High-sensitivity Resolution Mechanisms And Applications Of The CRISPR-Cas12 System For Microbial Diagnosis

Pathogenic microorganisms have caused great harm to the aquaculture and related industries.Thus,accurate and rapid detection of pathogenic microorganisms is essential for the prevention and control of possible zoonosis.Traditional diagnostic methods for pathogenic microorganism cannot meet the needs of clinical diagnosis due to the drawbacks of long detection time,requirement of expensive instruments and possible false positives.Recently,CRISPR-Cas system has gained much concern due to its highly specific activity of nucleic acids cleavage.CRISPR-Cas12 system,which belongs to class Ⅱ of CRISPR-Cas systems,has become a research hotspot in nucleic acid diagnostic technologies because of its specific/non-specific cleavage activity for DNA/single strand DNA(ssDNA).In this study,three diagnostic methods of high specificity,high sensitivity and high accuracy for pathogenic microorganisms were constructed by using the nucleic acid cleavage properties of CRISPR-Un1Cas12f1 system and CRISPR-LbCas12a system,combining with in vitro amplification technology and nucleic acid aptamer technology.They provide new alternatives for clinical diagnosis of pathogenic microorganisms.The main research contents are as follows:1.RNA-activated CRISPR-Un1Cas12f1 system in the diagnosis of microorganisms.This study firstly discovered that RNA could activate the CRISPR-Un 1 Cas 12fl system and trigger its non-specific ssDNA cleavage activity,but not the cis cleavage of RNA.The length of the target RNA sequence also had a certain influence on the cleavage activity of the CRISPR-Un 1 Cas 12fl system.The lacking of the 3’ end of the target RNA severely inhibited the cleavage activity of the CRISPR-Un1Cas12f1 system compared to that of the 5’ end.Meanwhile,Un1Cas12f1 protein had excellent ability to recognize base mismatch of target RNA.Based on these findings,we further developed a highly sensitive and highly specific method by combining RT-PCR amplification and RNA in vitro transcription technology for the detection of Salmonella typhi(ATCas-RNA).And a sensitivity of 1 aM for ATCas-RNA method was obtained.Importantly,100%accuracy was achieved in simulated sample diagnosis.The ATCas-RNA method thus has the potential to be applied to clinical diagnosis.2.Nucleic acid aptamer-based CRISPR-Un 1 Cas 12fl system in the diagnosis of microorganisms.Aiming to achieve the directly detection of pathogenic microorganisms without the need of nucleic acid extraction and pcr-based amplification procedures for the CRISPR-Un1Cas12fl system,this study developed a nucleic acid aptamer-Un1Cas12fl biosensor(ACasB)with high specificity and sensitivity for the detection of staphylococcus aureus.Firstly,the specific nucleic acid aptamer of S.aureus was muted by pairing with a dual function blocker to form a hybridization chain(Hy-dsDNA),which can be released upon bacteria-aptamer binding.Then,the released blocker would bind to sgRNA to activate the cleavage ability of Un1Cas12f1,which thus generated the fluorescence signal.ACasB biosensor can directly detect 400 CFU/mL live S.aureus cells.Furthermore,100%accuracy was achieved for this platform in complex samples,compared with qPCR.Therefore,this ACasB has the promise to serve the tool for the on-site detection of live S.aureus in food safety and environmental monitoring.3.In vitro amplifation-based CRISPR-LbCasl2a self-driven catalytic system for microorganisms diagmosis.In this project,we developed a self-driven catalytic network of CRISPR-Cas12a system in order to meet the requirements of high sensitivity and simple-to-operate.This platform was capable of directly detecting 1 fM level double stranded DNA(dsDNA)target,which increased by 4 orders of magnitude compared with that of the CRISPR-Cas 12a system.This sensor was further combined with in vitro amplification technology to develop an ultra-sensitive and accurate method for the diagnosis of Salmonella typhi(SfCas-P)with its diagnostic sensitivity of 1 CFU/mL was achieved.Moreover,compared with the qPCR diagnostic method,SfCas-P method reached 100%accuracy for the diagnosis of simulated samples.Therefore,the SfCas-P diagnostic method offers a new alternative for clinical diagnosis approaches.