Lipid Nanoparticles Enable High-Efficiency CRISPR HDR-Mediated Gene Insertions In Primary Human T Cells

By R Geczy, Cytiva; M Swaminathan, Cytiva; HH Ly, Cytiva; M Novin, Cytiva; A Thomas, Cytiva; D Sirskyj, Cytiva; P Marcus, Cytiva; BC Thommandru, IDT; SH Kassim, Danaher; and SJ Clarke, Cytiva

Non-viral delivery methods are transforming cell and gene therapy by addressing the limitations of viral vectors and electroporation. Lipid nanoparticles (LNPs) offer a scalable, chemically defined approach for precise gene editing in primary human T cells while maintaining high cell viability. In recent evaluations, LNPs achieved an average of 31% homology-directed repair (HDR) at the CD5 locus with 96% relative viability across multiple donors—without HDR enhancers. This streamlined, one-step workflow integrates seamlessly into large-scale manufacturing and supports co-delivery of Cas9 mRNA, sgRNA, and ssODN donors for targeted insertions. Variables such as nucleic acid ratios, basal media, and HDR enhancers were explored, reveaaling opportunities to further boost efficiency up to 47%. For researchers seeking safer, cost-effective alternatives to viral systems, LNPs present a compelling solution for next-generation cell therapy development.

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