A M~6A Sensing Method By Its Impact On The Stability Of RNA Double Helix Based On CRISPR-Cas13a

N6-methyladenosine(m~6A)is one of the most important RNA modifications in epigenetics.As one of the most abundant internal modifications in m RNA,m~6A has received extensive attention for its dynamic and reversible feature.The development of detection method for m~6A is limited by its abundance and structure.At present,some m~6A sequencing methods have been developed to identify m~6A in transcriptome,and many disease-related genes and m~6A modification sites located on these genes have been reported.Therefore,it is of great significance to detect m~6A modification at the specified sites on specific genes.In the meantime,CRISPR–Cas systems have been well-studied because of their specific functions especially in genetic editing.Among them,the Cas13a(once known as C2c2)is guided by cr RNA and can be programmed to cleave single-stranded RNA(ss RNA)targets,which has been used for nucleic acid detection.Although it has been previously reported that m~6A has an impact on the complementary pairing of RNA,few assays have been developed using this finding.We used this discovery and designed a detection method based on Cas13a system,which has different fluorescence signals for target RNAs containing m~6A modification and target RNAs without m~6A modification.We verified the fact that the presence of m~6A could cause the instability of ds RNA using the Cas13a system and provided a new direction and strategy for the development of m~6A detection methods in the future.