Homology-directed repair (HDR) DNA template design

DNA templates were designed in silico and synthesized by GeneArt and presented in pMK vectors (Life Technologies, Thermo Fisher Scientific). The structure of the HDR template was designed following the previously published method (79). The full length of α- and β-chains of the TCR to be introduced, self-cleaving peptides P2 to ensure the separation of both TCR-chains, and a poly-A tail (bGHpA) were flanked by left and right homology arms (LHA and RHA). Both α- and β-chains consist of the human variable regions and the murine constant region with an additional disulfide bond (80), to facilitate the identification of re-expressed transgenic TCR with anti-mouse TCRβ antibody.