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. 2020 Jun 1;9(6):1367. doi: 10.3390/cells9061367

Figure 2.

Figure 2

Schematic of orthotopic TCR replacement through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. The shown homology-directed repair (HDR) template is designed in a manner that the complete αβ TCR is integrated into the endogenous T-cell receptor alpha constant (TRAC) gene locus upon a CRISPR/Cas9-mediated double-strand break. Simultaneously, T-cell receptor beta constant (TRBC) is edited, and non-homologous end joining leads to knock-out of the endogenous TCR β-chain. Cleavage of 2A elements leads to intracellular expression of transgenic TCR chains and a truncated endogenous TCR α-chain; thus, at the surface, only the transgenic αβ TCR is expressed. LHA, left homology arm. RHA, right homology arm. P2A, T2A, self-cleaving peptides. VJ/VDJ, variable parts of TCR. Cα, TRAC. Cβ, TRBC. For more information, see References [26,31].