Construction,Activity Regulation And Biosensing Application Of CRISPR/Cas12a System

The CRISPR/Cas system,used to resist foreign invading nucleic acids,is presented widely in bacteria or archaea as an adaptive secondary immune system.It has critical application potential in the fields of disease treatment,gene editing or regulation and biosensing.The CRISPR/Cas12 a,a Class II type V CRISPR/Cas system,is guided by42-44 nt long cr RNA.It can degrade the single-stranded DNA after making specific cleavage of the target DNA.The CRISPR/Cas12 a system has been used to construct a variety tools for DNA recognition,editing and detection systems with excellent capabilities of target recognition and signal amplification.Therefore,it also has great potential in biosensing field.However,the CRISPR/Cas12 a biosensor still has these following bottlenecks:(1)The nucleic acids are the mainly detection target,but there is few other types of targets such as small molecules and proteins,so the types of targets are limited.(2)The detection system is not too sensitive enough to quantify the lowabundance biomarkers in organisms.Above all,this thesis utilizes gene recombination technology to construct the CRISPR/Cas12 a system,and then develops a new strategy to regulate the activity of Cas12 a in response to small molecules.Finally,we design a CRISPR/Cas12 a fluorescent sensor to achieve highly sensitive biosensing for micro RNA.This thesis includes specifically the following aspects:In Chapter 2,we first established the CRISPR/Cas12 a system in the laboratory,including Cas12 a protein and cr RNA,which lays the foundation for subsequent experiments.Using gene recombination technology,we construct the As Cas12 ap ET28a-TEV plasmid vector containing the target gene of the As Cas12 a protein and prepare the As Cas12 a protein successfully,with the activity equivalent to the commercial protein.through the steps of induced expression,extraction,and purification.Meanwhile,the cr RNA with recognition activity is obtained by the process of transcription and purification,and then a complete set of CRISPR/Cas12 a system is initially obtained.In order to optimize the activity of CRISPR/Cas12 a system,we have systematically optimized experimental conditions such as the reaction time,the amount of As Cas12 a and cr RNA,and the ion concentration.These conditions improve the sensing performance of CRISPR/Cas12 a and provide the necessary experimental basis for subsequent experiments.In Chapter 3,a new strategy is developed to regulate the activity of CRISPR/Cas12 a system responsed to small molecular,to solve the problem of limited target types.Inspired by sulfhydryl-disulfide exchange reaction,DNA strand(c DNA)complementary to c RNA is designed and modified with disulfide bond.CRISPR/Cas12 a system responding to GSH is constructed successfully,through a series of optimization of c DNA design,providing a new idea for broadening the detection target types of CRISPR/Cas12 a system.In order to improve the detection sensitivity of the CRISPR/Cas12 a biosensor,we combine the rolling circle amplification method with the CRISPR/Cas12 a system in Chapter 4.This biosensing platform can detect tumor marker micro RNA with high sensitivity and specificity.At the same time,this system can achieve spike recovery in normal human serum with high-accuracy,and subsequently identificate the actual breast cancer patient samples and determinate of mi RNA21 expression levels in related cell lines,providing a new idea to construct the CRISPR/Cas12 a fluorescence sensor for point-of-care testing applications.