Molecular AND logic gate for multiple single-nucleotide mutations detection based on CRISPR/Cas9n system-trigged signal amplification

Precise detection of single-nucleotide mutations (SNMs) is extremely important in various biomedical applications, but the simultaneous detection of multiple SNMs remains a great challenge. Herein, we developed a new method based on CRISPR/Cas9 system for multiple SNMs detection. The CRISPR/Cas9 system transduces the nucleic acid into an intermediate trigger to initiate the isothermal amplification reaction and further form fluorescence signals. According to this strategy, we established nucleic acid bio-computing operations-molecule logic gate in simultaneous distinction of the genetic locus. We demonstrate that the fluorescence signals generated from different input combinations can be used to discriminate the multiple genetic locus, and the molecular logic gate has great potential in single-base mismatch detection. In addition, the successful assay of real samples indicates that the novel strategy could further adapt for the pathogenic monitoring and biomedical research.