Research On Methods For Point-of-care Testing Of Nucleic Acid Based On CRISPR-Cas12a-mediated Enzymatic Catalysis

The precise detection of disease-related nucleic acid biomarkers will contribute to the early diagnosis,intervention and treatment of diseases,thereby reducing the harm of diseases to human body.Currently,polymerase chain reaction(PCR)is still the gold-standard method for nucleic acid detection.However,due to the reliance on the thermal-cycling process for amplification,PCR is confronted with the challenge of the requirements for expensive instruments,skilled personnel and laboratory environment,which limits its on-site detection application.Based on this situation,some isothermal nucleic acid amplification methods,such as recombinase polymerase amplification(RPA),loop-mediated isothermal amplification(LAMP),have been explored to solve the thermal-cycling problem of PCR.Although these methods can achieve isothermal and exponential amplification for nucleic acid in a short time,the problems of complicated primer design and nonspecific amplification can not be neglected.It is still a key issue for researchers to achieve point-of-care testing of diease-related nucleic acid with high detection sensitivity and specificity.Clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPRassociated(Cas)is an adaptive immunity system of bacteria and archaea for defense of foreign nucleic acid invasion.After years of research,CRISPR-Cas has been extensively applied to different fields including gene editing,transcription regulation,gene imaging and in vitro diagnosis.For in vitro diagnosis application,CRISPR-Cas system can accurately recognize and cleave the nucleic acid with short sequence.Regarding CRISPR-Cas12 a and CRISPR-Cas13 a,they can precisely recognize and degrade corresponding target nucleic acid,and then use transcleavage activity to cleave ss DNA reporter and ss RNA reporter respectively.Combining with isothermal nucleic acid amplication methods,CRISPR-Cas system can achieve comparable detection sensitivity of PCR and can solve the nonspecific amplification problem of isothermal nucleic acid amplification methods.However,in the aspect of signal detection,many developed methods based on CRISPR-Cas system require large-scale equipment(such as fluorescence spectrometer,electrochemical workstation)for signal detection,which restrains the application of CRISPR-Cas system for point-of-care testing of nucleic acid.Based on the property of CRISPR-Cas12 a,we designed and synthesized the enzymelabeled ss DNA reporter.By combining CRISPR-Cas12a-mediated enzymatic cleavage with the labled enzyme-mediated catalytic reaction,we achieve sensitive and specific disease-related nucleic acid biomarkers detection at the point-of-care without need of large-scale instrument.1.We have developed a strategy that combined duplex-specific nuclease(DSN)with CRISPR-Cas12 a.Personal glucose meter was utilized for quantitative detection of mi RNA in the strategy.Trigger DNA that could trigger the trans-cleavage activity of CRISPR-Cas12 a was generated via DSN in the presence of target mi RNA.The activated CRISPR-Cas12 a was used to cleave the sucrase-labeled ss DNA reporter on magnetic beads(MB),leading to the release of sucrase.The sucrase was able to catalyze sucrose into glucose.And the generated glucose signal could be quantitatively detected by a personal glucose meter.The strategy was simple-to-operate and could detect different mi RNAs by changing the template DNA.The detection sensitivity,specificity and anti-interference of the strategy were also good.The strategy provided an efficient tool for mi RNA detection at the point-of-care.2.We have designed an AND logic gate-based CRISPR-Cas12 a method for visual detection of two mi RNAs.AND logic gate was utilized for recognizing the signal output of mi RNAs and performing DNA computation.DNA logic gate would not perform and output trigger DNA when one kind of mi RNA was lacked.And DNA logic gate performed well and outputted trigger DNA in the presence of dual mi RNAs.The trigger DNA could activate the trans-cleavage activity of CRISPR-Cas12 a to cleave the glucose oxidase(GOx)-labeled ss DNA reporter on MB,resulting the release of GOx.GOx could catalyze the substrate to generate color change,thereby enabling visual detection of dual mi RNAs.The method was sensitive and could be used to detect mi R-205 and mi R-944 in lung cancer patients and healthy people,thereby realizing the differentiation of serum samples of lung cancer patients and healthy people.The method provided a facile platform for detection of disease-related nucleic acid biomarkers.3.We developed a strand displacement amplification(SDA)-assisted CRISPR-Cas12 a strategy for colorimetric detection of hepatitis B virus(HBV)DNA.In the strategy,HBV DNA could hybridize with template DNA and thus trigger SDA reaction.The product of SDA could initiate the trans-cleavage activity of CRISPR-Cas12 a,leading to the degradation of GOxlabeled ss DNA reporter on MB.GOx was released from MB and catalyzed the substrate to generate color signal change.The strategy could be used to sensitively detect HBV DNA with a limit of detection(LOD)of 41.8 f M,and could discriminate the DNA with single-base mutation.The strategy provided a new insight for point-of-care testing of nucleic acids of infection disease.4.We have designed a method that integrated RPA with CRISPR-Cas12 a for ultrasensitive and visual detection of SARS-Co V-2.In this approach,SARS-Co V-2 RNA could be efficiently amplified with RT-RPA.The amplicons could activate CRISPR-Cas12 a for cleavage of GOxlabeled ss DNA reporter on MB.GOx was capable of catalyzing the substrate to generate visual color change.The method was ultrasensitive and specific for target nucleic acid detection.The LOD was 2.86 a M(～2 copies/μL),which was comparable to that of PCR.No expensive equipment involved in the detection procedures and the operation could be finished within 50 min.The method is promising to be assembled into detection kit for SARS-Co V-2 nucleic acid analysis.