 2018;4:46.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Lomova A, Clark D, Campo‐Fernandez B, Flores‐Bjurström C, Kaufman M, Fitz-Gibbon S, et al. Improving gene editing outcomes in human hematopoietic stem and progenitor cells by temporal control of DNA repair. Stem Cells. 2018;37:284–94.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Yang D, Scavuzzo MA, Chmielowiec J, Sharp R, Bajic A, Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep. 2016;6:21264.

    Article  CAS  PubMed  Google Scholar 

  68. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015;33:538–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33:543–8.

    Article  CAS  PubMed  Google Scholar 

  70. Robert F, Barbeau M, Éthier S, Dostie J, Pelletier J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 2015;7:93.

    Article  PubMed  Google Scholar 

  71. Riesenberg S, Maricic T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun. 2018;9:2164.

    Article  PubMed  Google Scholar 

  72. Jayavaradhan R, Pillis DM, Malik P. A versatile tool for the quantification of CRISPR/Cas9-induced genome editing events in human hematopoietic cell lines and hematopoietic stem/progenitor cells. J Mol Biol. 2018;431:102–10.

    Article  PubMed  Google Scholar 

  73. Paulsen BS, Mandal PK, Frock RL, Boyraz B, Yadav R, Upadhyayula S, et al. Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing. Nat Biomed Eng. 2017;1:878–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Cuella-Martin R, Oliveira C, Lockstone HE, Snellenberg S, Grolmusova N, Chapman JR. 53BP1 integrates DNA repair and p53-dependent cell fate decisions via distinct mechanisms. Mol Cell. 2016;64:51–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the patients and healthy donors for their contribution to this study, the Department of Transfusion Medicine at the National Institutes of Health Clinical Center for their collection and processing of CD34+ HSPCs, and the CCR Genomics Core of the National Cancer Institute for their assistance in DNA sequencing and ddPCR analyses.

Funding

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) under intramural project numbers Z01-Al-00644 and Z01-Al-00988, as well as funding to MHP under NIH research grant R01-AI097320 and philanthropic gift funds from the Amon G. Carter Foundation.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Matthew H. Porteus or Suk See De Ravin.

Ethics declarations

Conflict of interest

LL was a full-time employee of MaxCyte Biosystems at the time of this study; RJM and GAD are full-time employees of CELLSCRIPT, LLC; MHP holds equity in CRISPR Tx and Allogene Tx and serves on the Scientific Advisory Board for Allogene Tx; the remaining authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sweeney, C.L., Pavel-Dinu, M., Choi, U. et al. Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair. Gene Ther 28, 373–390 (2021). https://doi.org/10.1038/s41434-021-00251-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-021-00251-z

Search

Quick links