Virus-Assisted Directed Evolution of Trnas (VADER) in Mammalian Cells

Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins in living cells, using engineered orthogonal aminoacyl-tRNA synthetase/tRNA pairs, has emerged as a powerful method to probe and engineer protein function. Despite its boundless potential in both in academia and industry, application of this technology in mammalian cells is currently limited by the poor intrinsic performance of the nonsense suppressor tRNA, which interfaces suboptimally with the heterologous translation system of the host cell. Directed evolution, a revolutionary method for the creation of biomolecules with novel and improved activities, has the potential to overcome this limitation. Indeed, improved suppressor tRNAs have been developed for enhanced ncAA incorporation in E. coli through directed evolution. However, the ability to perform similar directed evolution experiments in the context of mammalian cells remains poorly developed due to numerous technical challenges. By coupling the activity of the suppressor tRNA to the replication of a human virus, we have developed a platform for ‘virus-assisted’ directed evolution of tRNAs (VADER) that yielded tRNA variants with remarkably improved suppression efficiency in mammalian cells. This platform is currently being further extended to enable the directed evolution of a broad class of genetic parts in mammalian cells to advance the scope of synthetic biology in these cells.