An Optimized Workflow for <i>In Vitro</i> Transcription of Single Guide RNAs Minimizes Innate Immune Activation

transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.