Improved method for making guide RNAs reduces immune response in lab tests

Supplementation with 0.15 M NaCl during the in vitro transcription process significantly reduces immune activation triggered by CRISPR interference guide RNAs.

• Innate immune responses can be activated by single guide RNAs generated through in vitro transcription.
• Simply removing the 5'-triphosphate group from these RNAs does not reliably eliminate the immune response.
• Adjusting IVT reaction conditions, particularly by adding sodium chloride or urea, can further reduce immune activation.
• The optimized IVT condition with 0.15 M NaCl resulted in lower concentrations of double-stranded RNA by-products.
• Integrating NaCl-supplemented IVT with phosphatase treatment leads to an efficient workflow that minimizes immune responses while retaining target knockdown effectiveness.

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