Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

Dana V Foss; Joseph J Muldoon; David N Nguyen; Daniel Carr; Srishti U Sahu; John M Hunsinger; Stacia K Wyman; Netravathi Krishnappa; Rima Mendonsa; Elaine V Schanzer; Brian R Shy; Vivasvan S Vykunta; Vincent Allain; Zhongmei Li; Alexander Marson; Justin Eyquem; Ross C Wilson

doi:10.1038/s41551-023-01032-2

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

Nat Biomed Eng. 2023 May;7(5):647-660. doi: 10.1038/s41551-023-01032-2. Epub 2023 Apr 25.

Authors

Dana V Foss^#^{1

2

3}, Joseph J Muldoon^#^{4

5}, David N Nguyen^#^{1

2

4

5}, Daniel Carr^{1

4

5}, Srishti U Sahu^{1

2

3}, John M Hunsinger^{1

2

3}, Stacia K Wyman¹, Netravathi Krishnappa¹, Rima Mendonsa^{1

2

3}, Elaine V Schanzer^{1

2}, Brian R Shy^{4

5

6}, Vivasvan S Vykunta^{4

5}, Vincent Allain^{4

5

7}, Zhongmei Li⁴, Alexander Marson^{8

9

10

11

12

13

14

15}, Justin Eyquem^{16

17

18

19

20

21}, Ross C Wilson^{22

23

24}

Affiliations

¹ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
² Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
³ California Institute for Quantitative Biosciences at University of California Berkeley, Berkeley, CA, USA.
⁴ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁵ Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
⁶ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.
⁷ Université de Paris, INSERM UMR976, Hôpital Saint-Louis, Paris, France.
⁸ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. alexander.marson@ucsf.edu.
⁹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹⁰ Department of Medicine, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹¹ Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹² Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹³ Diabetes Center, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹⁴ UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹⁵ Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
¹⁶ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
¹⁷ Department of Medicine, University of California San Francisco, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
¹⁸ Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
¹⁹ Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
²⁰ UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
²¹ Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA. justin.eyquem@ucsf.edu.
²² Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. rosswilson@berkeley.edu.
²³ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA. rosswilson@berkeley.edu.
²⁴ California Institute for Quantitative Biosciences at University of California Berkeley, Berkeley, CA, USA. rosswilson@berkeley.edu.

^# Contributed equally.

Abstract

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Animals
CRISPR-Cas Systems*
Gene Editing* / methods
Humans
Mice
Peptides / genetics
Ribonucleoproteins
T-Lymphocytes / metabolism

Substances

Peptides
Ribonucleoproteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding