Research On The Application Of CRISPR/Cas12a System In The Detection Of Non-nucleic Acid Analytes

CRISPR/Cas is an RNA-guided adaptive immune system in archaea and bacteria,which can help archaea and bacteria to resist phage invasion.The CRISPR/Cas system can identify and cleavage target DNA at a specific site,so it is widely used in the field of gene editing and provides a promising development direction for disease treatment.Cas12a（Cpf1）belongs to the type V CRISPR/Cas system,which can recognize and cleavage double-stranded DNA with a specific sequence under the guidance of a single RNA.With the development of related research,the trans-cleavage activity of Cas12a was discovered,which can non-specifically cleavage single-stranded DNA.Using the quenched short single-stranded fluorescent DNA as a reporter,the trans-cleavage activity can be output as an amplified fluorescent signal.Therefore,Cas12a can not only be used for nucleic acid recognition,but also has the function of signal amplification,which has attracted widespread attention in the field of biosensing.Combined with isothermal nucleic acid amplification technology,CRISPR-based diagnostics,such as DETECTR,SHERLOCK and HOLMES have been developed for the detection of nucleic acid targets with attomolar sensitivity and single-base specificity,and they have been applied to the detection of infectious disease nucleic acid.CRISPR/Cas system has revolutionized nucleic acid molecular diagnostic technology to a certain extent,and its application in the sensing of non-nucleic acid analytes（such as small molecules and proteins）is also worthy of attention,but related research is still in its infancy,so it is of great significance to develop a non-nucleic acid biosensing platform based on CRISPR/Cas.In this paper,a new biosensing method based on CRISPR/Cas is constructed for the detection of non-nucleic acid analytes such as protease and T4 PNK kinase.The main points are summarized as follows:1、The design and construction of recombinant LbCas12a elements in response to protease activity.TEV protease specific substrate sequence was used as the linking peptide to connect LbCas12a and its inhibitor Acr VA4 to obtain recombinant LbCas12a protein.In the recombinant LbCas12a protein,the linking peptide makes LbCas12a and Acr VA4 form an intramolecular complex,so the LbCas12a nuclease activity is inhibited and in an inactive state.When the linking peptide is cleavaged by the target protease,the intramolecular effect is destroyed and the nuclease activity of LbCas12a is restored,so this element can realize the response to protease activity through the activation of LbCas12a.This chapter mainly constructed the expression vector of recombinant LbCas12a,obtained the recombinant protein after protein expression and purification,and conducted preliminary verification of the response performance of the recombinant LbCas12a protein.The specific process is as follows:constructed the recombinant LbCas12a plasmid by homologous recombination molecular cloning method,expressed protein in the E.coli expression system,and obtained high purity recombinant LbCas12a protein through purification and desalting.At the same time,the specificity and activability of recombinant LbCas12a element for target recognition were verified by enzyme digestion reaction and Cas12a nucleic acid hydrolysis reaction,providing a basis for the subsequent development of new biosensing platform.2、Constructed highly sensitive protease fluorescence sensing platform based on recombinant LbCas12a elements.In the presence of the target protease,the linking peptide in the recombinant LbCas12a was cleavaged and the intramolecular inhibition was destroyed,thereby activating the nuclease activity of LbCas12a.The cleavage of the fluorescent ss DNA reporter by the trans-cleavage activity of LbCas12a can convert the protease activity into an amplified fluorescent signal.In this chapter,the recombinant LbCas12a that responds to protease activity is used as a tool protein to construct a highly sensitive fluorescent biosensing platform for detecting protease activity.Feasibility exploration and detection conditions optimization was performed for the sensing platform.Under optimized reaction conditions,the detection limit of this sensing platform for TEV protease is as low as 0.66 p M,and it has good selectivity for the detection target.In addition,because the linking peptide between Acr VA4 and LbCas12a can be redesigned,and the protease specific substrate sequence can also be replaced,a modular and programmable biosensing platform for detecting different proteases can be developed based on this sensing method,which reflects the application potential of the sensing method.3、Constructed the CRISPR/Cas12a sensing platform triggered by the DNA ligation reaction to detect T4 polynucleotide kinase（T4 PNK）.Target double-stranded DNA with a gap loses the ability to activate the trans-cleavage activity of LbCas12a,DNA ligase can connect the gap to restore the activation of LbCas12a.DNA ligase can connect 5’-PO₄ and3’-OH to form a phosphodiester bond,rather than 5’-OH and 3’-OH.The phosphorylation of5’-OH by T4 PNK can make the ligation reaction proceed,therefore,T4 PNK can trigger the trans-cleavage activity of LbCas12a through the DNA ligation reaction.Based on this principle,this chapter constructed a CRISPR/Cas12a sensing platform based on DNA ligation reaction to detect T4 PNK,and conducted feasibility exploration and detection conditions optimization for the sensing system.Under optimized conditions,the sensing platform can detect T4 PNK kinase activity with high sensitivity（LOD=0.0005 U/m L）and high specificity.This method can be used for the analysis and screening of T4 PNK inhibitor,and can also realize the analysis of T4 PNK activity in Hela cells,which shows great application potentials in disease diagnosis.