Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Nature biotechnology, 2024-02

Ort / Verlag

United States

Erscheinungsjahr

2024

Beschreibungen/Notizen

• 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.