Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
20 Feb 2024
20 Feb 2024
Historique:
received:
14
12
2022
accepted:
10
01
2024
medline:
21
2
2024
pubmed:
21
2
2024
entrez:
21
2
2024
Statut:
aheadofprint
Résumé
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.
Identifiants
pubmed: 38379101
doi: 10.1038/s41587-024-02137-y
pii: 10.1038/s41587-024-02137-y
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NHLBI NIH HHS
ID : DP1 HL156819
Pays : United States
Informations de copyright
© 2024. The Author(s).
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