Figure 5.

Genetic engineering of T cells via CRISPR-Cas9-mediated TCR KI

Activated T cells with endogenous TCRs (orange) are required for successful editing. In preparation for electroporation, the linearized double-stranded DNA template is mixed with hTRAC and hTRBC RNPs. T cells resuspended in electroporation buffer are mixed with the DNA-RNPs mixture and electroporated. Respective crRNAs direct the Cas9 nuclease either to the TRAC or TRBC locus and allow for CRISPR-Cas9-mediated DNA double-strand breaks (right). At the TRAC locus, homology-directed repair (HDR) results in the targeted KI of the HDR template DNA encoding the full transgenic αβ TCR which leads to a simultaneous KO of the endogenous TRAC (black triangle). At the TRBC locus, the double-strand break is resolved by non-homologous end-joining leading to the KO of the endogenous TRBC (black cross). On the surface of edited T cells only the transgenic TCR (blue) is expressed.