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

Carolin Moosmann,* Thomas R. Mueller, Dirk H. Busch, and Kilian Schober
Source: STAR Protoc
Publication Date: (2022)
Issue: 3: 101031
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
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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 a- and b-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 a- and b-chains.
For complete details on the use and execution of this protocol, please refer to Schober et al. (2019) and Mueller et al. (2021).