Non-viral intron knock-ins for targeted gene integration into human T cells and for T-cell selection.

ABSTRACT: Current methods for the precise integration of DNA sequences into the genome of human T cells predominantly target exonic regions, which limits the choice of integration site and requires complex cell-selection strategies. Here we show that non-viral intron knock-ins for incorporating synthetic exons into endogenous introns enable efficient gene targeting and selective gene knockout in successfully edited cells. In primary human T cells, the knock-in of a chimaeric antigen receptor (CAR) into the T-cell receptor alpha constant locus facilitated the purification of more than 90% CAR⁺ T cells via the negative selection of T-cell-receptor-negative cells. The method is scalable, applicable across intronic sites, as we show for introns within four distinct endogenous surface-receptor

SUBMITTER: Roth TL 

PROVIDER: S-EPMC12261302 | biostudies-literature | 2025 Aug

REPOSITORIES: biostudies-literature