Engineered T7 RNA polymerase reduces dsRNA formation by lowering terminal transferase and RNA‐dependent RNA polymerase activities

T7 RNA polymerase (RNAP), the preferred tool for in vitro transcription (IVT), can generate double‐stranded RNA (dsRNA) by‐products that elicit immune stress and pose safety concerns. By combining the molecular beacon‐based fluorescence‐activated droplet sorting (FADS) utilized for random library screening with site‐directed mutagenesis aimed at facilitating conformational changes in T7 RNAP, we successfully identified four mutants that exhibit reduced dsRNA content: M1 (V214A), M7 (F162S/A247T), M11 (K180E) and M14 (A70Q). Furthermore, the combinatorial mutant M17 (A70Q/F162S/K180E) exhibited significantly reduced dsRNA production under various conditions. Cellular experiments confirm the application potential of the mutants, displaying mitigated immune stress responses and enhanced protein translation compared to the wild‐type protein. We then observed a close correlation between the production of dsRNA and the terminal transferase and RNA‐dependent RNAP (RDRP) activities of T7 RNAP. The terminal transferase activity adds several nucleotides to the terminus of RNAs, while the RDRP activity extends the complementary region formed by self‐pairing. In summary, we developed a novel approach for engineering T7 RNAP and demonstrated its potential in screening for T7 RNAP variants with reduced dsRNA production or improved product integrity.