Robust genome and cell engineering via in vitro and in situ circularized RNAs.
Journal
Nature biomedical engineering
ISSN: 2157-846X
Titre abrégé: Nat Biomed Eng
Pays: England
ID NLM: 101696896
Informations de publication
Date de publication:
26 Aug 2024
26 Aug 2024
Historique:
received:
29
01
2023
accepted:
24
07
2024
medline:
27
8
2024
pubmed:
27
8
2024
entrez:
26
8
2024
Statut:
aheadofprint
Résumé
Circularization can improve RNA persistence, yet simple and scalable approaches to achieve this are lacking. Here we report two methods that facilitate the pursuit of circular RNAs (cRNAs): cRNAs developed via in vitro circularization using group II introns, and cRNAs developed via in-cell circularization by the ubiquitously expressed RtcB protein. We also report simple purification protocols that enable high cRNA yields (40-75%) while maintaining low immune responses. These methods and protocols facilitate a broad range of applications in stem cell engineering as well as robust genome and epigenome targeting via zinc finger proteins and CRISPR-Cas9. Notably, cRNAs bearing the encephalomyocarditis internal ribosome entry enabled robust expression and persistence compared with linear capped RNAs in cardiomyocytes and neurons, which highlights the utility of cRNAs in these non-dividing cells. We also describe genome targeting via deimmunized Cas9 delivered as cRNA and a long-range multiplexed protein engineering methodology for the combinatorial screening of deimmunized protein variants that enables compatibility between persistence of expression and immunogenicity in cRNA-delivered proteins. The cRNA toolset will aid research and the development of therapeutics.
Identifiants
pubmed: 39187662
doi: 10.1038/s41551-024-01245-z
pii: 10.1038/s41551-024-01245-z
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : U.S. Department of Health & Human Services | National Institutes of Health (NIH)
ID : OT2OD032742
Organisme : U.S. Department of Health & Human Services | National Institutes of Health (NIH)
ID : DP2NS111507
Organisme : U.S. Department of Defense (United States Department of Defense)
ID : W81XWH-22-1-0401
Organisme : U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)
ID : U54CA274502
Organisme : American Heart Association (American Heart Association, Inc.)
ID : AHA 916973
Informations de copyright
© 2024. The Author(s), under exclusive licence to Springer Nature Limited.
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