An Optimized Workflow for In Vitro Transcription of Single Guide RNAs Minimizes Innate Immune Activation

Area of Science:

• Molecular Biology
• Immunology
• Gene Editing

Background:

• CRISPR interference (CRISPRi) relies on single guide RNAs (sgRNAs).
• sgRNAs produced via in vitro transcription (IVT) can activate innate immune responses, complicating functional studies.
• Standard methods like 5'-triphosphate removal are insufficient to fully suppress immune activation.

Purpose of the Study:

• To evaluate and mitigate innate immune activation caused by IVT-derived sgRNAs in CRISPRi.
• To optimize the IVT process for producing immunologically inert sgRNAs without compromising knockdown efficiency.

Main Methods:

• Assessed innate immune activation by IVT sgRNAs in CRISPRi systems.
• Tested modifications to IVT reaction conditions, including sodium chloride and urea supplementation.
• Evaluated sgRNA yield, knockdown efficiency, and double-stranded RNA (dsRNA) by-product levels.
• Integrated optimized IVT with phosphatase treatment for a comprehensive workflow.

Main Results:

• Enzymatic removal of 5'-triphosphate groups alone did not consistently prevent immune activation.
• Supplementation with sodium chloride or urea significantly attenuated immune responses.
• An optimized condition using 0.15 M NaCl reduced dsRNA by-products and immune activation.
• The optimized workflow maintained high sgRNA-mediated target knockdown efficiency.

Conclusions:

• Optimized IVT conditions, particularly with NaCl supplementation, effectively minimize innate immune activation from sgRNAs.
• A combined approach of optimized IVT and phosphatase treatment provides a scalable workflow for producing immunologically silent sgRNAs.
• This optimized workflow preserves the functional efficiency of CRISPRi gene knockdown.