The CRISPR-Cas system enables the insertion of DNA transgenes at specific genomic sites through homology-directed repair (HDR). Gene editing has been utilized to redirect T cells towards tumor cells by introducing a chimeric antigen receptor (CAR). Additional genetic modifications (knock-out, epitope editing) can enhance the ability of CAR T cells to resist exhaustion and immunosuppressive environments. Base editing (BE) is a precise tool for gene disruption via splice site inactivation or stop codon introduction, minimizing genotoxicity associated with DNA double-strand breaks.

In this study, we propose a system for base editing mediated knock-in (BEKI) that utilizes the nCas9 domain of the base editor to introduce paired nicks on opposite DNA strands, promoting HDR-mediated transgene insertion. Across different base editors, we demonstrate precise CAR knock-in into a variety of genes (such as TRAC, CD3ζ and B2M). We found that guide RNA orientation, specific base changes within the editing window and nick positioning impacted the BEKI efficiency. Furthermore, pharmacological HDR enhancement improved CAR knock-in, leading to CAR⁺ rates exceeding 50%.

Due to the favorable safety profile of base editors, the BEKI system is well-suited for clinical applications requring transgene knock-in with simultaneous (multiplex) gene knock-outs. Using BEKI, we generated quintuple gene-edited CAR T cells to enhance cytokine secretion, mediate selective drug resistance and allow off-the shelf use. Multiplex BEKI engineered CAR T cells did not show chromosomal translocations between the targeted loci. This streamlined approach, using a single gene editing tool for multiplex editing, offers significant potential for clinical translation.