Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair
Moosmann, C., Muller, T. R., Busch, D. H., and Schober, K. (2022). STAR Protoc 3, 101031. doi: 10.1016/j.xpro.2021.101031
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).