Robust genome and cell engineering via in vitro and in situ circularized RNAs.
Publication Year:
2024
PubMed ID:
39187662
Funding Grants:
Public Summary:
Circular RNAs (cRNAs) can enhance RNA stability and persistence, but effective and scalable methods to create them have been limited. In this study, they present two new methods for generating cRNAs: one using in vitro circularization with group II introns, and another using in-cell circularization with the RtcB protein. They also developed simple purification protocols that produce high yields of cRNAs (40-75%) with minimal immune response. These methods have broad applications in stem cell engineering and genome targeting, including with CRISPR-Cas9 and zinc finger proteins. Notably, cRNAs showed improved expression and stability compared to traditional linear RNAs in non-dividing cells like heart and nerve cells, making them valuable for gene therapies. Also demonstrated how cRNAs can be used for more effective gene editing and protein engineering while reducing immune reactions. This new cRNA toolkit has great potential for advancing scientific research and developing new therapeutics.
Scientific 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.
