RCA-Assisted CRISPR/Cas9Cleavage(RACE) For Highly Specific Detection Of Multiple Extracellular Vesicle MicroRNAs

MicroRNAs(miRNA)play an important role in the development,metabolism and disease process of many plants and animals.They are very useful biomarkers in the process of physiology and pathology.Extracellular vesicles(EV)also play an important regulatory role in various biological activities.EV-derived miRNAs express more direct and specific information about cell status,especially in the process of tumor occurrence and development.As for in vitro diagnosis,EV-derived mi RNAs are more appealing than plasma-derived circulating miRNAs,mainly because the EVs provide higher mi RNAs abundance than their counterparts.Therefore,the study of EV-derived miRNAs will provide much more important clinical reference value for the diagnosis and prognosis of the disease.CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases)system has been exploited for accurate nucleic acid detection owing to its high efficiency and rapidness.In a typical CRISPR/Cas9 system,the Cas9 nuclease efficiently shears exogenous double-stranded DNA(dsDNA)sequence upon the recognition of specific complementary dsDNA containing protospacer adjacent motif(PAM)sequences in the presence of a single-stranded guiding RNA(sgRNA).Rolling circular amplification(RCA)has emerged as a highly specific isothermal gene amplification approach that could be performed at a constant temperature(at 30 �C or even room temperature)in the presence of thermally stable DNA polymerase without sophisticated instruments.The high specificity of the RCA reaction is mainly attributed to its rigid parameters requiring a completed complementary between the primer and a circular DNA template for initiation,which makes it particularly useful for the analysis of single-nucleotide polymorphisms.In this study,we integrated the advantages of RCA and CRISPR/Cas9,and built a multi-functional isothermal multi-target nucleic acid detection platform�RACE,which can be used for the isothermal quantitative detection of multiple miRNAs in extracellular vesicles,which is of great significance for disease diagnosis and prognosis evaluation.In this study,a specific padlock probe was designed to identify target miRNA,which was used in RCA action under the help of HiFi Taq DNA ligase and Phi29 DNA polymerase.After amplification,a large number of repeated target sequences on the long single-stranded DNA(ssDNA)which can combine with TaqMan probe to form a large number of PAM structures,which can be specifically recognized by sgRNA.The powerful exonuclease activity of Cas9 protein is used to cut the target sequence and TaqMan probe so that the fluorescence can be "turned on",which can be easily measured by spectroscopy.For multiple detection,a multicolor TaqMan probe complementary to the specific target miRNA can be introduced to start the multicolor detection.RCA is a commonly used technique of miRNA isothermal amplification,which can amplify the signal of target sequence,and also can be used as the first recognition of target miRNA.The cleverness of the invention in combining CRISPR / Cas system with RCA lies in that a large number of repeated target sequences on the amplified ssDNA are hybridized with TaqMan probe to form PAM structure,and sgRNA realizes the specific recognition of target sequences through PAM structure,which is the second recognition of target mi RNA.After double recognition,Cas9 protein sheared the target sequence which hybridized with TaqMan probe,and the fluorescence recovered.RCA is used to amplify signals and CRISPR / Cas9 is used to identify and cut target sequence.The combination of RCA and CRISPR / Cas9 technology realizes signal amplification and double recognition of target miRNA,improves the sensitivity and specificity of miRNA detection,and avoids the false-positive results of traditional fluorescent probes.The results show that RACE has the performance of single base recognition and multiplexed detection.RACE not only successfully realizes multiplexed detection of EV-derived miRNAs from A549 cells,but also successfully detects multiple EV-derived mi RNAs from plasma in clinical nonsmall cell lung cancer(NSCLC)patients,which proves that RACE is suitable for all sources of miRNA detection.The comparative results of RACE and RT-qPCR showed that the two methods have a high consistency in detecting the abundance of intracellular miRNAs from A549 cells cultured in vitro and plasma from NSCLC patients.The feasibility and stability of RACE were verified,and the potential ability of RACE in screening,diagnosis and prognosis of various diseases was revealed.In a word,RACE is a powerful nucleic acid detection tool,which has the characteristics of single base recognition,and the detection sensitivity reaches the detection limit of 90 fM.It can realize the simultaneous detection of multi-target miRNA at room temperature.It is stable,fast and portable,and can be used for multi-target and specific detection of nucleic acids in real-time diagnosis.