Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering

The toolbox for genome editing in basic research and therapeutic applications is rapidly expanding. While efficient targeted gene ablation using nuclease editors has been demonstrated from bench to bedside, precise transgene integration remains a technical challenge. AAV6 has been a prevalent donor carrier for homology-directed repair (HDR) mediated genome engineering but has reported safety issues, manufacturing constraints, and restricted applications due to its 4.5 Kb packaging limit. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors. Both dsDNA and lssDNA have been previously demonstrated to have low efficiency and cytotoxicity. Here, we developed a non-viral genome writing catalyst (GATALYST™) system which allows production of ultrapure, minicircle single-stranded DNAs (cssDNAs) up to ∼20 Kb 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), superior efficiency in multiple clinically relevant primary cell types, and at multiple genomic loci implicated for clinical applications with various nuclease editor systems. When applied to immune cell engineering, cssDNA engineered CAR-T cells exhibit more potent and durable anti-tumor efficacy than those engineered from AAV6 viral vectors. The exceptional precision and efficiency, improved safety, payload flexibility, and scalable manufacturability of cssDNA unlocks the full potential of genome engineering with broad applications in therapeutic development, disease modeling and other research areas. Highlights Scalable production of minicircle ssDNA (cssDNA) with highly engineered phagemid system Genome writing catalyst (GATALYST™) system with cssDNA donor template demonstrates superior efficiency and safety in various cell types and genomic loci GATALYST gene writing system enables ultra-large transgene integration cssDNA engineered CAR-T outperforms AAV engineered CAR-T with superior anti-tumor function