Development And Agricultural Applications Of CRISPR/Cas-Based Novel Nucleic Acid Detection Technologies

CRISPR/Cas systems that can accurately modify target DNA or RNA have become the frontier and hot-spot of biological sciences and technologies.Among them,"trans-cleavage" activities based on Type V or Type VI Cas nuclease had also been developed and applied into nucleic acid detection.Compared with traditional detection technologies,CRISPR/Cas based nucleic acid detection technologies do not rely on highly specific antibody screening,expensive instruments or well-trained technicians.These technologies do have the advantages due to the simple designs,flexibility,low-cost development,and high detection sensitivity and specificity,and possess the technical potential for multiplex detection and broad agricultural applications.The Rfx Cas13 d nuclease from Ruminococcus flavefaciens XPD3002 has the advantages of small molecular size and high targeting RNA activity in cell.Therefore,it has important prospect and value for target gene editing and derivative technology development.However,CRISPR/Rfx Cas13d-based nucleic acid detection technology has not been reported.One objective of this study is to establish nucleic acid detection technology based on CRISPR/Rfx Cas13 d.Moreover,combined with the existed CRISPR effector nuclease based detection technologies such as As Cas12 a,Lb Cas12 a and Lwa Cas13 a,we aimed developed a set of plant pathogens detection methods targeting pathogens of major disease in current agricultural production.The main research and results are as follows :1.Recombinant Cas proteins were employed and purified in this study.The As Cas12 a,Lb Cas12 a,Lwa Cas13 a and Rfx Cas13 d proteins were expressed in E.coli.The proteins were purified by Ni affinity chromatography,cation exchange chromatography and gel filtration chromatography.Thus,high-purified CRISPR effector proteins were prepared for the establishment and application of subsequent nucleic acid detection system.2.CRISPR/Rfx Cas13 d nucleic acid detection technologies were developed and optimized.Screened from a variety of artificially synthesized probes,our data showed that poly r U-linked reporter molecule could be used as a fluorescent probe for nucleic acid detection technology using CRISPR/Rfx Cas13 d.By introducing a series of designed mismatched bases in crRNAs and/or target sequences,our data showed that this system can specifically distinguish target samples with only as few as a single mismatch.A novel nucleic acid detection technique with high specificity based on CRISPR/Rfx Cas13 d has been established.3.The nucleic acid detection technologies of As Cas12 a,Lb Cas12 a and Lwa Cas13 a were established in our own lab.The sensitivities of Rfx Cas13 d,As Cas12 a,Lb Cas12 a,and Lwa Cas13a-based nucleic acid detection systems were further improved using a coupled target molecules thermostatic amplification treatment.The results indicated that qualitative and quantitative detection and analysis on one or multiplex tagetes can be achieved in a single reaction.4.Multiplex nucleic acid target assay in a single reaction was established using a system combined CRISPR/Lb Cas12 a with CRISPR/Lwa Cas13 according to their different probe preferences.The multiplex assay in one reaction could be applied into both DNA and/or RNA detection.5.In order to improve usability,crude extract samples for easy preparation were tested their sensitivity and specificity.Four Cas-based nucleic acid detection systems were developed on detection of maize ear rot major pathogens of Fusarium graminearum and Fusarium verticillioides.The whole detection process from sampling to result readout could be completed within 30-40 min.The combined detection system could accurately differentiate Fusarium graminearum and Fusarium verticillioides pathogens in the crude extract samples.6.Four sets of Cas-based nucleic acid detection techniques were also verified on detecting RNA virus of Rice black-streaked dwarf virus(RBSDV),which can infect a variety of cereal crops including rice,wheat,maize.The results showed that the samples harboring RBSDV could be sensitively detected.In addition,our data also showed the detection system could be also used into crude extract samples.7.The strategy of CRISPR/Lwa Cas13 a and CRISPR/Rfx Cas13 d based technology for generating RBSDV-resistant germplasm in maize was also performed.Using conventional RNA interference(RNAi)technology as a comparing system,stable transformants of both CRISPR systems and RNAi had been had been obtained to meet this end.These efforts had laid the material basis for novel diseaseresistant approach.In summary,we had developed a series of highly sensitive,specific,and convenient nucleic acid detection technology tools.They were tested on several major diseases of major crops.These technologies provide an easy and low cost way to develop sensitive detection methods for those pathogens,which detection methods were needed to be developed.This study also laid the initial research foundation for CRISPR/Cas13-based RNA virus-disease resistant breeding.