Advances in evolved T7 RNA polymerases for expanding the frontiers of enzymatic nucleic acid synthesis

Abstract In vitro RNA synthesis technologies are crucial in developing therapeutic RNA drugs, such as mRNA vaccines and RNA interference (RNAi) therapies. Enzymatic RNA synthesis, recognized for its sustainability and efficiency, enables the production of extensive RNA sequences under mild conditions. Among the enzymes utilized, T7 RNA polymerase is distinguished by its exceptional catalytic efficiency, enabling the precise and rapid transcription of RNA from DNA templates by recognizing the specific T7 promoter sequence. With the advancement in clinical applications of RNA‐based drugs, there is an increasing demand for the synthesis of chemically modified RNAs that are stable and resistant to nuclease degradation. To this end, researchers have applied directed evolution to broaden the enzyme's substrate scope, enhancing its compatibility with non‐canonical substrates and reducing the formation of by‐products. This review summarizes the progress in engineering T7 RNA polymerase for these purposes and explores prospective developments in the field.