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:

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing

Nature Biotechnology volume 42, pages 305–315 (2024)Cite this article

Subjects

Abstract

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR–Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Development of PAGE CRISPR–Cas9 system.
Fig. 2: Development of Cas9-PAGE genome editing in clinically relevant models of mouse primary T cells.
Fig. 3: Development of CRISPR-RNP-PAGE genome editing in human CAR T cells.
Fig. 4: PAGE-mediated multiplex genome editing in human CAR T cells and HSPCs.

Similar content being viewed by others

Data availability

The accession numbers for the RNA-seq dataset in this study is GSE223805(ref. 55). The GRCh38/hg38 human reference genome is publicly available. Key plasmids, Cas9-T6N and Cas12a-T8N have been deposited at Addgene (plasmid ID, 199604–199605). Source data are provided with this paper, including unprocessed Western blots.

References

  1. Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).

    CAS  PubMed  Google Scholar 

  4. June, C. H., O’Connor, R. S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. Science 359, 1361–1365 (2018).

    CAS  PubMed  ADS  Google Scholar 

  5. Rosenberg, S. A., Restifo, N. P., Yang, J. C., Morgan, R. A. & Dudley, M. E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat. Rev. Cancer 8, 299–308 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).

    CAS  PubMed  Google Scholar 

  7. Atsavapranee, E. S., Billingsley, M. M. & Mitchell, M. J. Delivery technologies for T cell gene editing: applications in cancer immunotherapy. EBioMedicine 67, 103354 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 16, 387–399 (2017).

    CAS  PubMed  Google Scholar 

  9. Chen, Z. et al. In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer. Cell 184, 1262–1280 (2021).

  10. Dong, M. B. et al. Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell 178, 1189–1204 (2019).

  11. Wei, J. et al. Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature 576, 471–476 (2019).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  12. Huang, H. et al. In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T-cell fate decisions. Cell 184, 1245–1261 (2021).

  13. LaFleur, M. W. et al. A CRISPR–Cas9 delivery system for in vivo screening of genes in the immune system. Nat. Commun. 10, 1668 (2019).

  14. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Staahl, B. T. et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotechnol. 35, 431–434 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Erazo-Oliveras, A., Muthukrishnan, N., Baker, R., Wang, T. Y. & Pellois, J. P. Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel) 5, 1177–1209 (2012).

    CAS  PubMed  Google Scholar 

  18. Heitz, F., Morris, M. C. & Divita, G. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 157, 195–206 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Varkouhi, A. K., Scholte, M., Storm, G. & Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J. Control. Release 151, 220–228 (2011).

    CAS  PubMed  Google Scholar 

  20. Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193 (1988).

    CAS  PubMed  Google Scholar 

  21. Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).

    CAS  PubMed  Google Scholar 

  22. Wherry, E. J. T cell exhaustion. Nat. Immunol. 12, 492–499 (2011).

    CAS  PubMed  Google Scholar 

  23. Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat. Rev. Immunol. 15, 486–499 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Kurachi, M. et al. Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function. Nat. Protoc. 12, 1980–1998 (2017).

  25. Barber, D. L. et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682–687 (2006).

    CAS  PubMed  ADS  Google Scholar 

  26. Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015).

    CAS  PubMed  ADS  Google Scholar 

  27. Odorizzi, P. M., Pauken, K. E., Paley, M. A., Sharpe, A. & Wherry, E. J. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. J. Exp. Med. 212, 1125–1137 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Gier, R. A. et al. High-performance CRISPR–Cas12a genome editing for combinatorial genetic screening. Nat. Commun. 11, 3455 (2020).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  29. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. DeWeirdt, P. C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat. Biotechnol. 39, 94–104 (2021).

    CAS  PubMed  Google Scholar 

  31. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).

    CAS  PubMed  ADS  Google Scholar 

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

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  34. Weissman, I. L. & Shizuru, J. A. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood 112, 3543–3553 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Grevet, J. D. et al. Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361, 285–290 (2018).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  36. Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and beta-thalassemia. N. Engl. J. Med. 384, 252–260 (2021).

    CAS  PubMed  Google Scholar 

  37. Bauer, D. E. et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342, 253–257 (2013).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  38. Qin, K. et al. Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells. Nat. Genet. 54, 874–884 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Shmakov, S. et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  42. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  43. Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. (in press).

  44. Tou, C. J., Orr, B. & Kleinstiver, B. P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat. Biotechnol. (in press).

  45. Durrant, M. G. et al. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat. Biotechnol. (in press).

  46. Pauken, K. E. et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165 (2016).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  47. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  48. Cao, Z. et al. ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency. Mol Cell 81, 3604–3622 (2021).

  49. Milone, M. C. et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 17, 1453–1464 (2009).

  50. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  51. Ritchie, M. E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Google Scholar 

  52. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 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).

    PubMed  PubMed Central  Google Scholar 

  54. Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Zhang, Z. et al. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. NCBI. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223805 (2023).

Download references

Acknowledgements

We thank M. Szurgot and R. Marmorstein (Department of Biochemistry and Biophysics, University of Pennsylvania) for sharing the protease ULP1 expression vector and purification protocol. We also thank the staff at the Flow Cytometry Core Laboratory of Children’s Hospital of Philadelphia. G.A.B. acknowledges NIH/NHLBI (R01-HL119479). R.M.K. acknowledges NIH (R01-GM138908). E.J.W. acknowledges support from the NIH (AI105343, AI082630, AI108545, AI155577, AI149680 and U19AI082630), funding from the Allen Institute for Immunology and the Parker Institute for Cancer Immunotherapy. Work in the Wherry lab is supported by the Parker Institute for Cancer Immunotherapy. S.L.B. acknowledges NIH/NCI (R35-CA263922). J.S. acknowledges NIH/NCI (R01-CA258904).

Author information

Authors and Affiliations

  1. Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA

    Zhen Zhang, Diqiu Ren, Kunhua Qin, Sierra M. Collins, Hua Huang, Chad A. Komar, Peter F. Bailer, Gerd A. Blobel, Rahul M. Kohli, Shelley L. Berger & Junwei Shi

  2. Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA

    Zhen Zhang, Sierra M. Collins, Hua Huang & Shelley L. Berger

  3. Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA

    Amy E. Baxter, Zeyu Chen, Hua Huang & E. John Wherry

  4. Institute for Immunology and Immune Health, University of Pennsylvania, Philadelphia, PA, USA

    Amy E. Baxter, Zeyu Chen & E. John Wherry

  5. Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA

    Diqiu Ren, Chad A. Komar & Junwei Shi

  6. Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA

    Diqiu Ren, Chad A. Komar & Junwei Shi

  7. Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Kunhua Qin & Gerd A. Blobel

  8. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Peter F. Bailer, Jared B. Parker & Rahul M. Kohli

  9. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA

    Peter F. Bailer & Rahul M. Kohli

  10. Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    E. John Wherry

Authors
  1. Zhen Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Amy E. Baxter
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Diqiu Ren
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Kunhua Qin
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Zeyu Chen
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Sierra M. Collins
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Hua Huang
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Chad A. Komar
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Peter F. Bailer
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Jared B. Parker
    View author publications

    You can also search for this author inPubMed Google Scholar

  11. Gerd A. Blobel
    View author publications

    You can also search for this author inPubMed Google Scholar

  12. Rahul M. Kohli
    View author publications

    You can also search for this author inPubMed Google Scholar

  13. E. John Wherry
    View author publications

    You can also search for this author inPubMed Google Scholar

  14. Shelley L. Berger
    View author publications

    You can also search for this author inPubMed Google Scholar

  15. Junwei Shi
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Z.Z., E.J.W., S.L.B. and J.S. conceived and developed the Peptide-Assisted Genome Editing (PAGE) approach and designed the research. Z.Z., A.E.B., D.R., K.Q., Z.C., S.M., H.H., C.A.K., P.F.B. and J.B.P. performed experiments and analyzed the data. G.A.B., R.M.K., E.J.W., S.L.B. and J.S. supervised the research. Z.Z. and J.S. drafted the manuscript. Z.Z., A.E.B., G.A.B., R.M.K., E.J.W., S.L.B. and J.S. reviewed and edited the manuscript with input from all authors. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to E. John Wherry, Shelley L. Berger or Junwei Shi.

Ethics declarations

Competing interests

Z.Z., A.E.B., Z.C., J.B.P., R.M.K., E.J.W., S.L.B. and J.S. through the University of Pennsylvania have filed a patent application on aspects of this work. E.J.W. is a member of the Parker Institute for Cancer Immunotherapy which supported this study. E.J.W. is an advisor for Danger Bio, Janssen, New Limit, Marengo, Pluto Immunotherapeutics Related Sciences, Santa Ana Bio, Synthekine and Surface Oncology. E.J.W. is a founder of and holds stock in Surface Oncology, Danger Bio and Arsenal Biosciences. R.M.K. is on the Scientific Advisory Board for Life Edit, Inc.

Peer review

Peer review information

Nature Biotechnology thanks Meisam Kararoudi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–22.

Reporting Summary

Supplementary Tables

Table 1–Sequences of guide RNA used in this study; Table 2–Sequences of primers used in this study.

Source data

Source Data Fig. 1

Unprocessed Western Blots

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, Z., Baxter, A.E., Ren, D. et al. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat Biotechnol 42, 305–315 (2024). https://doi.org/10.1038/s41587-023-01756-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-023-01756-1

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research