Abstract：
All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.
摘要：
全RNA介导的靶向基因整合方法,降低免疫原性，非病毒载体的有效交付能力，和低风险的随机诱变，迫切需要下一代基因添加技术。天然存在的R2反转录转座子由于其位点特异性整合概况而在这种情况下具有希望。这里,我们系统分析了R2元件的生物多样性，并在哺乳动物细胞中筛选了几个能够全长基因插入的R2直向同源物。使用组合的供体RNA和蛋白质工程获得了稳健的R2系统基因整合效率。重要的是,全RNA递送的工程R2系统显示出有效的整合活性，在小鼠胚胎中效率超过60%。无偏高通量测序表明工程化的R2系统表现出高的靶整合特异性(99%)。总之,我们的研究为基于命中并运行的靶向DNA整合和进一步优化反转录转座子系统的见解的应用提供了工程化的R2工具。
