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.

使用腺相关病毒 （AAV） 作为同源定向修复 （HDR） 介导的基因组工程的供体受到安全问题、制造限制和限制包装限制的限制。非病毒靶向基因敲入主要依赖于双链 DNA （dsDNA） 和线性单链 DNA （lssDNA） 供体。已知 dsDNA 具有低效率和高细胞毒性，而 lssDNA 难以进行规模化生产。在这项研究中，我们开发了一种非病毒基因组写入催化剂 （GATALYST） 系统，该系统允许生产高达约 20 kb 的环状单链 DNA （cssDNA） 作为供体模板，以实现高效、精确的转基因整合。cssDNA 供体可在诱导多能干细胞 （iPSC） 中实现高达 70% 的敲入效率，并提高多种临床相关的原代免疫细胞类型和与各种核酸酶编辑器系统临床应用相关的多个基因组位点的效率。嵌合抗原受体 （CAR）-T 和自然杀伤 （NK） 细胞的高精度和高效性、cssDNA 的安全性、有效载荷灵活性的提高和可扩展的可制造性显示了基因组工程未来应用的潜力。