N6 methyladenine (m6A) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (C-SDA) and CRISPR/Cas12a to detect m6A FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to m6A RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for m6A RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 × 10-4 U mL-1. More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting m6A FzD5 mRNA and MazF activity.
Keywords: CRISPR/Cas12a; Cascaded strand displacement amplification (SDA); Frizzled class receptor 5 (FzD5); MazF; N(6) methyladenine (m(6)A).
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