2024-08-26 カリフォルニア大学サンディエゴ校(UCSD)
<関連情報>
- https://today.ucsd.edu/story/closing-the-rna-loop-holds-promise-for-more-stable-effective-rna-therapies
- https://www.nature.com/articles/s41551-024-01245-z
試験管内および人工的に環状化されたRNAを用いた頑健なゲノムおよび細胞工学 Robust genome and cell engineering via in vitro and in situ circularized RNAs
Michael Tong,Nathan Palmer,Amir Dailamy,Aditya Kumar,Hammza Khaliq,Sangwoo Han,Emma Finburgh,Madeleine Wing,Camilla Hong,Yichen Xiang,Katelyn Miyasaki,Andrew Portell,Joseph Rainaldi,Amanda Suhardjo,Sami Nourreddine,Wei Leong Chew,Ester J. Kwon & Prashant Mali
Nature Biomedical Engineering Published:26 August 2024
DOI:https://doi.org/10.1038/s41551-024-01245-z
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.
