Abstract
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.
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Data availability
All key reagents will be made available via Addgene. Source data are provided with this paper.
Code availability
The code is available at https://github.com/natepalmer/lorax.
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Acknowledgements
We thank members of the Mali lab for discussions, advice and help with experiments. We also thank T. Long, S. Brightman and A. Sutherland for their advice on performing the ELISpot assay. This work was generously supported by UCSD Institutional Funds, NIH grants (R01HG012351, OT2OD032742, R01NS131560, U54CA274502 and DP2NS111507), Department of Defense Grant (W81XWH-22-1-0401), a Longevity Impetus Grant from Norn Group, a UC San Diego Gene Therapy Initiative Grant (GTI-2024-018), and an American Heart Association Postdoctoral Fellowship (AHA 916973). This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (S10 OD026929). This work was performed in part at the San Diego Nanotechnology Infrastructure (SDNI) of UCSD, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (grant ECCS-2025752). Some schematics were created using BioRender.
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Conceptualization: M.T., N.P., A.K. and P.M. Experiments: M.T., N.P., A.K., A.D., H.K., S.H., E.F., M.W., C.H., Y.X., K.M., A.P., J.R., A.S., S.N. and P.M. Computational analyses: M.T. and N.P. Design: M.T., N.P., W.L.C., E.J.K. and P.M. Writing: M.T., N.P., A.K. and P.M. with input from all authors.
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The authors have filed patents based on this work. P.M. is a scientific co-founder of Shape Therapeutics, Navega Therapeutics, Pi Bio, Boundless Biosciences and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies. The other authors declare no competing interests.
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Tong, M., Palmer, N., Dailamy, A. et al. Robust genome and cell engineering via in vitro and in situ circularized RNAs. Nat. Biomed. Eng 9, 109–126 (2025). https://doi.org/10.1038/s41551-024-01245-z
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DOI: https://doi.org/10.1038/s41551-024-01245-z
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