Development Of DNA Substrate-engineered CRISPR-Cas Biosensing

The CRISPR-Cas systems are RNA-guided nucleases to degrade foreign nucleic acids in bacteria and archaea,which have been redesigned as programmable biogenetic research,disease diagnosis,and disease treatment tools due to their unique target recognition and nucleic acid cleavage functions.By rationally integrating the advantages of proteins and nucleic acids,the CRISPR-Cas system can construct a generalized stimulus-responsive switch mechanism with a high spatial and temporal resolution,significantly enriching the application of CRISPR-Cas systems in the field of biosensing.In addition,by fusing nuclease-deficient Cas protein（d Cas）with fluorescent tags or engineering guide RNA（g RNA）with fluorescent labels,the CRISPR-Cas system has been repurposed as a powerful imaging tool for tracking the dynamics of genomic loci containing protospacer-adjacent motifs（PAMs）and RNA in living cells,which contribute to gain better insights into the dynamics of nuclear organization,gene regulation,and viral infection.Given the significance of this progress,we envision the deployment of the CRISPR-Cas system as a universal live-cell biosensing tool for a variety of intracellular biomolecules rather than just for the nucleic acid targets of interest.Herein,we take advantage of the precise nucleic acid binding and high-turnover nuclease capabilities to engineer the CRISPR-Cas12a system with a generalizable stimulus-responsive mechanism,and then deploy it to monitor various biomolecules in living cells.1.Construction of PAM-less DNA substrates（pDNA）and Engineering pDNA with a signal transduction mechanism.Single g RNA-guided CRISPR-Cas12a can specifically recognize and cleaves the target nucleic acid with a T-rich PAM motif and possesses high-turnover random ss DNA cleavage activity after activation（trans-cleavage）.The programmability and signal amplification function of CRISPR-Cas12a makes it a great advantage of flexibility and sensitivity in biosensing applications.In this chapter,based on the reported importance of the seed region in the unwinding of substrate dsDNA,we introduced a bubble structure into the seed region to eliminate the PAM preference of Cas12a toward the DNA substrates,termed PAM-less substrate dsDNA（pDNA）.We assessed the effect of bubble size,the number of flanking bases,and the PAM integrity of pDNA on activating Cas12a,thereby identifying the critical role of bubble structure in maintaining pDNA function.Given that the no PAM requirement and the bubble structure confer the pDNAs better flexibility and freedom,we proposed a general strategy for constructing stimulus-responsive DNA substrates（pcDNAs）,providing basises for the development of novel CRISPR-Cas biosensing systems.2.Construction of pcDNA_Tel-Cas12a system for telomerase activity assay.Based on the assumption in the previous chapter that enzymatic chain elongation-responsive pcDNA can be constructed by truncating pDNA,we rationally integrated the DNA polymerase activity of telomerase and created a telomerase-responsive pcDNA_Tel.Then,we constructed pcDNA_Tel-Cas12a biosensing platform by combining the signal amplification function of Cas12a to achieve sensitive analysis of telomerase activity.Benefiting from the non-specific PAM motif dependence of pDNA,telomerase-responsive pcDNA_Tel can be achieved by using telomerase primer as truncated TS.The optimized pcDNA_Tel-Cas12a system is sensitive to telomerase with a detection limit of up to 8 cells.In addition,we verified the trans-cleavage activity of Cas12a in living cells for the first time and successfully realized live-cell telomerase biosensing based on the pcDNA_Tel-Cas12a system.The application of the CRISPR system to endogenous protease markers further broadens the potential of the CRISPR system,which dramatically expands the toolbox of CRISPR imaging systems.Meanwhile,visual sensing of key intracellular biomolecules using trans-cleavage of Cas12a provides a new direction for live-cell bioimaging.3.Fabrication of pcDNA_ATP-Cas12a system for ATP biosensing.Aptamers are nucleic acid sequences with specific binding to their cognate ligands,which provide powerful tools for biological recognition and biosensing analysis due to their ease of design,target specificity,and versatility.In this chapter,we made full use of the high flexibility of base complementation to rationally incorporate ATP aptamer into pDNA as a blocking sequence,thereby constructing an ATP-responsive pcDNA_ATP-Cas12a live-cell sensing system.Under the binding of aptamer,the released pDNA can activate the trans-cleavage activity of Cas12a to generate fluorescent signals with high specificity for ATP.By determining the fluorescence responses toward ATP at different concentrations,the calibration curve for ATP detection was found to be linear over a concentration range of 2.5-800μM with a limit of detection of 0.47μM.Furthermore,the pcDNA_ATP-Cas12a system was demonstrated to be successfully used for visual sensing of ATP in living cells.Due to the diversity of aptamers,the simple replacement of aptamer sequences is expected to achieve biosensing of other key biomolecules in cells,which will further expand the potential of the CRISPR system in intracellular biomolecule imaging.4.Estabilishment of pcDNA_miRNA-Cas12a system for miRNAs biosensing.Nucleic acid strand displacement technology,a subfield of dynamic nucleic acid nanotechnology,utilizes sequence complementarity differences between multiple DNA strands.Based on Watson-Crick base pairing,this technology is highly predictable and dynamically programmable,and can be used in combination with other enzyme-driven processes to build integrated and efficient biosensing systems.In this chapter,by rationally combining the design flexibility and predictability of nucleic acid strand displacement with a high-performance Cas12a stimulus-responsive mechanism,we developed a disease-associated miRNAs-responsive pcDNA_miRNA-Cas12a biosensing system.With the design advantages provided by the PAM-less and bubble structure in pDNA,we successfully constructed a miRNA-responsive pcDNA by designing the miRNA-complementary sequence as a toehold-containing pDNA blocking sequence.Under the optimal conditions,the fluorescence response of pcDNA_miRNA-Cas12a system showed a linear relationship between miRNA-21 concentration in the range of1-250 n M with a limit of detection of 0.25 n M.In addition,based on the high specificity and scalability of base complementation,an orthogonal different target miRNAs-responsive pcDNA_miRNA-Cas12a system can be realized by simply replacing the blocking sequences.This study provides a versatile,easily target-replaceable CRISPR-Cas imaging system that provides a powerful tool for basic biochemical research and disease diagnosis.