美国加州大学伯克利分校Kathleen Collins小组利用真核逆转录元件蛋白将转基因插入人类安全港基因座。这一研究成果于2024年2月20日在线发表在国际学术期刊《自然—生物技术》上。

研究人员描述了精确的RNA介导的转基因插入（PRINT），这是一种特定位点先导反转录的方法，可将转基因合成直接导入多拷贝安全港位点的基因组中。PRINT使用两种体外转录的RNA：编码禽类R2逆转录素蛋白的信使RNA和编码经验证长度达4kb的转基因模板RNA。R2蛋白协同识别目标位点，在精确的位置切断一条链，并启动互补DNA合成，以实现稳定的转基因插入。

在培养的人类原代细胞系中，50%以上的细胞可以获得多个2kb的转基因，其中50%以上是全长转基因。PRINT的优势包括无基因组外DNA，限制了有害突变和先天免疫反应的风险，以及纯RNA运送的相对低成本、快速生产和可扩展性。

据了解，目前将自主转基因插入基因组的方法，如CRISPR-Cas9或基于病毒的策略，都有其局限性，包括效率低和高风险的非靶向基因组突变。

附：英文原文

Title: Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci

Author: Zhang, Xiaozhu, Van Treeck, Briana, Horton, Connor A., McIntyre, Jeremy J. R., Palm, Sarah M., Shumate, Justin L., Collins, Kathleen

Issue&Volume: 2024-02-20

Abstract: 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 4kb. 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 2kb 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.

DOI: 10.1038/s41587-024-02137-y

Source: https://www.nature.com/articles/s41587-024-02137-y