Direct quantification of N-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated CRISPR-Cas12a platform.

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N-methyladenosine (mA) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the mA modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify mA fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only mA-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of mA at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of mA-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine mA fractions in real biological samples, even in low abundance RNA biomarkers.