Abstract

Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

References

  1. 1.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  2. 2.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  3. 3.

    Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

  4. 4.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

  5. 5.

    Dagdas, Y. S. et al. A conformational checkpoint between DNA binding and cleavage by CRISPR–Cas9. Sci. Adv. 3, eaao0027 (2017).

  6. 6.

    Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015).

  7. 7.

    Kass, E. M. & Jasin, M. Collaboration and competition between DNA double-strand break repair pathways. FEBS Lett. 584, 3703–3708 (2010).

  8. 8.

    Porteus, M. H. Towards a new era in medicine: therapeutic genome editing. Genome Biol. 16, 286 (2015).

  9. 9.

    Dever, D. P. & Porteus, M. H. The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation. Curr. Opin. Hematol. 24, 481–488 (2017).

  10. 10.

    Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

  11. 11.

    Cameron, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 14, 600–606 (2017).

  12. 12.

    Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR–Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

  13. 13.

    Gundry, M. C. et al. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9. Cell Reports 17, 1453–1461 (2016).

  14. 14.

    Gundry, M. C. et al. Technical considerations for the use of CRISPR/Cas9 in hematology research. Exp. Hematol. 54, 4–11 (2017).

  15. 15.

    Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

  16. 16.

    DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016).

  17. 17.

    Hultquist, J. F. et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV–host interactions in primary human t cells. Cell Reports 17, 1438–1452 (2016).

  18. 18.

    Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015).

  19. 19.

    Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

  20. 20.

    Gatti, R. A., Meuwissen, H. J., Allen, H. D., Hong, R. & Good, R. A. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 2, 1366–1369 (1968).

  21. 21.

    Johnson, F. L. et al. Bone-marrow transplantation in a patient with sickle-cell anemia. N. Engl. J. Med. 311, 780–783 (1984).

  22. 22.

    Lucarelli, G. et al. Allogeneic marrow transplantation for thalassemia. Exp. Hematol. 12, 676–681 (1984).

  23. 23.

    Naldini, L. Gene therapy returns to centre stage. Nature 526, 351–360 (2015).

  24. 24.

    De Ravin, S. S. et al. CRISPR–Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci. Transl. Med. 9, eaah3480 (2017).

  25. 25.

    Traxler, E. A. et al. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat. Med. 22, 987–990 (2016).

  26. 26.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

  27. 27.

    Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

  28. 28.

    Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).

  29. 29.

    Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).

  30. 30.

    Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

  31. 31.

    Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

  32. 32.

    Chen, Z. & Zhao, H. A highly sensitive selection method for directed evolution of homing endonucleases. Nucleic Acids Res. 33, e154 (2005).

  33. 33.

    Dobosy, J. R. et al. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol. 11, 80 (2011).

  34. 34.

    Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

  35. 35.

    Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015).

  36. 36.

    Wang, J. et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. 44, e30 (2016).

  37. 37.

    De Ravin, S. S. et al. Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat. Biotechnol. 34, 424–429 (2016).

  38. 38.

    Sather, B. D. et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci. Transl. Med. 7, 307ra156 (2015).

  39. 39.

    Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

  40. 40.

    Schiroli, G. et al. Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Sci. Transl. Med. 9, eaan0820 (2017).

  41. 41.

    Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).

  42. 42.

    Osborn, M. J. et al. Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases. Mol. Ther. 24, 570–581 (2016).

  43. 43.

    Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).

  44. 44.

    MacLeod, D. T. et al. Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol. Ther. 25, 949–961 (2017).

  45. 45.

    Hoban, M. D. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 125, 2597–2604 (2015).

  46. 46.

    Hoban, M. D. et al. CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells. Mol. Ther. 24, 1561–1569 (2016).

  47. 47.

    Dulmovits, B. M. et al. Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors. Blood 127, 1481–1492 (2016).

  48. 48.

    Romero, Z. et al. β-globin gene transfer to human bone marrow for sickle cell disease. J. Clin. Invest. https://doi.org/10.1172/JCI67930 (2013).

  49. 49.

    Hu, J. et al. Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo. Blood 121, 3246–3253 (2013).

  50. 50.

    Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

  51. 51.

    Miller, J. B. et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Edn Engl. 56, 1059–1063 (2017).

  52. 52.

    Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).

  53. 53.

    Wang, M. et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. USA 113, 2868–2873 (2016).

  54. 54.

    Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

  55. 55.

    Haldimann, A., Daniels, L. L. & Wanner, B. L. Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J. Bacteriol. 180, 1277–1286 (1998).

  56. 56.

    Jacobi, A. M. et al. Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes. Methods 121-122, 16–28 (2017).

  57. 57.

    Williams, M., Rainville, I. R. & Nicklas, J. A. Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations. Environ. Mol. Mutagen. 39, 22–32 (2002).

  58. 58.

    Lennox, K. A., Vakulskas, C. A. & Behlke, M. A. Non-nucleotide modification of anti-miRNA oligonucleotides. Methods Mol. Biol. 1517, 51–69 (2017).

  59. 59.

    Munteanu, B., Braun, M. & Boonrod, K. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids. J. Zhejiang Univ. Sci. B 13, 244–247 (2012).

  60. 60.

    Karvelis, T. et al. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10, 841–851 (2013).

  61. 61.

    Magoč, T. & Salzberg, S. L. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011).

  62. 62.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  63. 63.

    Bak, R. O. et al. Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. eLife 6, e27873 (2017).

  64. 64.

    Bak, R. O. & Porteus, M. H. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Reports 20, 750–756 (2017).

  65. 65.

    Khan, I. F., Hirata, R. K. & Russell, D. W. AAV-mediated gene targeting methods for human cells. Nat. Protoc. 6, 482–501 (2011).

  66. 66.

    Aurnhammer, C. et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2–derived inverted terminal repeat sequences. Hum. Gene Ther. Methods 23, 18–28 (2012).

  67. 67.

    Chicaybam, L., Sodre, A. L., Curzio, B. A. & Bonamino, M. H. An efficient low cost method for gene transfer to T lymphocytes. PLoS One 8, e60298 (2013).

  68. 68.

    Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

  69. 69.

    Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J. & Bao, G. COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol. Ther. Nucleic Acids 3, e214 (2014).

  70. 70.

    Lee, C. M., Cradick, T. J. & Bao, G. The Neisseria meningitidis CRISPR–Cas9 system enables specific genome editing in mammalian cells. Mol. Ther. 24, 645–654 (2016).

  71. 71.

    Ross, M. G. et al. Characterizing and measuring bias in sequence data. Genome Biol. 14, R51 (2013).

  72. 72.

    Quail, M. A. et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13, 341 (2012).

  73. 73.

    Güell, M., Yang, L. & Church, G. M. Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics 30, 2968–2970 (2014).

Download references

Acknowledgements

We thank S. Mantri for collecting and purifying the HSPCs. We thank K. Lennox for critical review of the manuscript. The Band3 APC antibody was a kind gift from A. Narla and M. Narla (Stanford University). M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research and NIH grant support R01-AI097320 and R01-AI120766. G.B. acknowledges support from the Cancer Prevention and Research Institute of Texas (RR14008 and RP170721). We thank the Binns Program for Cord Blood Research at Stanford University for cord-blood-derived CD34+ HSPCs and also for SCD-HSPCs. Patients with SCD consented to the use of CD34+ HSPCs for research with the accompanying IRB approval.

Author information

Author notes

  1. These authors contributed equally: Christopher A. Vakulskas, Daniel P. Dever.

Affiliations

  1. Integrated DNA Technologies, Inc., Coralville, IA, USA

    • Christopher A. Vakulskas
    • , Garrett R. Rettig
    • , Rolf Turk
    • , Ashley M. Jacobi
    • , Michael A. Collingwood
    • , Nicole M. Bode
    • , Matthew S. McNeill
    • , Shuqi Yan
    •  & Mark A. Behlke
  2. Department of Pediatrics, Stanford University, Stanford, CA, USA

    • Daniel P. Dever
    • , Joab Camarena
    • , Volker Wiebking
    • , Natalia Gomez-Ospina
    • , Mara Pavel-Dinu
    •  & Matthew H. Porteus
  3. Department of Bioengineering, Rice University, Houston, TX, USA

    • Ciaran M. Lee
    • , So Hyun Park
    •  & Gang Bao
  4. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    • Rasmus O. Bak
  5. Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Aarhus, Denmark

    • Rasmus O. Bak
  6. Biomaterials and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Palo Alto, CA, USA

    • Wenchao Sun

Authors

  1. Search for Christopher A. Vakulskas in:

  2. Search for Daniel P. Dever in:

  3. Search for Garrett R. Rettig in:

  4. Search for Rolf Turk in:

  5. Search for Ashley M. Jacobi in:

  6. Search for Michael A. Collingwood in:

  7. Search for Nicole M. Bode in:

  8. Search for Matthew S. McNeill in:

  9. Search for Shuqi Yan in:

  10. Search for Joab Camarena in:

  11. Search for Ciaran M. Lee in:

  12. Search for So Hyun Park in:

  13. Search for Volker Wiebking in:

  14. Search for Rasmus O. Bak in:

  15. Search for Natalia Gomez-Ospina in:

  16. Search for Mara Pavel-Dinu in:

  17. Search for Wenchao Sun in:

  18. Search for Gang Bao in:

  19. Search for Matthew H. Porteus in:

  20. Search for Mark A. Behlke in:

Contributions

C.A.V. performed and designed the bacterial screen and subsequent identification of the HiFi mutants. C.A.V., M.A.C., and N.M.B. cloned and purified all proteins examined in this study. C.A.V., G.R.R., R.T., A.M.J., and M.S.M. performed NGS on-target and off-target editing experiments with RNP in human cells. C.A.V., M.A.C., and S.Y. created and characterized the HEK293 Cas9 stable cell lines. D.P.D., J.C., R.O.B., V.W., M.P.-D., and N.G.-O. carried out experiments related to HSPC and T cell gene editing. G.B., C.M.L., and S.H.P. carried out the NGS analysis of HSPC HBB editing events. W.S. carried out the HPLC analysis of hemoglobin tetramers. M.A.B. and M.H.P. directed the research and participated in the design and interpretation of the experiments and the writing of the manuscript. C.A.V., D.P.D., M.H.P., and M.A.B. wrote the manuscript with assistance from all authors.

Competing interests

M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input or opinions on the subject matter described in this manuscript. C.A.V., G.R.R., R.T., A.M.J., M.A.C., N.M.B., M.S.M., S.Y., and M.A.B. are employees of Integrated DNA Technologies (IDT), which sells reagents similar to some described in the manuscript.

Corresponding authors

Correspondence to Matthew H. Porteus or Mark A. Behlke.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–5

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41591-018-0137-0