CRISPR RNAs trigger innate immune responses in human cells

Area of Science:

• Molecular Biology
• Immunology
• Gene Editing

Background:

• CRISPR-Cas9 and Cpf1 systems are powerful gene editing tools.
• In vitro transcribed guide RNAs (gRNAs) can elicit unintended cellular responses.
• The 5 -triphosphate group on gRNAs is implicated in triggering innate immunity.

Purpose of the Study:

• To investigate the immunogenicity of in vitro transcribed 5 -triphosphate gRNAs (5 -ppp gRNAs).
• To evaluate the efficiency and safety of 5 -hydroxyl gRNAs for gene editing.
• To establish a method for improving the efficacy of in vitro transcribed gRNAs.

Main Methods:

• Treatment of in vitro transcribed gRNAs with a phosphatase to remove the 5 -triphosphate group.
• Co-transfection of modified gRNAs with Cas9 or Cpf1 into human and murine cells.
• Assessment of cell viability and innate immune responses (Type I interferon).
• Quantification of targeted mutagenesis in primary human CD4+ T cells.

Main Results:

• 5 -ppp gRNAs induced significant cytotoxicity (up to 80% cell death) via DDX58-mediated Type I interferon response.
• Phosphatase treatment to generate 5 -hydroxyl gRNAs abrogated innate immune activation.
• 5 -hydroxyl gRNAs, complexed with Cas9 or Cpf1, achieved high-efficiency (95%) targeted mutagenesis in human CD4+ T cells.
• Phosphatase treatment offers a cost-effective method to enhance gRNA activity and safety.

Conclusions:

• The 5 -triphosphate group on in vitro transcribed gRNAs is a key trigger of innate immune responses and cytotoxicity.
• Generating 5 -hydroxyl gRNAs, through enzymatic dephosphorylation, significantly improves their safety and efficiency for gene editing.
• This phosphatase treatment method presents a practical and cost-effective strategy to optimize CRISPR-based gene editing in mammalian cells.