Current approaches for inserting autonomous transgenes into the genome, such as CRISPR–Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

目前将自主转基因插入基因组的方法（例如 CRISPR-Cas9 或基于病毒的策略）存在局限性，包括效率低下和非靶向基因组诱变的高风险。在这里，我们描述了精确的 RNA 介导的转基因插入 (PRINT)，这是一种位点特异性引发的逆转录方法，可将转基因合成直接引导到基因组的多拷贝安全港位点。PRINT 使用两种体外转录 RNA 的递送：编码禽类 R2 逆转录元件蛋白的信使 RNA 和编码经验证长度高达 4 kb 的转基因的模板 RNA。R2 蛋白协同识别靶位点，在精确位置切开一条链，并启动互补 DNA 合成以实现稳定的转基因插入。对于培养的人类原代细胞系，超过 50% 的细胞可以获得多个 2 kb 转基因，其中超过 50% 是全长的。PRINT 的优势包括不存在基因组外 DNA、限制有害突变和先天免疫反应的风险，以及仅 RNA 递送的成本相对较低、快速生产和可扩展性。