Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration

The use of adeno-associated viruses (AAVs) as donors for homology-directed repair (HDR)-mediated genome engineering is limited by safety issues, manufacturing constraints and restricted packaging limits. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors. dsDNA is known to have low efficiency and high cytotoxicity, while lssDNA is challenging for scaled manufacture. In this study, we developed a non-viral genome writing catalyst (GATALYST) system that allows production of circular single-stranded DNAs (cssDNAs) up to approximately 20 kilobases as donor templates for highly efficient precision transgene integration. cssDNA donors enable knock-in efficiency of up to 70% in induced pluripotent stem cells (iPSCs) and improved efficiency in multiple clinically relevant primary immune cell types and at multiple genomic loci implicated for clinical applications with various nuclease editor systems. The high precision and efficiency in chimeric antigen receptor (CAR)-T and natural killer (NK) cells, improved safety, payload flexibility and scalable manufacturability of cssDNA shows potential for future applications of genome engineering.