Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

由于精确操纵DNA修复途径存在困难，由双链断裂触发的大转基因的高保真靶向整合本质上是低效的。在这里，我们利用 Prime 编辑器设计了一种强大的敲入 (KI) 策略，称为引发微同源物辅助整合 (PAINT)，该策略利用逆转录单链微同源物来增强不同类型细胞中的靶向 KI。 PAINT 的改进版本（指定为 PAINT 3.0）可最大限度地提高编辑效率并最大限度地减少脱靶整合，特别是在处理无疤框内 KI 时。使用 PAINT 3.0，我们将报告转基因靶向看家基因，编辑效率高达 80%，比传统同源定向修复方法高 10 倍以上。此外，使用 PAINT 3.0 插入 2.5 kb 转基因，在几个治疗相关的基因组位点上实现了高达 85% 的 KI 频率，这表明其具有临床应用的潜力。最后，PAINT 3.0能够在原代T细胞中实现高效的非病毒基因组靶向，并产生具有特定肿瘤杀伤能力的功能性CAR-T细胞。因此，我们确定 PAINT 方法是用于大型转基因整合的强大基因编辑工具，并可能为细胞和基因治疗以及基因组写入技术开辟新途径。