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

Zhen Zhang; Amy E Baxter; Diqiu Ren; Kunhua Qin; Zeyu Chen; Sierra M Collins; Hua Huang; Chad A Komar; Peter F Bailer; Jared B Parker; Gerd A Blobel; Rahul M Kohli; E John Wherry; Shelley L Berger; Junwei Shi

doi:10.1038/s41587-023-01756-1

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

Nat Biotechnol. 2024 Feb;42(2):305-315. doi: 10.1038/s41587-023-01756-1. Epub 2023 Apr 24.

Authors

Zhen Zhang^{1

2}, Amy E Baxter^{3

4}, Diqiu Ren^{1

5

6}, Kunhua Qin^{1

7}, Zeyu Chen^{3

4}, Sierra M Collins^{1

2}, Hua Huang^{1

2

3}, Chad A Komar^{1

5

6}, Peter F Bailer^{1

8

9}, Jared B Parker⁸, Gerd A Blobel^{1

7}, Rahul M Kohli^{1

8

9}, E John Wherry^{10

11

12}, Shelley L Berger^{13

14}, Junwei Shi^{15

16

17}

Affiliations

¹ Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA.
² Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA.
⁴ Institute for Immunology and Immune Health, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA.
⁷ Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
⁸ Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁹ Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.
¹⁰ Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA. wherry@pennmedicine.upenn.edu.
¹¹ Institute for Immunology and Immune Health, University of Pennsylvania, Philadelphia, PA, USA. wherry@pennmedicine.upenn.edu.
¹² Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. wherry@pennmedicine.upenn.edu.
¹³ Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA. bergers@pennmedicine.upenn.edu.
¹⁴ Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA. bergers@pennmedicine.upenn.edu.
¹⁵ Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA. jushi@upenn.edu.
¹⁶ Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA. jushi@upenn.edu.
¹⁷ Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA. jushi@upenn.edu.

PMID: 37095348
DOI: 10.1038/s41587-023-01756-1

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.

MeSH terms

Animals
CRISPR-Cas Systems* / genetics
Electroporation
Gene Editing* / methods
Hematopoietic Stem Cells
Humans
Mice

Abstract

MeSH terms

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