Tong Michael, Palmer Nathan, Dailamy Amir, Kumar Aditya, Khaliq Hammza, Han Sangwoo, Finburgh Emma, Wing Madeleine, Hong Camilla, Xiang Yichen, Miyasaki Katelyn, Portell Andrew, Rainaldi Joseph, Suhardjo Amanda, Nourreddine Sami, Chew Wei Leong, Kwon Ester J, Mali Prashant
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Biological Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA.
Nat Biomed Eng. 2025 Jan;9(1):109-126. doi: 10.1038/s41551-024-01245-z. Epub 2024 Aug 26.
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.
环化可以提高RNA的稳定性，但目前缺乏实现这一目标的简单且可扩展的方法。在此，我们报告了两种有助于制备环状RNA（cRNA）的方法：通过使用II组内含子进行体外环化开发的cRNA，以及通过普遍表达的RtcB蛋白进行细胞内环化开发的cRNA。我们还报告了简单的纯化方案，可实现高cRNA产量（40-75%），同时保持低免疫反应。这些方法和方案有助于在干细胞工程中广泛应用，以及通过锌指蛋白和CRISPR-Cas9对基因组和表观基因组进行强力靶向。值得注意的是，与线性加帽RNA相比，携带脑心肌炎病毒内部核糖体进入位点的cRNA在心肌细胞和神经元中能够实现强劲的表达和稳定，这凸显了cRNA在这些非分裂细胞中的实用性。我们还描述了通过作为cRNA递送的去免疫化Cas9进行基因组靶向，以及一种用于组合筛选去免疫化蛋白变体的远程多重蛋白质工程方法，该方法能够使cRNA递送蛋白的表达稳定性与免疫原性之间实现兼容。cRNA工具集将有助于研究和治疗方法的开发。
