Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

中文翻译：

---

用于同时敲入和多基因敲除的适体介导的碱基编辑平台，用于同种异体 CAR-T 细胞的生成


基因编辑技术有望实现下一代过继性细胞疗法。在依赖核酸酶活性的传统基因编辑平台中，例如成簇的规则间隔的短回文重复序列 CRISPR 相关蛋白 9 (CRISPR-Cas9)，可以有效地引入基因修饰；然而，这些修饰是通过 DNA 双链断裂 (DSB) 的产生而发生的，并可能导致不必要的基因组改变和基因毒性。在这里，我们应用一种新型模块化RNA适体介导的Pin-point碱基编辑平台，在人类原代T细胞中同时引入多个基因敲除和转基因的位点特异性整合。与传统的 CRISPR-Cas9 系统相比，我们在所有靶位点都表现出高编辑效率和纯度，并且显着降低了染色体易位的频率。嵌合抗原受体的位点特异性敲入和多重基因敲除在单次干预内实现，无需额外的序列靶向成分。高效、精确地执行复杂基因组编辑的能力凸显了 Pin-point 平台在一系列先进细胞疗法中应用的潜力。