Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair

Carolin Moosmann; Thomas R Müller; Dirk H Busch; Kilian Schober

doi:10.1016/j.xpro.2021.101031

Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair

STAR Protoc. 2021 Dec 14;3(1):101031. doi: 10.1016/j.xpro.2021.101031. eCollection 2022 Mar 18.

Authors

Carolin Moosmann¹, Thomas R Müller², Dirk H Busch², Kilian Schober¹

Affiliations

¹ Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
² Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

Abstract

Adoptive T cell therapy using T-cell receptor (TCR)-engineered T cells allows to redirect T cell specificity and to target any antigen of interest. Here, we apply advanced genetic engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) for simultaneous editing of TCR α- and β-chains in primary human T cells. Together with non-virally delivered template DNA, this CRISPR-Cas9-system allows for elimination of the endogenous TCR and orthotopic placement of TCR α- and β-chains. For complete details on the use and execution of this protocol, please refer to Schober et al. (2019) and Müller et al. (2021).

Keywords: Biotechnology and bioengineering; CRISPR; Health Sciences; Immunology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
Gene Editing* / methods
Humans
Receptors, Antigen, T-Cell / genetics
Receptors, Antigen, T-Cell, alpha-beta / genetics
Recombinational DNA Repair
T-Lymphocytes

Substances

Receptors, Antigen, T-Cell
Receptors, Antigen, T-Cell, alpha-beta