Research On A New Method For Nucleic Acid Fluorescence Detection Based On CRISPR/Cas12a

Circulating tumor DNA(ctDNA)can be used as a biomarker for tumor progression and metastasis monitoring,medication guidance and efficacy evaluation.Accurate detection of ctDNA is a basic prerequisite for the realization of the above clinical applications.Traditional ctDNA detection technology relies heavily on large testing instruments and has high cost.Therefore,it is necessary to develop a low-cost,portable technique and method for detecting ctDNA to support cancer screening and diagnosis.CRISPR/Cas12a protein can specifically cleave double-stranded DNA(dsDNA)or single-stranded DNA(ssDNA)by using a specific guide RNA(crRNA),and has been used to explore a new method for nucleic acid detection.In this paper,the unique non-specific nucleic acid cleavage activity of CRISPR/Cas12a protein,combined with hybridization chain reaction and fluorescence sensing strategy,was used to study the detection of ctDNA.It mainly includes two aspects of research content:1.Nano-flow strategy based on CRISPR/Cas12a and hybridization chain reaction for detecting ctDNAA highly specific,sensitive,convenient and rapid ctDNA assay was developed by using CRISPR/Cas 12a protein in combination with hybridization chain reaction and flow cytometry.In the absence of the target,the Cas12a trans-cleavage activity is silenced,retaining the initiator which can trigger the subsequent HCR.After rounds of the HCR,the product will bind to the surface of the magnetic beads via the biotin-avidin recognition and then analyzed by flow cytometry.In the presence of the target,the binary complex of Cas12a/crRNA specifically recognize and cleave the target,the trans-cleavage activity of Cas12a is activated,cleaving the initiator,therefore decreasing the subsequent HCR and the fluorescence on the surface of magnetic beads.It was found that the sensitivity of ctDNA reached the level of pM.2.Detection of ctDNA Based on CRISPR/Cas12a and Fluorescence Resonance Energy TransferIn view of the foregoing,magnetic beads were used as a carrier for Flow cytometry bead assays(FCBA)to capture targets.However,the process was cumbersome.We consider the possibility of further enhancing the sensitivity of the detection by means of a non-carrier fluorescence sensing strategy.Fluorescence resonance energy transfer combined with CRISPR-Cas 12a and HCR are introduced as a rapid method for detecting ctDNA.In this part of the study,nucleic acid hairpin probes(H1 and H2),which were labelled with carboxyfluorescein(FAM)as the donor and tetramethylrhodamine(TAMRA)as the acceptor respectively,can coexist metastably in solution.In the absence of the target,the Cas12a trans-cleavage activity is silenced,retaining the initiator which can trigger the subsequent HCR process.And that leads to FRET.In the presence of the target,the binary complex of Cas12a/crRNA specifically recognize and cleave the target,the trans-cleavage activity of Cas12a is activated,cleaving the initiator,therefore inhibiting the subsequent HCR and FRET process.The relative fluorescence intensity of the recipient and donor can be used to quantitatively detect the target DNA.It was found that compared with the flow cytometry in the previous section,the detection limit of ctDNA was increased about 4 times.The results of this paper indicate that nucleic acid detection strategies designed based on CRISPR/Cas12a and HCR can provide meaningful attempts and explorations for new clinical detection methods of ctDNA.