CRISPR-based Nucleic Acid Test Strip To Achieve Multi-gene Accurate Diagnosis Of Prostate Cancer

【Background】Prostate cancer is the most common type of cancer and cause of cancer-related death in men,with 1.4 million new cases and 400 000 deaths worldwide.Although there is evidence that PSA testing is effective in reducing the mortality rate of prostate cancer,due to its low tumor specificity,some patients with prostatitis or benign prostatic hyperplasia(BPH)are misdiagnosed as prostate cancer.Prostate cancer antigen 3(PCA3),as a specific biomarker of prostate cancer,has been confirmed by the US Food and Drug Administration to be used for the specific diagnosis of prostate cancer.However,the current PCA3 gene detection technology represented by RTq PCR often has high equipment and technical requirements.Different from RT-q PCR,reverse transcription-polymerase recombinase-mediated nucleic acid amplification(RT-RAA)can complete a large amount of amplification in 25 minutes at 37 °C,so it has great application potential in the field of in vitro diagnosis.In addition,paperbased nucleic acid test strips can be used for rapid visualization of test results in vitro without device dependence.Therefore,it is very necessary to develop a prostate cancer detection platform based on the above technology for specific in vitro diagnosis.【Purpose】The purpose of this study is to construct a platform for early detection of prostate cancer based on reverse transcriptase-recombinant enzyme-mediated amplification(RT-RAA),regular clustering interval short palindromic repeat sequence(CRISPR)-Cas9 and nucleic acid test strip.At the same time,through the detection of the actual clinical samples,to analyze its clinical guidance and application value.【Methods】1.TCGA database was used to verify the diagnostic specificity of PCA3 gene and KLK3 gene for prostate cancer.The data of 497 primary prostate cancer cases and 52 non-tumor cases in the Cancer Genome Atlas(TCGA)database were analyzed.Pan-tumor analysis of PCA3 revealed a high degree of organ specificity.Differential expression analysis of PCA3 and KLK3 genes revealed their tumor specificity.The differential expression of PCA3 gene in different Gleason groups and different races revealed its distribution characteristics.2.RT-RAA and CRISPR-Cas9 were used to complete the rapid amplification and identification of the target gene.After designing the primers modified with biotin and digoxin groups,the RTRAA method was used to amplify the RNA as a substrate for a short time.After the amplification was completed,Cas9 in the CRISPR-Cas9 system was used to unwind the double strand of the amplified product.Sgrnas were used as complementary base pairs to accurately bind the amplified products.The combination of RT-RAA and CRISPR-Cas9 can achieve the purpose of rapid amplification and recognition.3.Rapid detection of the tested samples was completed by using double gene test strips.The necessity of each part of the test strip was proved by component deletion experiment.The specificity of the platform for PCA3 and KLK3 genes was verified by agarose gel electrophoresis and dipstick single gene detection.The lowest detection limit of this platform was demonstrated by dipstick detection experiments using a set of gradient diluted prostate cancer cell RNA as the substrate.Finally,the ability of the platform to distinguish prostate cancer from non-prostate cancer was verified by actual clinical samples.【Results】1.TCGA analysis showed that PCA3 gene and KLK3 gene had sufficient specificity for the rapid detection of prostate cancer,and had a very broad range of target population.2.By agarose gel electrophoresis and sequencing analysis of the amplified products,it was proved that the RT-RAA assay could amplify the target gene rapidly and accurately.In addition,the in vitro cleavage test and single gene detection of the test strip also confirmed the specificity of the designed sg RNA and the formation of the ternary complex of Cas9-sgr NA-target gene.3.The detection limit of PCA3 and KLK3 gene was 500 fg/μL and 50 fg/μL,respectively.Clinical samples have also demonstrated that the developed dual-gene detection platform can specifically identify prostate cancer samples.【Conclusion】In this study,CRISPR-Cas9 was used to identify and combine with RT-RAA amplification products to construct a prostate cancer double gene test strip for the early detection of prostate cancer.PCA3 gene and KLK3 gene can be used for accurate diagnosis of prostate cancer.RT-RAA amplification of target genes can get rid of large instruments and reduce operation steps and time,and improve the application ability of gene detection in resource-poor areas.CRISPR-Cas9 system can not only specifically identify the target gene,but also act as a bridge to connect the target gene with gold nanoparticles to form a complex.The dual-gene test strip can be used for rapid detection of the above complexes in vitro and the detection results can be directly observed by the naked eye.These advantages highlight the potential of this platform for prostate-cancer genetic testing in low-resource region or in point-of-care testing.