A scalable pipeline for SARS-CoV-2 replicon construction based on de-novo synthesis

Replicons are synthetic viral RNA molecules that recapitulate the self-replicating activities of the virus but are missing its infectivity potential. Here, we report on a scalable pipeline to generate a replicon of any SARS-CoV-2 strain using de-novo synthesis. Our pipeline relies only on publicly available sequencing data without requiring access to any material, simplifying logistical and bureaucratic issues of sample acquisition. In addition, our system retains the nucleotide sequence of most of the SARS-CoV-2 full genome and therefore better captures its underlying genomic and biological functions as compared to the popular pseudotypes or any replicon system published to-date. We utilized our system to synthesize a SARS-CoV-2 non-infectious version of the Beta strain. We then confirmed that the resulting RNA molecules are non-infectious and safe to handle in a BSL2/CL2 facility. Finally, we show that our replicon can be specifically inhibited by molnupiravir and RNAi treatments, demonstrating its utility for drug research and development. ### Competing Interest Statement A.C.B, O.W., R.R., I.F., and Y.E. are employees of Eleven Therapeutics. E.L. and S.C. are employees of Twist Bioscience. Y.E. is a part time employee of the Reichman University (IDC Herzliya) and of MyHeritage LTD. He serves as an SAB member of ArcBio.