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 genes, and supports the integration of large synthetic exons (longer than 5 kb), of alternative splicing architectures that preserve endogenous gene expression, and of synthetic promoters allowing for endogenous or user-defined gene regulation. Non-viral intron knock-ins expand the range of targetable genomic sites and provide a simplified and high-throughput strategy for selecting edited primary human T cells.