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Journal Article Ppublish 26 citations

PubMed: 38379101 DOI: 10.1038/s41587-024-02137-y

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

Nat Biotechnol 43(1):42-51

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

Affiliations

  • Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.
  • Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.

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 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.

MeSH

Mutagenesis, Risk, Biotechnology, Rna, Retroelements, Cells, Dna, Complementary, Genome, Transgenes

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