Directed Evolution of T7 RNA Polymerase Minimizes dsRNA By-product and Enables High-Fidelity mRNA Synthesis for Demanding Therapeutic Applications.

T7 RNA polymerase (T7 RNAP) is the most widely used enzyme for synthesizing therapeutic mRNA. However, RNA transcribed by T7 RNAP often contains double-stranded RNA (dsRNA) by-products that trigger innate immune responses and complicate purification. Here, we report an engineered T7 RNAP variant, M30, which exhibits higher catalytic efficiency and reduced dsRNA by-product formation. M30 was developed through 4 rounds of directed evolution using an ultrahigh-throughput aptamer-based fluorescence-activated droplet sorting system. M30 displays a 10-fold increase in catalytic efficiency over wild-type T7 RNAP at 37 °C, along with markedly enhanced thermostability and approximately 10-fold lower production of dsRNA by-products. mRNAs synthesized with M30 achieve efficient protein expression in human cells and in mice, while eliciting reduced immunogenicity compared with mRNAs produced by wild-type T7 RNAP. Biophysical assays and structural analyses suggest that these improvements result from increased DNA template binding affinity and decreased RNA binding affinity. Together, these features make M30 a promising catalyst for high-quality therapeutic mRNA production.