Multiplex Genome Editing to Dissect Complex Viral Phenotypes

Functional cDNA clones have been incredibly useful for performing “reverse genetic” experiments on (+)-strand RNA viruses. However, considerable effort is usually put into manipulating a viral cDNA clone in vitro to create a genotype of interest before the phenotype could be examined in cell culture or in vivo. In collaboration with Dr. Farren Isaacs (Yale Systems Biology Institute and Department of Molecular, Cellular and Developmental Biology), we have been novel applying synthetic biology approaches to rewrite the standard workflow for (+)-strand RNA virus reverse genetics, helping to move us from the molecular biology era into a high-throughput genomics era. Now, by offloading the cDNA editing process to yeast via a technique called eukaryotic multiplex automated genome editing (eMAGE), we have shifted this paradigm. With this set of programmable, in vivo genome-editing tools, it becomes trivial to generate a single mutation or dozens of mutations, or all combinations thereof (i.e., multiplex combinatorial genomics), simultaneously.

These improvements to the standard workflow of viral reverse genetics means that a researcher can focus their attention on what matters: the phenotype. As proof of concept, we have developed an eMAGE platform to rapidly edit the SARS-CoV-2 cDNA and have rescued numerous mutant viruses and non-infectious replicons. We are utilizing these tools to examine how mutations outside of the viral Spike gene contribute to improved fitness of SARS-CoV-2 variants of concern, as well as examining the genetic barriers to drug resistance. This powerful technology is also being applied to other (+)-strand RNA viruses that we study.