Novel CRISPR/Cas12a Based Methods For Bioanalysis

Highly sensitive and highly specific bioassays are of great significance for life science research and early diagnosis of diseases.Traditional detection techniques cannot meet the needs of scientific research or clinical laboratory due to low sensitivity,complex operation procedures and high cost.Presently,CRISPR technology has developed rapidly in recent years and has been widely used in gene editing,biological imaging,nucleic acid detection and other fields,becoming a hotspot of life science research.Due to its programmability,high sensitivity and specificity,the detection technology based on CRISPR/Cas system has a very broad application prospect and is known as the "next generation molecular diagnostic technology".CRISPR/Cas12 a is the most commonly used CRISPR/Cas system in nucleic acid detection.Cas12 a specifically recognizes and cleaves double-strandedDNA under the guidance of CRISPR RNA(crRNA),simultaneously releasing highly efficient trans-cleavage activity.Based on this characteristic,CRISPR/Cas12 a nucleic acid amplification detection system was established by cleaving single-strandedDNA reporter.In this paper,the cleavage performance and working mechanism of CRISPR/Cas12 a are deeply discussed.The new CRISPR/Cas12 a universal detection platforms were be developed for the detection and imaging of nucleic acid and protein markers,providing technical support for life science research and early diagnosis of diseases,and providing new ideas and experimental basis for the development of biological analysis methods.This study mainly includes the following two parts:1.CRISPR/Cas12a-Mediated Interfacial Cleaving of HairpinDNA Reporter for Electrochemical Nucleic Acids SensingRapid and sensitive isothermal method is crucial for point-of-care nucleic acids testing.Recently,RNA-guided CRISPR/Cas12 a proteins were discovered to exhibit target triggered nonspecific single-stranded deoxyribonuclease(ss DNase)activity.Herein,the ss DNase cleavage capacity of CRISPR/Cas12 a system for interfacial hairpinDNA(hpDNA)and linearDNA was investigated detailedly.A novel electrochemicalDNA biosensor was then developed via target-induced Cas12 a cleaving interfacial hpDNA.In this strategy,the recognition of targetDNA can morphologically active the robust Cas12 a ss DNase activity.The immobilized hpDNA electrochemical reporters with low surface coverage and incompact morphological structure present accessible substrates for high-efficient Cas12 a cleavage,leading to highly sensitive electrochemicalDNA biosensor.Under the optimal conditions,as low as 30 pM of targetDNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 n M.Furthermore,the practical application ability of the established biosensor for detection of targetDNA in complex matrices was also demonstrated.The proposed strategy enables rapid and sensitive detection of nucleic acids,providing a potential tool for point-of-care molecular diagnostics.2.Controlling the trans-Cleavage of CRISPR-Cas12 a with Nicked PAM: Universal Platform for BiosensingCRISPR/Cas12 a is able to precisely recognize double-strandedDNA(dsDNA)containing PAM sequence under the guidance of CRISPR RNA(crRNA)and release indiscriminate trans-cleavage capability,providing an efficient signal amplification tool for molecular diagnostics.In this study,we investigated the trans-cleavage activity of CRISPR/Cas12 a in detail by engineering nicked dsDNA activators with different nucleotide defective modes.The influence of PAM nucleotides on the activation of CRISPR/Cas12 a trans-cleavage activity and the process by which Cas12a-enhanced strand displacement mechanism to promote crRNA invasion have been revealed.Meanwhile,we demonstrated that dsDNA with a nicked PAM could be specifically recognized and initiate the trans-cleavage activity of CRISPR/Cas12 a efficiently.Based on binding-induced PAM assembly,we developed a modular CRISPR/Cas12a-based signal amplification system for universal biosensing.Since different kinds of targets can be recognized with corresponding affinity probes,this system allows the general detection of a wide range of analytes.As a proof of concept,the system was employed for intracellular BCR/ABL1 mRNA imaging and PDGF-BB detection with high sensitivity and specificity.This work is of great importance for the fundamental understanding of CRISPR/Cas12 a system and provides significant improvements for the rational design of CRISPR/Cas12a-based sensing platform for molecular diagnostic applications.