Robust genome and cell engineering via in vitro and in situ circularized RNAs.

Autor: Tong M; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Palmer N; Biological Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA., Dailamy A; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Kumar A; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Khaliq H; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Han S; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Finburgh E; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Wing M; Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA., Hong C; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Xiang Y; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Miyasaki K; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Portell A; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Rainaldi J; Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA., Suhardjo A; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Nourreddine S; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Chew WL; Genome Institute of Singapore (GIS), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore., Kwon EJ; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA., Mali P; Department of Bioengineering, University of California San Diego, La Jolla, CA, USA. pmali@ucsd.edu.
Jazyk: angličtina
Zdroj: Nature biomedical engineering [Nat Biomed Eng] 2024 Aug 26. Date of Electronic Publication: 2024 Aug 26.
DOI: 10.1038/s41551-024-01245-z
Abstrakt: 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.
(© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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