Site-specific genome engineering of primary human natural killer cells for programmable anti-tumor function

Allain, V.; Rothrock, A. G.; Bernard, P.-L.; Nyberg, W. A.; Talbot, A.; Muldoon, J. J.; Chung, J.-Y.; To, A.; Chang, C. R.; Kimmerly, G. R.; Liu, C.; Tsao, T.; Mortazavi, Y.; Seo, J.; Li, Z.; Shemesh, A.; Schmidt, R.; Ward, C. C.; Marson, A.; Lanier, L. L.; Aguilar, O. A.; Eyquem, J.

doi:10.1101/2025.10.05.680386

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bioRxiv · 10.1101/2025.10.05.680386

Site-specific genome engineering of primary human natural killer cells for programmable anti-tumor function

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Abstract

Natural killer (NK) cells are emerging as a promising platform for engineered adoptive cell therapies. However, gene editing in NK cells remains challenging, and more effective strategies are needed. Here, we established a robust, feeder-free, and modular workflow for genome engineering in primary human NK cells, combining CRISPR/Cas9 with AAV6-mediated transgene delivery. Efficient site-specific transgene integration was achieved at various loci and can be coupled with concurrent disruption of the target locus in a single editing step. Furthermore, transgene expression was tunable according to the integration site and promoter. We applied this strategy to target a chimeric antigen receptor (CAR) transgene to a panel of inhibitory NK receptor loci, establishing a synergistic approach to enhance anti-tumor activity and facilitate the reliable comparison of CAR variants without expression bias. We identified TIGIT as an ideal locus that supports strong CAR expression and anti-tumor function. This genome engineering framework, which leverages multiple, complementary and precisely controlled genetic edits, can support the rational design of future NK-cell therapies tailored to overcome cell-intrinsic limitations and tumor-specific barriers.

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Allain, V., Rothrock, A. G., Bernard, P.-L., Nyberg, W. A., Talbot, A., Muldoon, J. J., Chung, J.-Y., To, A., Chang, C. R., Kimmerly, G. R., Liu, C., Tsao, T., Mortazavi, Y., Seo, J., Li, Z., Shemesh, A., Schmidt, R., Ward, C. C., Marson, A., Lanier, L. L., Aguilar, O. A., Eyquem, J.. 2025-10-06. Site-specific genome engineering of primary human natural killer cells for programmable anti-tumor function. https://doi.org/10.1101/2025.10.05.680386

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