Novel Methods For Protease Detection Based On CRISPR-Cas And Activatable RNA Polymerase

As an important class of biomarkers,proteases involve in multiple processes of cancer progression including tumor development and metastasis.The abnormal changes of protease activity and expression level are related to the development of tumors,and thus they have been widely used as measurable indicators of tumor pathogenesis and prognosis in clinical diagnosis.Although many methods have been developed for the protease biomarker detection,most of them are unable to meet the requirements of clinical diagnosis.Therefore,there is still a need to develop more effective strategies for protease detection.Recently,several class 2 CRISPR-Cas systems,including Cas12a,Cas12b,Cas13a and Cas14a,have shown a powerful signal amplification capability due to their extraordinarily high trans-cleavage activity toward single-stranded DNA(ss DNA)or RNA.Here we are envisaged to develop a CRISPR-based method for the sensitive activity assay of protease biomarkers.Given that the protease target cannot be directedly activate the trans-cleavage activity of CRISPR-Cas,we introduced protease-activatable RNA polymerases(PR)as a signal transduction platform,and successfully developed a novel assay(PR-Cas)for the sensitive analysis of protease biomarkers based on PR and CRISPR-Cas.This PR-Cas system not only expands the application scope of CRISPR-Cas into protease detection,but also provides a method for the ultra-sensitive detection of protease biomarkers in clinical samples.The research work is as follows:1.Expression and purification of PR_Thr,PR_MMP-2and Lb Cas12a proteins.The PR_Thr,PR_MMP-2and Lb Cas12a proteins that induced and expressed in E.coli,were purified by Ni-NTA affinity chromatography.And then the signal transduction capabilities of PR_Thrand PR_MMP-2and the cleavage activity of Lb Cas12a were verified,respectively.These experiments could lay the foundation for the subsequent coupling of PR and Lb Cas12a,and their application in protease detection,cancer cell differentiation and cancer diagnosis.2.Construction of a novel methods for protease detection based on CRISPR-Cas and activatable RNA Polymerase.Firstly,to ensure the effective coupling of PR and Lb Cas12a,the effect of the length of the cr RNA spacer sequence on the activity of CRISPR-Cas was investigated.Using thrombin as the protease model,the existence of target protease is programmatically transduced and amplified into multiple cr RNA inputs through the PR_Thrconstructed in the previous chapter,which subsequently activates the trans-cleavage activity of Cas12a,and thereby realizing amplified detection of thrombin(LOD=31.6 f M).This PR_Thr-Cas not only provides a method for the ultra-sensitive detection of protease biomarkers,but also expands the application scope of CRISPR-Cas into protease detection,which provides a new platform for subsequent protease detection in clinical diagnosis.3.Detection of MMP-2 based on CRISPR-Cas and protease activated RNA polymerase.Encouraged by the above successful construction and application of PR_Thr-Cas in previous chapter,we have designed a PR(PR_MMP-2)by tuning the linker peptide between the lysozyme and the T7 RNAP,making it can only respond to MMP-2.Owing to the rational combination of the signal transduction and amplification capability of PR and the signal amplification feature of CRISPR-Cas,the PR_MMP-2-Cas system enables ultra-sensitive detection of MMP-2 with a LOD of5.4 f M.In addition,according to the differences in MMP-2 expression levels,PR_MMP-2-Cas can be further used for cell discrimination and the detection of MMP-2in serum samples.