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

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...

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Bibliographic Details
Published in:	Nature biomedical engineering Vol. 7; no. 5; pp. 647 - 660
Main Authors:	Foss, Dana V., Muldoon, Joseph J., Nguyen, David N., Carr, Daniel, Sahu, Srishti U., Hunsinger, John M., Wyman, Stacia K., Krishnappa, Netravathi, Mendonsa, Rima, Schanzer, Elaine V., Shy, Brian R., Vykunta, Vivasvan S., Allain, Vincent, Li, Zhongmei, Marson, Alexander, Eyquem, Justin, Wilson, Ross C.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 01-05-2023 Nature Publishing Group
Subjects:	13/107 13/109 42/41 42/44 631/61/201 631/61/2297 631/67/1059/2325 96/106 96/31 Adenine Animals Antigens Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Chimeric antigen receptors CRISPR CRISPR-Cas Systems Cytotoxicity Editing Electroporation Gene Editing - methods Genomes Genotoxicity Homology Humans Lymphocytes Lymphocytes B Lymphocytes T Mice Natural killer cells Peptides Peptides - genetics Receptors Ribonucleoproteins Screening Sequential delivery T cell receptors T-Lymphocytes - metabolism
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Summary:	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. The yields of edited primary human lymphocytes can be increased substantially, with respect to those obtained via electroporation, by delivering a CRISPR ribonucleoprotein alongside an amphiphilic peptide identified via screening.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2157-846X 2157-846X
DOI:	10.1038/s41551-023-01032-2