A digital PCR-based protocol to detect and quantify RNA editing events at hotspots

Programmable RNA base editors are excellent tools to modify RNA transcripts at precise locations or designated hotspots. Post editing, it is critical to provide precise and accurate quantification for the successful editing events to determine the activity and efficiency of the RNA base editor enzymes used. Digital PCR is a specialized tool which facilitates accurate absolute quantification of nucleic acid targets. By splitting a bulk PCR reaction across many micro-reactions, robust and reproducible absolute quantification is possible without the need for a standard curve. APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare RNA mutations induced by APOBEC3A by digitizing a bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events caused by APOBEC3A in cell lines or patient samples.

 

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