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 持久性，但缺乏简单且可扩展的方法来实现这一目标。在这里，我们报告了两种促进寻找环状 RNA （cRNA） 的方法：通过使用 II 组内含子的体外环化开发的 cRNA，以及通过普遍表达的 RtcB 蛋白的细胞内环化开发的 cRNA。我们还报道了简单的纯化方案，可实现高 cRNA 产量 （40-75%），同时保持低免疫反应。这些方法和方案促进了干细胞工程的广泛应用，以及通过锌指蛋白和 CRISPR-Cas9 进行稳健的基因组和表观基因组靶向。值得注意的是，与线性加帽 RNA 相比，携带脑心肌炎内部核糖体进入的 cRNA 在心肌细胞和神经元中实现了稳健的表达和持久性，这突出了 cRNA 在这些非分裂细胞中的效用。我们还描述了通过以 cRNA 形式递送的去免疫 Cas9 和用于去免疫蛋白变体组合筛选的长程多重蛋白质工程方法进行基因组靶向，该方法实现了 cRNA 递送蛋白中表达持久性和免疫原性之间的兼容性。cRNA 工具集将有助于治疗学的研究和开发。