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

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

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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.

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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.

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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.

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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).

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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

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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.

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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.

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

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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

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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

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