New CRISPR-based Nucleic Acid Detection Technique

In recent years,CRISPR technology is the most popular gene editing technology.Although Cas9/sgRNA has been widely used in gene editing and regulation,it has not yet fully developed its application in nucleic acid detection.Cas9/sgRNA has a strong cutting ability to specific sequence DNA,and can even distinguish the sequence DNA of single base mismatch,showing the potential of good detection and typing DNA.However,there is no detection and typing technology directly targeting DNA in the nucleic acid detection method based on Cas9.In order to fully develop the potential of the Cas9/sgRNA system to detect nucleic acid,this study attempted to use Cas9/sgRNA to detect and type HPV DNA in vivo and in vitro.In this study,total 12 sgRNAs were designed for four kinds of HPV(two low-risk HPV: HPV6,HPV11;two high-risk HPV: HPV16,HPV18).Plasmids targeted with different kinds of HPV were constructed.After transformed into the E.coli,Cas9 protein and sgRNA were expressed under the control of promoter J23110 and J23119 respectively.The growth of E.coli were effected by Cas9/sgRNA complex which cut the target sequence specifically.Bacterial growth indicated that the sgRNA could specifically target DNA and correctly guide Cas9.The Cas9/sgRNA system can be used for the detection and typing of HPV.To explore the effect of Cas9/sgRNA in vitro,sgRNAs were prepared through in vitro transcription and were used to cut pHPV plasmid in vitro with the Cas9 protein.The results showed that the pHPV plasmids was cut to the fragments with expected sizes successfully,and the Cas9/sgRNA system could specifically cut the target DNA in vitro.In order to improve the sensitivity of the detection,we established the Cas9/sgRNA-typing PCR(ctPCR)method to detect HPV DNA.This technique detects target DNA in three steps:(1)amplifying target DNA with PCR by using a pair of universal primers(PCR1);(2)treating PCR1 products with a process referred to as CAT,representing Cas9 cutting,A tailing and T adaptor ligation;(3)amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers(gs-primers)(PCR2).The technique was verified by detecting HPV16 and HPV18 L1 gene in 11 different high-risk human papillomavirus(HPV)subtypes.The technique was also detected two high-risk HPVs(HPV16 and HPV18)in cervical carcinoma cells(HeLa and SiHa)by detecting the L1 and E6/E7 genes,respectively.In addition,the HPV18 L1 gene was amplified by qPCR1 from as low as 0.005 ng HeLa gDNA.Especially,even the CAT-treated qPCR1 product of 0.005 ng gDNA template was diluted 10000 fold,qPCR2 could amplify HPV18 L1 gene using 1 ?L of the diluted solution.These results indicated that both PCR1 and PCR2 can be realized with qPCR in high sensitivity,suggesting that the qPCR-based ctPCR is favorable to clinical application.This method was used to detect eight clinical samples.The results of qPCR1 indicated that two samples were HPV positive and the other six were HPV negative.After CAT treatment and qPCR2,HPV positive samples were typed,and were infected by HPV16 and HPV18 respectively.Therefore,the ctPCR method established in this study can detect and type HPV DNA successfully.In this method,PCR1 was performed to determine if the detected DNA sample contained the target DNA(such as virus infection),while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample(such as virus subtypes).This method provides new ideas and methods for nucleic acid detection and typing based on CRISPR technology.