Construction Of Biosensors Based On CRISPR/Cas And Bioamplification For Biomolecules Detection

Accurate and reliable diagnosis of biomolecules is essential for early treatment of diseases and clarification of various physiological and pathological processes.Personalized medicine based on the detection and recognition of molecular information makes the treatment of many rare or common diseases a reality.After decades of development,a variety of biomolecular detection platforms have been proposed,but the rapid detection of low-abundance biomolecules in complex samples and global outbreaks of diseases is still challenging.In addition,traditional molecular diagnostic techniques that rely on large-scale equipment involve in the disadvantages of tedious operation,high costs,complex sample pretreatment and the requirement of professional operators,making them difficult for large-scale deployment and on-site detection.Since the emergence of CRISPR/Cas technology,it has obtained breakthrough applications in the field of biomolecular detection and is considered as the next generation of molecular diagnostic technology.High sensitivity and specific recognition are the most critical factors in the construction of biosensors.The specificity of target detection is ensured by sequence-dependent recognition of CRISPR/Cas system.In addition,trans-cleavage activity of CRISPR/Cas12 and CRISPR/Cas13 system can be activated after recognizing target nucleic acids,endowing CRISPR/Cas become a signal amplification and readout system.The combination of CRISPR/Cas technology and biological amplification will further improve the detection sensitivity,achieving highly sensitive,specific,rapid,and cost-effective detection of target nucleic acids,which also supports on-site use without the need for professional operators and complex equipment.It is suitable for on-site deployment and timely monitoring.I view of these,five CRISPR/Cas biosensors are established for detection of single-stranded nucleotides,DNA methylation,alkaline phosphatase,staphylococcus aureus and ATP.The specific work is as follows:We develop a CRISPR/Cas9 system mediated G4-EXPAR(Cas-G4EX)strategy for site-specific detection of ss RNA and ssDNA.By introducing a short-strand PAMmer sequence,CRISPR/Cas9 executes the site-specific cleavage of target ss RNA or ssDNA and releases product fragments with the desired sequence at the 3'-terminal.The product fragments serve as the primer activated subsequent sequence-dependent exponential amplification reaction(EXPAR).The EXPAR amplification products assembles with hemin to form the structure of G-Quadruplex(G4/hemin).The peroxide mimic enzyme of G4/hemin catalyzes ABTS-H₂O₂ system accompanied with the appearance of green color,realizing the naked-eye detection of ss RNA and ssDNA.The Cas-G4EX strategy integrates the superiority of CRISPR/Cas9 and EXPAR,which presents outstanding site-specific recognition and high-performance amplification efficiency.The Cas-G4EX reveals the real detection limit of 250 aM for ss RNA and 100 aM for ssDNA,respectively.More importantly,the proposed method exhibits an excellent discrimination for single-base mutation of single-stranded nucleic acids.Additionally,the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection platform for any ss RNA or ssDNA.A digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with RPA-assisted CRISPR/Cas13a system(DESCS)is developed for accurate and sensitive determination of DNA methylation.This dual methylation-sensitive restriction endonuclease selectively digests the unmethylated DNA but exhibits no response to methylated target.Therefore,the intact methylated DNA target triggers the RPA reaction for rapid signal amplification.RPA products with a T7 promoter can execute the T7 transcription to generate a mass of ss RNA.These ss RNA can be recognized by CRISPR/Cas13a to induce the ss RNase activity of Cas13a,showing the indiscriminate cleavage(namely“trans-cleavage”)of collateral RNA FQ-reporter(a short ss RNA labeled with 5'FAM and 3'BHQ1)to release the fluorescence signal.With such a design,the DESCS system presents the rapid and powerful signal amplification for determination of CpG methylation in human SEPT9 gene.The linear range is 200aM?10 fM and 10 fM?20 pM,respectively,and the detection limit is 86.4 aM.More importantly,0.01%methylation level with the existence of excess unmethylated DNA can be effectively distinguished.In addition,the DESCS assay is integrated in the lateral flow biosensor(LFB)for point-of-care determination of DNA methylation.Ultra-sensitive determination of phosphatase(ALP)activity is realized by target-induced transcription amplification to trigger the trans-cleavage activity of Cas13a(referred as“TITAC-Cas”).T7 promoter/template DNA duplex with5'-phosphate in T7 promoter serves as a simple and stable substrate.In the presence of ALP,ALP-dephosphorylation reaction provides the protection for T7 promoter against the digestion from?exo.The intact T7 promoter/template DNA duplex executes T7transcription to generate a mass ss RNA,which possesses a full complementarity to the spacer of crRNA.Subsequently,the ss RNase activity of CRISPR/Cas13a is activated by the ss RNA products and exhibits the indiscriminate cleavage of collateral RNA FQ-reporter to release significant fluorescence signal.This TITAC-Cas assay based on dephosphorylation-initiated T7 transcription-mediated CRISPR/Cas13a system for ALP activity detection involves in triple signal amplification,which includes self-enzymatic amplification of ALP,T7 transcription amplification,and trans-cleavage of CRISPR/Cas13a system.Due to the triple signal amplification,ultra-sensitive determination of ALP activity can be realized with a wide linear range(about four orders of magnitude from 0.008 to 250 U·L^-1)and the LOD of 0.006 U·L^-1.In addition,the TITAC-Cas assay is successfully used for the determination of ALP activity in Hep G2 cell lysate with high fidelity.More importantly,it also exhibits the capacity for screening of enzyme inhibitor.These results suggest the TITAC-Cas assay provides great potentials for diagnosis of ALP-related diseases.We present a CRISPR/Cas12a system for detection of staphylococcus aureus and identification of relevant drug resistance genes.Genus-specific gene of staphylococcus aureus(spec gene)and methicillin-resistant gene(mecA gene)are amplified by polymerase chain reaction(PCR).According to the sequences of spec gene and mecA gene,corresponding programmable crRNAs are designed and prepared in vitro.The PCR products are complementary with crRNA and recognized by CRISPR/Cas12a.Subsequently,the trans-cleavage activity of Cas12a is activated to indiscriminately digest surrounding DNA FQ-reporter(a short ssDNA modified with 5'HEX and 3'BHQ1)and release fluorescence signal.In addition,a AuNPs-DNA probe pair is also designed and prepared for colorimetric identification of staphylococcus aureus.Target-triggered trans-ssDNA cleavage of linker probe leads to the non-aggregation of AuNPs-DNA probes pair.The obvious color change AuNPs-DNA probes pair presents the naked-eye gene detection.With such a design,the detection of nucleic acid ofMRSA presents the sensitivity of about 33 cfu/mL with high selectivity.Successful identification ofMRSA in complex clinical samples indicates the real applications of proposed CRISPR/Cas12a assay,which provides a universal platform for diagnosis of pathogenic microorganisms with universality and compatibility.A four-stage signal amplification is established for ATP detection based on an allosteric probe-conjugated strand displacement amplification(SDA)integrated with CRISPR/Cas12a system(ASD-Cas).An allosteric probe(AH)with a hairpin structure is designed to reduce background interference from self-extension and non-specific amplification during the SDA reaction.ATP binds with the aptamer segment at5'-terminal of AH probe and subsequently opens the AH probe to activate the SDA reaction.The amplification products are recognized by CRISPR/Cas12a system and trigger the ssDNase activity of Cas12a,resulting in robust cleavage of surrounding DNA FQ-reporter.With this four-step amplification,ATP is measured with high sensitivity and low background interference.Under the optimal conditions,a linear relationship is obtained between the fluorescence response and ATP concentrations ranging from 2 pM to 10?M with a limit of detection(LOD)as low as 1.8 pM.Successful detection of ATP in diluted human serum samples further indicates that ASD-Cas system provides a potential platform for detection of small molecules.