A New Method Based On CRISPR For Homogeneous Nucleic Acid Detection

The CRISPR/Cas9 system is a precisely targeted gene editing technology,which enables editing of multiple genes simply and effectively.With the powerful gene editing capability,this technique is widely applied to research in the prevention and treatment of human diseases.However,there are few studies about Cas9-based nucleic acid detection technology.The traditional nucleic acid detection technologies represented by DNA hybridization technology and DNA sequencing technology are costly and tedious.The Cas9-based nucleic acid detection method is with great potential to overcome these shortcomings.Combining the advantages of CRISPR and PCR,this study established a novel CRISPR-based nucleic acid detection method,named as Cas9/sgRNA-typing PCR(ctPCR)method,which could perform detection in a uniform condition.The principle of this method is that a pair of Cas9/sgRNA complexes cleave the target DNA strand,which releases a pair of 3? ends providing the annealing site for insertion primers and serves as amplifying template.After the extension with DNA polymerase,a pair of identical universal PCR primer annealing sites are generated at each end of the target DNA.Finally,a universal PCR primer can be used to amplify the target.The main steps of the method include:(1)preparation of the premix: the components consist of the assembled Cas9/sgRNA complexes,hot-starting high-fidelity DNA polymerase,a pair of insertion primers,universal PCR primer;(2)detection of the sample: mix the above components in a certain ratio to form a PCR detection reaction by setting a PCR temperature control program(37? for 30 min;72? for 10 min;30 PCR amplification cycles),and complete cutting,primers insertion,extension,amplification and other steps in only one PCR tube with the PCR instrument.In the whole reaction process,after mixing the required reaction components in the initial stage,there is no need to open the testing tube during the detection process.This method does not require specific primers that need to be designed in traditional PCR detection,and its specificity depends on the CRISPR system.The reliability and specificity of the technique were verified by accurate identifying and classifying 10 different human papilloma virus(HPV)subtype vectors.Subsequently,this method was used to detect the L1 gene of two high-risk HPV(HPV16 and HPV18)in two HPV-positive cervical cancer cell lines,SiHa and HeLa.The results indicated that the method was with high specificity and sensitivity even in the complex human genome context.Finally,this method was used to detect and classify 30 clinical samples.7 samples are successfully detected as HPV58 subtype,3 samples are HPV 52 subtype,2 samples are HPV 45 subtype,and 2 samples are HPV 18 subtype,2 samples are HPV 59 subtype,1 sample is HPV 16 subtype,1 sample is HPV 35 subtype,and 1 sample is HPV 56 subtype.In order to further verify the detection results,the gDNA of clinical sample was amplified with the primers MY09/11 that were designed for the L1 region,and the amplification products were sequenced by Sanger sequencing.The HPV subtypes contained in clinical samples detected by ctPCR were all matched with the subtypes identified by Sanger sequencing,and the reliability of the method is verified.The ctPCR method established in this study can be used to detect and classify DNA,and provides new idea and method for detecting the nucleic acid with CRISPR/Csa9 technology.