Team:NTU-Singapore/Measurement

Best measurement

The most straightforward method to measure RNA editing events is by traditional Sanger sequencing. However, Sanger sequencing has low sensitivity, low depth of coverage and low scalability. To circumvent these issues, we are exploring the use of next generation sequencing (NGS) methods such as amplicon sequencing to detect and quantify RNA editing events mediated by our CasRx-ADAR constructs. Although Sanger sequencing remains an important adjunct in our measurement methods, amplicon sequencing has higher depth of coverage enabling higher sensitivity and higher sample throughput as compared to Sanger sequencing. We also incorporated a luciferase reporter assay into our experimental design to measure the relative RNA editing efficiency of our CasRx-ADAR constructs. As part of our goal to develop a CRISPR-based RNA editing toolkit with high specificity (i.e. low off-targets) and editing efficiency, we utilised orthogonal methods to calculate the A-to-I editing. For Sanger sequencing we utilised the formula of RNA Editing% = Peak height of G/(G+A) x 100%, as published in various sources.