Poster Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

N6-methyladenosine (m⁶A) is one of the most abundant internal RNA modifications that regulates multiple aspects of RNA metabolism. To probe the functional significance of specific m⁶A sites, dead CRISPR Cas13 (dCas13)-based tools tethered to m⁶A writer (METTL3) or eraser (ALKBH5) have been developed for site-specific editing. However, accurate quantification of m⁶A levels following editing remains technically challenging. Among available detection methods, single-base elongation- and ligation-based qPCR amplification (SELECT) is widely used due to its simplicity and accessibility (1). Despite its utility, we discover that SELECT can produce misleading results when used with dCas13 systems that transiently express guide RNAs (gRNAs). Specifically, plasmids expressing gRNAs with spacer sequences overlapping the m⁶A target site can bind to SELECT probes, leading to artificial qPCR signals. Although gRNAs targeting non-overlapping regions yield more reliable results, interference with the assay can still occur in a site-dependent manner. To overcome these limitations, we employed a high-resolution quantitative method: eTAM-Sanger/Deep sequencing (2), that is capable of measuring absolute levels of m⁶A changes at specific sites following dCas13-ALKBH5-mediated removal of m⁶A. Similar to the concept of DNA methylation detection using bisulfite-sequencing, eTAM utilises an evolved deaminase enzyme, TadA8.20, that converts all unmethylated adenosine to inosine, while methylated adenosine remains unconverted. This allows accurate detection and measurement of site-specific m⁶A following PCR and sequencing as only methylated A will remain unchanged. Following editing using dCas13-ALKBH5, we successfully and accurately measured a decrease in m⁶A levels up to 74% at established sites, including ACTB A1216. To enable rapid quantification of m⁶A levels following editing, we further developed a new eTAM-pyrosequencing assay, based on the same principle behind the use of pyrosequencer for DNA methylation detection. In summary, we have established a highly precise and robust workflow for the detection of methylation changes following m⁶A editing, which is critical towards elucidating the functions of physiological- and pathological-relevant m⁶A sites.

References:

1. Xiao Y, Wang Y, Tang Q, Wei L, Zhang X, Jia G. An elongation‐and ligation‐based qPCR amplification method for the radiolabeling‐free detection of locus‐specific N6‐methyladenosine modification. Angewandte Chemie International Edition. 2018 Dec 3;57(49):15995-6000.
2. Xiao YL, Liu S, Ge R, Wu Y, He C, Chen M, Tang W. Transcriptome-wide profiling and quantification of N 6-methyladenosine by enzyme-assisted adenosine deamination. Nature biotechnology. 2023 Jul;41(7):993-1003.