Researches On Hypersensitive Biomarker Detection Technology Based On CRISPR-Cas System

Purpose:Exosomes and single-stranded nucleic acids are closely related to the pathogenesis of various diseases,especially cancer.Therefore,rapid,sensitive exosomes and single-stranded nucleic acid detectionnare of great significance for the diagnosis and prognosis of diseases.Method:1.Exosomes detection methods based on CD63 aptamer and CRISPR-Cas12a system mainly include two parts:exosomal membrane protein recognition based on CD63 aptamer and signal amplification based on CRISPR-Cas12a.We first designed exosome capture probes based on the CD63 aptamer,which is responsible for converting the number of exosomes into nucleic acid detection,while CRISPR-Cas12a is responsible for the amplification of highly specific nucleic acid signals.Then we optimized the detection conditions of the method and verified its application in the detection of exosomes.2.Single-stranded DNA binding protein(SSB)-EXPAR-assisted CRISPR-Cas12a ultra-sensitive molecular detection platform(SECURE),mainly by integrating EXPAR efficient exponential amplification of single-stranded nucleotides and CRISPR-Cas12a for trans-cutting The novelty of this method stems from its clever design,which ensures incredibly high specificity while maintaining excellent sensitivity.Results:1.Finally,the detection range of the established method was determined to be 3×10~3-6×10~7 particles/μL.In addition,we successfully applied this method to the detection of exosomes in clinical samples from healthy people and lung cancer patients,and the results were highly consistent with NTA technology.2.Using SSB,we delayed non-specific EXPAR amplification by 20 minutes by suppressing unwanted template-template hybridizations.At the same time,the CRISPR component was introduced within 10 minutes after specific replication was completed,and the secondary amplification of CRISPR-Cas12a-mediated specific amplicons could be completed within 8 minutes.This double amplification makes SECURE an ultra-sensitive method for detecting single-stranded nucleic acid sequences in exosomes with sensitivity of 19 aM from A549 cells and non-small lung cancer patients.Conclusion:CRISPR-Cas not only has a very high application value in the detection of nucleic acids,but also has a very broad application prospect in the detection of other biomarkers.It is expected to become a potential general molecular detection platform and can be used for screening of various diseases,diagnosis and prognosis.