Cascaded amplification CRISPR-Cas nucleic acid detection system and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a cascade amplification CRISPR-Cas nucleic acid detection system and application thereof. According to the closed chain based on chemical modification, a cascade amplification digital CRISPR-Cas detection system is realized, the sensitivity of the CRISPR-Cas detection system combined with polymerase amplification can be achieved, and meanwhile, the risk of cross contamination caused by amplification is avoided. According to the invention, a chemically modified closed chain is adopted as a trigger medium between two CRISPR-Cas systems, so that the stability of a cascade amplification system is improved, a detection background signal is reduced, and a scheme of the cascade amplification CRISPR-Cas detection system for DNA and RNA is realized respectively. Two cascade amplification systems based on Cas12 and Cas13 established by the invention are combined with liquid drop digital detection and chemically modified nucleic acid sealing to realize single-molecular-level DNA and RNA detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a cascade amplification CRISPR-Cas nucleic acid detection system and application thereof. BACKGROUND

[0002] Molecular diagnostic techniques based on nucleic acid detection are used for pathogen and various disease diagnosis. Among them, the real-time fluorescent quantitative Polymerase Chain Reaction (qPCR) method is widely used in clinical nucleic acid detection. The method replicates the target nucleic acid through polymerase, realizes signal amplification by detecting a large number of replication products, has high sensitivity and specificity, and is considered as the "gold standard" of nucleic acid detection. In addition, isothermal amplification technology has shown good application prospects in rapid diagnosis due to its independence on precise instruments. Commonly seen are recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), rolling circle amplification (RCA), etc. Digital nucleic acid detection method disperses the reaction solution containing target molecules into thousands of small chambers, so that each chamber contains one or zero target molecules. After the reaction, positive and negative chambers can be distinguished according to the fluorescence intensity. Through the total number of chambers, the total number and volume of positive chambers, absolute quantification can be realized. Due to the confinement effect, the concentration of target molecules in the chamber is increased, so that the digital detection method is more sensitive than the bulk detection. Its counting method enables it to realize absolute quantification of target nucleic acid.

[0003] The CRISPR / Cas system, due to its speed, portability, and accuracy, holds great potential for developing new methods for nucleic acid detection. CRISPR (clustered regularly spaced short palindromic repeats) are nucleic acid sequences in bacteria and archaea associated with the adaptive immune response system. CRISPR-associated proteins (Cas) are endonucleases that form a complex with crRNA (CRISPR RNA). The crRNA recognizes the target nucleic acid, guiding the Cas protein to recognize and cleave the target sequence; this enzymatic activity is called cis-cleavage activity. Class II CRISPR / Cas systems, such as Cas12a and Cas13a, exhibit trans-cleavage activity after cis-cleavage of the Cas / crRNA / target DNA ternary complex, enabling non-specific cleavage of nearby single-stranded DNA or RNA. Cas12a recognizes and cleaves DNA, while Cas13a recognizes and cleaves RNA. Combining this characteristic with FRET (fluorescence resonance transfer) technology enables nucleic acid detection. An oligonucleotide sequence (reporter) with fluorescent and fluorescent quencher groups added to its ends is cis-cleaved in a CRISPR / Cas system. The trans-cleavage of the reporter then generates a fluorescent signal. Since CRISPR / Cas recognizes one target DNA or RNA, it can trans-cleave thousands of reporters, achieving signal amplification and enabling nucleic acid detection. Current methods for directly detecting nucleic acids using the CRISPR / Cas system suffer from low sensitivity. New nucleic acid detection methods, SHERLOCK and DETECTR, developed using the CRISPR / Cas system combined with RPA isothermal amplification technology, offer high sensitivity and specificity. However, the addition of polymerase amplification introduces cumbersome procedures and a risk of cross-contamination. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a chemically modified cascaded amplification CRISPR-Cas nucleic acid detection system and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a cascaded signal amplification and detection system based on a CRISPR-Cas system, comprising:

[0007] (1) Target nucleic acid, CRISPR effector protein and its crRNA;

[0008] (2) Cascade amplification probes are complementary hybrid nucleic acid strands that block the triggering element of the next stage reaction; the triggering element of the next stage reaction includes cleaving crRNA or substrate DNA;

[0009] The complementary hybrid nucleic acid chain is a complementary hybrid double strand or a self-complementary single strand comprising an uncomplementary single-stranded bubble region;

[0010] The complementary hybrid nucleic acid chain comprises a chemical modification; the chemical modification comprises a locked nucleic acid modification, a 2'-fluoro nucleotide modification, and a 2'-methoxyethyl modification.

[0011] (3) an auxiliary signal amplification element, which releases a trigger element of a next-stage reaction through cleavage of a previous-stage CRISPR effector protein, forming a self-catalytic cycle.

[0012] After the CRISPR / Cas system is combined with a polymerase amplification method, there are problems such as complicated operation and easy cross contamination. The present application has found, through a large number of investigations, that the combination of digital nucleic acid detection and CRISPR / Cas can realize nucleic acid detection without amplification, with a sensitivity of aM-fM level, which is suitable for most detections, but the sensitivity is still not as good as that of nucleic acid detection based on enzyme amplification. Therefore, the present application further introduces a special nucleotide, locked nucleic acid (LNA), which is a synthetic nucleic acid analogue containing a bridged, bicyclic sugar moiety. The addition of a methylene group between the 2'-O- and 4'-positions "locks" the furanose ring into a 3'-endo conformation. LNA completely complies with the Watson-Crick base pairing principle, but the affinity of LNA:DNA hybrid is stronger than that of its corresponding DNA:DNA. By adopting the mode of locked nucleic acid modification, it can be ensured that the next-stage CRISPR / Cas system is not triggered in the case that the first-stage CRISPR / Cas system is not triggered, thereby reducing the background signal of the cascade amplification reaction and improving the detection sensitivity. Through this cascade amplification, the present application can detect 1aM level of target DNA and RNA, and reduce the background signal. At the same time, the chemical modification also includes 2'-fluoro nucleotide (2'-F) modification and 2'-methoxyethyl (2'-O-Me) modification; as long as the affinity with the complementary strand can be improved, the similar effect in the present application can also be achieved.

[0013] Preferably, the chemical modification is a locked nucleic acid modification.

[0014] The present application realizes a cascade amplification digital CRISPR-Cas detection system based on the blocking chain of locked nucleic acid modification. Compared with the sensitivity of a single CRISPR-Cas detection system, the present application has higher sensitivity, which can reach the sensitivity of a CRISPR-Cas detection system combined with a polymerase amplification, and at the same time avoids the cross contamination risk caused by amplification. On the other hand, the present application adopts the locked nucleic acid modification blocking chain as a trigger medium between two CRISPR-Cas systems, improves the stability of the cascade amplification system, reduces the detection background signal, and realizes the cascade amplification CRISPR-Cas detection system scheme for DNA and RNA respectively.

[0015] Preferably, when the target nucleic acid to be detected is DNA, the detection system satisfies the following conditions:

[0016] (a) the CRISPR effector protein is Cas12a protein; the crRNA specifically recognizes the target nucleic acid DNA; the Cas12a protein and the crRNA form a Cas12a-crRNA complex;

[0017] (b) the cascade amplification probe is a complementary hybrid double-stranded chain that encloses the split crRNA; the split crRNA hybridizes with the chemically modified closed chain to form a double-stranded structure containing a 5-7 nt single-stranded bubble region;

[0018] The split crRNA includes Split crRNA-handle and Split crRNA-spacer;

[0019] (c) the auxiliary signal amplification element is a pre-filled double-stranded DNA; when the target DNA exists, the Cas12a-crRNA complex recognizes the target DNA through the crRNA, activates the Cas12a enzyme activity and cuts the bubble region of the cascade amplification probe to release Split crRNA-handle and Split crRNA-spacer;

[0020] The Split crRNA-spacer recognizes and binds to the auxiliary signal amplification element; the Split crRNA-handle binds to the Cas12a protein through a displacement mechanism, and further activates the Cas12a enzyme activity in combination with the Split crRNA-spacer and the pre-filled double-stranded DNA to form a self-catalytic cycle.

[0021] Preferably, the Split crRNA-handle is a sequence that binds to the Cas12a protein; and the Split crRNA-spacer is a sequence that recognizes and binds to the auxiliary signal amplification element.

[0022] Preferably, the chemical modification is a locked nucleic acid modification.

[0023] The present application develops a cascade amplification system based on CRISPR-Cas12 for DNA detection, which mainly includes Cas12a enzyme, crRNA capable of specifically recognizing target DNA, complementary hybrid double-stranded chain enclosing split crRNA, and auxiliary signal amplification element-double-stranded DNA (Ancillary dsDNA).

[0024] The split crRNA (Split crRNA-handle and Split crRNA-spacer) can be hybridized with a lock DNA (Lock DNA) modified with a locked nucleic acid (LNA).

[0025] In the complementary hybridization chain formed by the Lock DNA and the split crRNA, the present application designs a 7nt non-complementary single-stranded DNA (bubble region) which can be cut by the activated Cas12-crRNA complex to open the double-stranded hybridization and release the Split crRNA-handle and Split crRNA-spacer, thereby initiating the next reaction. When the target nucleic acid activates the Cas12a-crRNA complex, the complex can efficiently cut the cascade probe (7nt bubble DNA region formed by the hybridization of the Lock DNA and the split crRNA) to quickly release the split crRNA fragments in the probe. These released split fragments can replace the full-length crRNA-DNA, and then target the pre-filled double-stranded DNA (Ancillary dsDNA, high concentration in the system) to reactivate Cas12a for secondary trans-cleavage reaction. Through this reaction cycle, the active Cas12a-crRNA complex can indirectly catalyze its own continuous generation, forming a self-catalytic circuit that significantly enhances signal amplification.

[0026] The binding affinity of the full-length crRNA to Cas12a is higher than that of the split fragment, and Cas12a will preferentially form a complex with the full-length crRNA, and the split fragment can replace the full-length crRNA and bind to Cas12a after Cas12a-full-length crRNA is activated. Such a crRNA replacement mechanism can effectively eliminate the background signal caused by the partial dissociation of the blocking chain, and the use of LNA modified Lock DNA structure can improve the blocking efficiency, further reduce the background signal, and improve the detection sensitivity.

[0027] Preferably, in the reaction system of the present application, the crRNA is designed according to the target gene. The Split crRNA-handle (the part recognizing Cas12) is a universal sequence (fixed sequence), and therefore the LNA Lock DNA-handle is a universal sequence. The auxiliary signal amplification element of the present application is a double-stranded DNA (Ancillary dsDNA), which can be set to any sequence that can be recognized by the Split crRNA-spacer for activating the Cas12-crRNA complex, and is added at a high concentration to accelerate the entire reaction and achieve signal amplification. The sequences of the Split crRNA-spacer, the LNA Lock DNA-spacer, and the Ancillary dsDNA need to be designed in coordination.

[0028] Preferably, the position of the chemical modification is 1-5 nucleotides at the 5' end and the 3' end of the closed strand.

[0029] Preferably, the nucleotide sequence of the bubble region is 5-7 consecutive C.

[0030] After the exploration experiments of different ATCG sequences, it is found that Cas12a has the highest trans-cleavage efficiency for consecutive 5-7C. The closed strand and the crRNA are complementary to the sequence other than the consecutive 5-7C, and after the complementation, a "bubble region" is generated in the middle due to the non-complementarity of 5-7C.

[0031] Preferably, when the target nucleic acid to be detected is RNA, the detection system satisfies the following conditions:

[0032] (d) The CRISPR effector protein and its crRNA include Cas12a protein and its crRNA, Cas13a protein and its crRNA;

[0033] The Cas12a protein and its crRNA form a Cas12a-crRNA complex;

[0034] The Cas13a protein and its crRNA form a Cas13a-crRNA complex;

[0035] (e) The cascade amplification probe is a self-complementary single strand of closed substrate DNA; the self-complementary single strand comprises a targeting DNA region and a closed RNA region; the targeting DNA region can activate Cas12a; the closed RNA region is closed to the targeting DNA region by reverse complementation, and comprises a 5-7nt non-complementary single-stranded bubble region;

[0036] The self-complementary single strand contains chemical modification;

[0037] (f) The auxiliary signal amplification element is the substrate DNA released by cleavage; when the target RNA exists, the Cas13a-crRNA complex can recognize the target RNA through the crRNA, activate the Cas13 enzyme activity and cleave the bubble region of the cascade amplification probe, release the substrate DNA in the probe, which is the trigger element of the next reaction, and further activate the Cas12a-crRNA complex for secondary signal amplification.

[0038] Preferably, the chemical modification is a locked nucleic acid modification.

[0039] The present application develops a cascade amplification system based on CRISPR-Cas13 and Cas12 for RNA detection, which includes Cas13a enzyme and Cas12a enzyme, crRNA of the two enzymes, and cascade amplification probe for amplifying signal.

[0040] The cascade amplification probe is LNA-CA-template, which is a nucleic acid chain containing a target DNA region capable of activating Cas12a and a blocking RNA region, and the blocking of DNA is realized by reverse complementation (containing a 5nt single-stranded RNA bubble region), and the cascade amplification probe contains a locked nucleic acid modification, which ensures that the RNA perfectly blocks the DNA and does not activate Cas12, thereby reducing the background signal.

[0041] The crRNA corresponding to the Cas13a protein forms a complex with the Cas13a to recognize the target gene RNA, and the crRNA corresponding to the Cas12a protein forms a complex with the Cas12a to recognize the first-stage CRISPR-Cas13a cut LNA-CA-template to release the DNA region.

[0042] The LNA-CA-template is a closed template chain with a locked nucleic acid modification, which has two complementary strands that can be self-complementary to form a double strand, but contains two RNA single-stranded loop structures, which can be cut by the first-stage activated CRISPR / Cas13a in trans to release the DNA region to trigger the second-stage CRISPR / Cas12a. The locked nucleic acid modification enhances the complementary strand closure at the 5' and 3' ends to solve the problem of poor closure, and ensures that the target DNA region can be released after the destruction of the loop structure to provide a binding site for the CRISPR / Cas12a-crRNA complex.

[0043] When the target RNA exists, the Cas13a-crRNA complex can recognize the RNA and be activated, at which time the Cas13a can efficiently cleave the two 5nt bubble RNA regions of the RNA blocking chain on the cascade probe, thereby releasing the target DNA region in the probe, further activating the Cas12a-crRNA complex, and performing a second trans-cleavage reaction to realize signal cascade amplification.

[0044] Preferably, the position of the chemical modification is 1-5 nucleotides from the 5' end and the 3' end of the self-complementary single strand.

[0045] The selected point of the locked nucleic acid modification is at the two ends (5' end and 3' end) away from the loop structure, which ensures that the double-stranded closure does not open before the loop structure is destroyed, and the blocked chain can be released after the loop structure is destroyed, so the locked nucleic acid modification is at the end.

[0046] In a second aspect, the present application provides a method for nucleic acid detection using the detection system, comprising the following steps:

[0047] (1) mixing the nucleic acid sample with the components of the detection system;

[0048] (2) adding a fluorescent detection probe, performing a Cas enzyme cutting reaction, detecting a fluorescent signal, and determining the presence and concentration of the target nucleic acid through the signal intensity.

[0049] Preferably, the nucleic acid sample comprises DNA or RNA extracted from a plasma sample.

[0050] Preferably, the step (2) is detected in combination with a droplet digital detection technology.

[0051] In a third aspect, the present application provides an application of the detection system in nucleic acid detection and / or preparation of a nucleic acid detection product.

[0052] In a fourth aspect, the present application provides a nucleic acid detection product comprising the detection system.

[0053] The present application has the following beneficial effects:

[0054] The detection method of the CRISPR / Cas system combined with isothermal amplification is prone to cross contamination and other problems, and the sensitivity of the CRISPR / Cas system without amplification is relatively low, and the present application establishes two cascade amplification systems based on Cas12 and Cas13, combines with droplet digital detection and locked nucleic acid blocking, realizes single molecule level DNA and RNA detection, and has the following advantages:

[0055] 1. The binding stability of the chemically modified, especially the locked nucleic acid modified blocking chain, is improved, the first level CRISPR-Cas system is not activated, and the second level CRISPR-Cas system is not triggered to produce background signals when there is no target nucleic acid. In the DNA detection system, the background signal is reduced by blocking and splitting crRNA, and Cas12a is activated faster to perform the second trans-cleavage reaction. The unmodified locked nucleic acid blocking chain has poor blocking effect, and even without target nucleic acid, the cascade probe blocking is not firm, which directly triggers the second level CRISPR-Cas system to produce a high background signal.

[0056] 2. The sensitivity of the cascade amplification CRISPR-Cas detection system based on the locked nucleic acid modified chain blocking is greatly improved compared with the single CRISPR / Cas detection system, the detection limit is improved from pM level to aM level, and the detection sensitivity is improved.

[0057] 3. The cascade amplification CRISPR-Cas detection method developed in this research can be applied in Cas12 and Cas13 systems, and can be used for detecting DNA and RNA. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1Schematic diagram of DNA detection by cascade amplification digital CRISPR-Cas12 modified by locked nucleic acid.

[0059] Figure 2 Schematic diagram of RNA detection by cascade amplification digital CRISPR-Cas13 modified by locked nucleic acid.

[0060] Figure 3 Performance comparison results of different locked nucleic acid detection systems in cascade amplification CRISPR-Cas12 detection.

[0061] Figure 4 Performance comparison results of different locked nucleic acid modified sequence blocking chains in cascade amplification CRISPR-Cas13 detection.

[0062] Figure 5 Performance comparison results of different chemically modified blocking chains in cascade amplification CRISPR-Cas13 detection.

[0063] Figure 6 Effect comparison results of locked nucleic acid modification before and after droplet cascade amplification CRISPR-Cas12 detection; A figure is a representative fluorescence droplet chart of blank and positive groups before and after modification of locked nucleic acid, and B figure is a corresponding positive droplet quantity statistical chart; Blank group is a blank control group without target, and Target group is a positive group with target nucleic acid concentration of 100 pM.

[0064] Figure 7 Effect comparison results of locked nucleic acid modification before and after droplet cascade amplification CRISPR-Cas13 detection; A figure is a representative fluorescence droplet chart of blank and positive groups before and after modification of locked nucleic acid, and B figure is a corresponding positive droplet quantity statistical chart; Blank group is a blank control group without target, and Target group is a positive group with target nucleic acid concentration of 100 pM.

[0065] Figure 8 Fluorescence growth curve diagram of cascade amplification CRISPR-Cas12 detection and conventional CRISPR-Cas12 detection (target concentration is 10 pM).

[0066] Figure 9 Fluorescence growth curve diagram of cascade amplification CRISPR-Cas13 detection and conventional CRISPR-Cas13 detection (target concentration is 10 pM).

[0067] Figure 10 Droplet statistical curve diagram of droplet cascade amplification CRISPR-Cas12 detection of different concentrations of samples.

[0068] Figure 11The droplet statistical curve diagram for amplifying the CRISPR-Cas13 detection of different concentrations of samples by the droplet cascade is shown. DETAILED DESCRIPTION

[0069] For the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific embodiments.

[0070] The LNA-modified sequence designed in the present application is synthesized in Kings River Company and Shanghai Biotech Company.

[0071] The oil phase used in the droplet digital detection technology includes mineral oil, palm oil, fluorine oil, carbon oil or oil phase compounded by surfactants; the surfactants are non-ionic surfactants, including span 80, tween 20 or Abil 90.

[0072] The target nucleic acid used in the experiment of the present application, the designed crRNA, the probe and the sequence of the blocking chain are shown in Table 1.

[0073] Table 1

[0074]

[0075]

[0076] Example 1: CRISPR-Cas12-based cascade amplification system

[0077] The CRISPR-Cas12-based cascade amplification system developed in the present embodiment is for DNA detection, and the system mainly includes Cas12a enzyme and its crRNA, cascade probes formed by complementary hybridization of split crRNA (including Split crRNA-handle and Split crRNA-spacer) and LNA-modified blocking chain, and ancillary double-stranded DNA (Ancillary dsDNA) for amplifying signals.

[0078] The Cas12a enzyme and its crRNA form a Cas12a-crRNA complex; the crRNA can specifically recognize the target DNA.

[0079] The Split crRNA-handle and Split crRNA-spacer in the split crRNA can be complementary hybridized with the LNA-modified blocking chain (Lock DNA).

[0080] In the hybridization chain of the Lock DNA and the split crRNA, the present application designs a 7nt single-stranded DNA (bubble region) which is not complementary, so that it can be cut by the activated Cas12 to open the double-stranded hybridization, release the split fragments of the split crRNA, and start the next reaction.

[0081] When the target nucleic acid activates the Cas12a-crRNA complex, the complex can efficiently cleave the cascade probe (7nt bubble DNA region formed by the hybridization of Lock DNA and split crRNA), quickly release the Split crRNA-handle and Split crRNA-spacer fragments in the probe. These released crRNA fragments can replace the full-length crRNA, and then target Ancillary dsDNA (high concentration in the system), re-activate Cas12a for secondary trans-cleavage reaction. Through this reaction cycle, active Cas12a can indirectly catalyze its own continuous generation, forming a self-catalytic loop that significantly enhances signal amplification. Figure 1 ).

[0082] Example 2: CRISPR-Cas13-based cascade amplification system

[0083] This embodiment develops a CRISPR-Cas13 and Cas12-based cascade amplification system for RNA detection, which includes Cas13a enzyme and Cas12a enzyme, crRNA of the two enzymes, and cascade amplification probe for signal amplification.

[0084] Among them, the cascade amplification probe is LNA-CA-template, which contains a nucleic acid chain that targets the DNA region (which can activate Cas12a) and the closed RNA region, and realizes the closure of DNA by reverse complement (contains a 5nt single-stranded RNA bubble region), and contains a locked nucleic acid modification, which ensures that the RNA region perfectly closes the DNA region and does not activate Cas12, reducing the background signal.

[0085] The crRNA corresponding to the Cas13a protein forms a complex with the Cas13a to recognize the target gene RNA.

[0086] The crRNA corresponding to the Cas12a protein forms a complex with the Cas12a to recognize the first-stage CRISPR-Cas13a-cleaved LNA-CA-template-released DNA region.

[0087] LNA-CA-template is a closed template chain with locked nucleic acid modification, which has two DNA and RNA complementary chains that can be closed by itself in single strand, and can be cut by the first level of CRISPR / Cas13a trans to two exposed RNA stem-loop structures, releasing the DNA region to trigger the second level of CRISPR / Cas12a. The locked nucleic acid modification enhances the complementary chain closure at the 5' and 3' ends to solve the problem of poor closure, and ensures that the DNA structure can be released after the stem-loop structure is destroyed, providing a binding site for the CRISPR / Cas12a-crRNA complex.

[0088] When the target RNA exists, the Cas13a-crRNA complex can recognize the RNA and be activated, at which time the Cas13a can efficiently cleave the two 5nt bubble RNA regions of the RNA closed chain on the cascade probe, thereby releasing the DNA in the probe, further activating the Cas12a-crRNA complex, and performing a second trans cleavage reaction Figure 2 ), realizing signal cascade amplification.

[0089] Test Example 1: Performance comparison experiment of cascade amplification CRISPR-Cas12 detection system of different sequences of Lock DNA

[0090] Prepare the cascade amplification CRISPR-Cas12 detection DNA reaction system:

[0091] 1. Add Cas12a with a final concentration of 50-200nM, corresponding fullcrRNA (crRNA targeting target DNA) with a final concentration of 100-250nM, 1x reaction buffer and 0.5-2μM DNA reporter1) to the bottom of a 96-well plate, respectively, and stand for 15 minutes;

[0092] 2. Add Ancillary dsDNA with a final concentration of 100-200nM, 10pM of dsDNA1 target gene, 50-100nM of split crRNA and closed chain mixture: including split crRNA-handle and lock DNA-handle closed mixture (molar concentration ratio of split crRNA-handle: lock DNA-handle = 1:1.2, at this concentration, it can be ensured that the hybridization chain is fully closed), split crRNA-spacer and lock DNA-spacer closed mixture (molar concentration ratio of split crRNA-spacer: lock DNA-spacer = 1:1.2);

[0093] Four groups of Lock DNA-spacer, Lock DNA-handle sequences were set up for comparison in this example. The nucleotide sequences of split crRNA (including Split crRNA-handle, Split crRNA-spacer) and blocking chain (including Lock DNA-handle, Lock DNA-spacer) are shown in Table 2.

[0094] 3. Add DEPC water to make the reaction system to 10-20 μL and mix to get the reaction mixture.

[0095] 4. Transfer the reaction mixture to the PCR instrument and incubate at 37-42 °C for 1-2 hours, detecting the fluorescence intensity (Hex: λex: 535 / 20 nm, λem: 556 / 30 nm) every minute.

[0096] After the reaction, the final fluorescence intensity of the fluorescence curve of the cascade amplification CRISPR-Cas12a of different Lock DNA was analyzed, and the performance differences of the cascade detection system of different Lock DNA were compared.

[0097] Table 2

[0098]

[0099] Note: In the above table, A: adenine deoxyribonucleotide; T: thymine deoxyribonucleotide; C: cytosine deoxyribonucleotide; G: guanine deoxyribonucleotide; *: ribonucleotide modified with locked nucleic acid.

[0100] The results are shown in Figure 3 Among the three groups of Lock DNA with different base mismatches, the Lock DNA 3 group (i.e. SEQ ID NO. 12, SEQ ID NO. 13) not only achieved self-catalytic cycle, but also successfully distinguished positive samples from negative controls, and showed lower background signal and higher signal-to-noise ratio in negative controls, so this group of sequences was selected for subsequent detection.

[0101] Test Example 2: Cascade amplification CRISPR-Cas13 detection system performance comparison experiment of different sequences of locked nucleic acid modified blocking chain

[0102] Prepare the reaction system:

[0103] 1. Add Cas12a with a final concentration of 100-200 nM, crRNA (targeting DNA) with a final concentration of 150-250 nM, Cas13a with a final concentration of 100-200 nM, crRNA (targeting RNA) with a final concentration of 150 nM-250 nM, DNA reporter2 with a final concentration of 0.5-2 μM, 1x reaction buffer, and 100 pM of RNA target gene to the bottom of a 96-well plate, and stand for 15 minutes;

[0104] 2. To explore the difference in blocking effect between self-complementary single-stranded and complementary hybrid strands (double-stranded), the following reagents were added to the mixture in step (1) in groups:

[0105] Group ①: LNA-CA-template (lock nucleic acid modified self-complementary single-stranded, in which RNA blocks DNA-template sequence, preventing DNA-template from activating Cas12) with a final concentration of 20-200 nM;

[0106] Group ②: Lock DNA-template-1 and DNA-template annealing blocked template mixture;

[0107] Group ③: Lock DNA-template-2 and DNA-template annealing blocked template mixture;

[0108] Group ④: Lock DNA-template-3 and DNA-template annealing blocked template mixture.

[0109] This example sets up three groups of Lock DNA-template (template DNA) sequences to verify the blocking effect of LNA-CA-template. Lock DNA-template and DNA-template form double-stranded complementary strands, preventing DNA-template from activating Cas12.

[0110] 3. Add DEPC water to make the reaction system 10-20 μL and mix well to obtain the reaction mixture.

[0111] 4. Transfer the reaction mixture to a PCR instrument and incubate at 37-42 °C for 1-2 hours, detecting the fluorescence intensity (FAM: λex: 494 / 20 nm, λem: 522 / 30 nm) every minute.

[0112] After the reaction, the final fluorescence intensity of the fluorescence curve of the cascade amplification CRISPR-Cas12a of the different locked nucleic acid modified blocking chains was analyzed, and the performance differences of the cascade detection systems of different locked nucleic acid modified blocking chains were compared. The sequences of LNA-CA-template, DNA-template, and Lock DNA-template are shown in Table 3.

[0113] Table 3

[0114]

[0115]

[0116] In the above table, rA: adenine ribonucleotide, rU: uracil ribonucleotide, rC: cytosine ribonucleotide; rG: guanine ribonucleotide, A: adenine deoxyribonucleotide, T: thymine deoxyribonucleotide, C: cytosine deoxyribonucleotide, G: guanine deoxyribonucleotide, *: using locked nucleic acid modified ribonucleotide.

[0117] The results are shown in Table 4. Figure 4 The blocking effect of the single-stranded locked nucleic acid blocking scheme is the best, which can ensure that each template DNA can be blocked, and shows a lower background signal in the negative control, and has a higher signal-to-noise ratio, so this sequence is selected for subsequent detection.

[0118] Test Example 3: Performance comparison experiment of cascade amplification CRISPR-Cas13 detection system of different chemically modified blocking chains

[0119] Prepare the reaction system:

[0120] 1. Add 100-200 nM of Cas12a, 150-250 nM of crRNA (targeting DNA), 100-200 nM of Cas13a, 150 nM-250 nM of crRNA (targeting RNA), 0.5-2 μM of DNA reporter2, 1×reactionbuffer and 100 pM of RNA target gene) to the bottom of the 96-well plate, respectively, and stand for 15 minutes;

[0121] 2. In order to explore the difference in blocking effect of different chemical modifications (including locked nucleic acid modification, 2'-fluoro nucleotide (2'-F) modification, 2'-methoxy ethyl (2'-O-Me) modification), the following reagents were added to the mixture in step (1) in groups:

[0122] Group 1: LNA-CA-template (self-complementary single strand modified by LNA, which blocks the DNA-template sequence to prevent the activation of Cas12) with final concentration of 20-200 nM;

[0123] Group 2: 2’-F-CA-template (self-complementary single strand modified by 2’-fluorine, which blocks the DNA-template sequence to prevent the activation of Cas12) with final concentration of 20-200 nM;

[0124] Group 3: 2’-OMe-CA-template (self-complementary single strand modified by 2’-methoxy, which blocks the DNA-template sequence to prevent the activation of Cas12) with final concentration of 20-200 nM;

[0125] Three groups of chemically modified DNA-template sequences are set up in this example to verify the blocking effect of different chemical modification methods.

[0126] 3. Add DEPC water to make the reaction system 10-20 μL and mix to get the reaction mixture.

[0127] 4. Transfer the reaction mixture to the PCR instrument and incubate at 37-42 °C for 1-2 hours, detecting the fluorescence intensity (FAM: λex: 494 / 20 nm, λem: 522 / 30 nm) every minute.

[0128] After the reaction, the final fluorescence intensity of the fluorescence curve of the cascade amplification CRISPR-Cas12a of different nucleic acid modification blocking chains is analyzed, and the performance differences of the cascade detection system of different chemical modification blocking chains are compared.

[0129] The sequences of LNA-CA-template, 2F-CA-template and 2OMe-CA-template are shown in Table 4.

[0130] Table 4

[0131]

[0132] In the above table, rA: adenine ribonucleotide, rU: uracil ribonucleotide, rC: cytosine ribonucleotide; rG: guanine ribonucleotide, A: adenine deoxyribonucleotide, T: thymine deoxyribonucleotide, C: cytosine deoxyribonucleotide, G: guanine deoxyribonucleotide, *: ribonucleotide modified with a locked nucleic acid, #: ribonucleotide modified with a 2'-fluoro nucleotide (2'-F), &: ribonucleotide modified with a 2'-methoxyethyl (2'-O-Me).

[0133] The results are shown in Figure 5 The locked nucleic acid modification, 2'-fluoro nucleotide modification, and 2'-methoxyethyl modification all have blocking effects, among which the locked nucleic acid modification of LNA-CA-template has the best blocking effect, which can ensure that each template DNA is blocked, and shows lower background signal and higher signal-to-noise ratio in the negative control. The 2'-fluoro nucleotide modified 2'-F-CA-template and the 2'-methoxyethyl (2'-O-Me) modified 2'-OMe-CA-template have higher background signal in the negative control and lower signal-to-noise ratio than the locked nucleic acid modification, so the locked nucleic acid modification of LNA-CA-template is selected for subsequent detection.

[0134] Test Example 4:

[0135] The droplet cascade amplification CRISPR-Cas12 detection utilizes locked nucleic acid modification to compare the blocking effects before and after blocking the chain. The steps are as follows:

[0136] 1. Add a total volume of 3-4 μL of cascade amplification CRISPR-Cas12 system premix to the EP tube:

[0137] containing Cas12a at a final concentration of 100-200 nM, full crRNA (targeting DNA) at a final concentration of 150-250 nM, Ancillary dsDNA at a final concentration of 100-200 nM, DNA reporter1 at a final concentration of 0.5-2 μM, 1x reaction buffer, and 100 pM of dsDNA1 target gene (DEPC water is used instead of the blank group).

[0138] 2. On the basis of the premix, add different locked nucleic acid modified / unmodified blocking chains and split crRNA (including Split crRNA-handle and Split crRNA-spacer) mixtures to different tubes, add DEPC water to 4-5 μL and mix well.

[0139] ① Tube 1: mixture of locked nucleic acid modified blocking chain and split crRNA:

[0140] Split crRNA-handle and LNA Lock DNA-handle closed mixture a (molar concentration ratio of Split crRNA-handle: LNA Lock DNA-handle = 1: 1.2);

[0141] Split crRNA-spacer and LNA Lock DNA-spacer closed mixture b (molar concentration ratio of Split crRNA-spacer: LNA Lock DNA-spacer = 1: 1.2);

[0142] The final concentrations of mixtures a and b are equal, which are 50-100 nM respectively.

[0143] 2. Tube 1: Split crRNA mixed with closed chain without locked nucleic acid modification:

[0144] Split crRNA-handle and Lock DNA-handle (sequence reference LNA Lock DNA-handle, Lock DNA-handle without locked nucleic acid modification) closed mixture c (molar concentration ratio of Split crRNA-handle: Lock DNA-handle = 1: 1.2);

[0145] Split crRNA-spacer and Lock DNA-spacer (sequence reference LNA Lock DNA-spacer, Lock DNA-spacer without locked nucleic acid modification) closed mixture d (molar concentration ratio of Split crRNA-spacer: Lock DNA-spacer = 1: 1.2);

[0146] The final concentrations of mixtures c and d are equal, which are 50-100 nM respectively.

[0147] 3. Add 100-200 μL of oil phase to cover the tube, and shake on a vortex shaker for 1-5 s or blow and suck 4-7 times with a pipette to quickly divide the reagents into polydisperse droplets (the length of shaking time is related to the shaking frequency, and the number of blowing and sucking is related to the gun head of different scales);

[0148] 4. Transfer the generated droplets (emulsion) to a temperature control device (which can be a PCR instrument, a constant temperature instrument, a constant temperature incubator, etc.) for CRISPR-Cas cutting (incubate at 37-42°C for 1-2 hours);

[0149] 5、After the reaction is completed, the signals of the positive droplets are collected by using a fluorescence microscope or a camera / mobile phone with a magnifying lens. The average fluorescence intensity of the blank control group droplets is set as the threshold value of the positive droplets, and the droplets exceeding the threshold value are identified as positive droplets. The number of positive droplets is counted.

[0150] The results are shown in Table 1. Figure 6 As shown in Table 1, the number of droplets in the blank group after blocking the modified locked nucleic acid in the droplet cascade amplification CRISPR-Cas12 detection is the lowest, and the background signal is obviously reduced; while the number of droplets in the positive group after modification of the locked nucleic acid does not decrease significantly, that is, the signal does not decrease significantly, indicating that the locked nucleic acid modification can reduce the background signal of the cascade amplification CRISPR-Cas12a and improve the signal-to-noise ratio.

[0151] Test Example 5:

[0152] The blocking effect comparison experiment before and after the locked nucleic acid modification in the droplet cascade amplification CRISPR-Cas13 detection is as follows:

[0153] 1. Add 3-4 μL of the total volume of the cascade amplification CRISPR-Cas13 system premix liquid to the EP tube:

[0154] containing 100-200 nM of Cas12a, 150-250 nM of crRNA (targeting DNA), 100-200 nM of Cas13a, 150-250 nM of crRNA (targeting RNA), 0.5-2 μM of DNA reporter2, 1x reaction buffer and 100 pM of RNA target gene (DEPC water is used instead of the blank group).

[0155] 2. On the basis of the premix liquid, add 20-200 nM of locked nucleic acid modified / unmodified LNA-CA-template / CA-template (the only difference between the two sequences is / without locked nucleic acid modification, and CA-template is a locked nucleic acid unmodified blocking template chain) to different tubes, add DEPC water to 4-5 μL and mix well.

[0156] 3. Add 100-200 μL of oil phase to cover the tube, and shake on the vortex shaker for 1-5 s or blow and suck 4-7 times with a pipette to quickly divide the reagents into polydisperse droplets (the length of the shaking time is related to the shaking frequency, and the number of blowing and sucking times is related to the gun head of different scales);

[0157] 4. Transfer the generated droplets to the CRISPR-Cas cutting (37-42°C incubation for 1-2 hours) in the temperature control device (which can be a PCR instrument, a constant temperature instrument, a constant temperature incubator, etc.);

[0158] 5、After the reaction is completed, the signals of the positive droplets are collected by a fluorescence microscope or a camera / mobile phone with a magnifying lens. The average fluorescence intensity of the blank control droplets is set as the threshold of the positive droplets, and the droplets exceeding the threshold are identified as positive droplets. The number of positive droplets is counted.

[0159] The results are shown in Table 1. Figure 7 As shown in Table 1, the number of blank droplets in the cascade amplification CRISPR-Cas13 detection after the template modification of the lock nucleic acid is the lowest, and the background signal is obviously reduced; while the number of positive droplets after the modification of the lock nucleic acid does not decrease significantly, that is, the signal does not decrease significantly, indicating that the modification of the lock nucleic acid can reduce the background signal of the cascade amplification CRISPR-Cas13 and improve the signal-to-noise ratio.

[0160] Test Example 6: Trans-cleavage efficiency

[0161] 1. Comparison of trans-cleavage efficiency of cascade amplification CRISPR-Cas12 detection and conventional CRISPR-Cas12 detection:

[0162] Cascade amplification CRISPR-Cas12 detection: The reaction system is the same as that in Test Example 1 (the concentration of the target gene is 10 pM), and the reaction mixture is transferred to a PCR instrument for incubation at 37-42°C for 1-2 hours, and the fluorescence intensity (Hex: λex: 535 / 20 nm, λem: 556 / 30 nm) is detected once every minute.

[0163] Conventional CRISPR-Cas12 detection includes the following steps:

[0164] (1) First, add the reagents required for CRISPR-Cas12 reaction (including components such as Cas12a with a final concentration of 50-200 nM, corresponding crRNA (targeting DNA) with a concentration of 100-250 nM, 1x reaction buffer, and 0.5-2 μM DNA reporter1) to the bottom of a 96-well plate, and stand for 15 minutes (the blank control group is replaced with DEPC water).

[0165] (2) Then add 10 pM of dsDNA1 target gene, and make the system constant volume to 10-20 μL by DEPC water and mix well, and then transfer the obtained mixture to a PCR instrument for incubation at 37-42°C for 1-2 hours, and detect the fluorescence intensity (Hex: λex: 535 / 20 nm, λem: 556 / 30 nm) once every minute.

[0166] After the reaction, the fluorescence growth curves of the conventional CRISPR-Cas12a and the cascade amplification CRISPR-Cas12a were obtained. The fluorescence curves obtained by analyzing different CRISPR-Cas12 reactions were compared to obtain the difference in transcleavage efficiency between the cascade amplification CRISPR-Cas12 detection and the conventional CRISPR-Cas12 detection.

[0167] By observing the fluorescence growth curves in Figure 8 , it can be seen that the transcleavage efficiency of the cascade amplification CRISPR-Cas12 is higher than that of the conventional CRISPR-Cas12 method, and the final fluorescence intensity is higher, while the blank background signal is equivalent.

[0168] 2. Comparison of transcleavage efficiency between cascade amplification CRISPR-Cas13 detection and conventional CRISPR-Cas13 detection:

[0169] Cascade amplification CRISPR-Cas13 detection:

[0170] (1) Add Cas13a with a final concentration of 50-200 nM, corresponding 100-250 nM crRNA (targeting RNA), 0.5-2 μM DNA reporter2 and 1×reaction buffer) to the bottom of a 96-well plate, and stand for 15 minutes.

[0171] (2) Add Cas12a with a final concentration of 50-200 nM, corresponding 100-250 nM crRNA (targeting DNA), 20-200 nM LNA-CA-template and 10 pM RNA target gene.

[0172] (3) Add DEPC water to make the reaction system 10-20 μL and mix to obtain the reaction mixture.

[0173] (4) Transfer the reaction mixture to a PCR instrument and incubate at 37-42°C for 1-2 hours, and detect the fluorescence intensity (FAM: λex: 494 / 20 nm, λem: 522 / 30 nm) every minute.

[0174] The conventional CRISPR-Cas13 detection includes the following steps:

[0175] (1) First, add the reagents required for CRISPR-Cas13 reaction (the reagents include Cas13a with a final concentration of 50-200 nM, corresponding 100-250 nM crRNA (targeting RNA), 0.5-2 μM RNA reporter and 1×reaction buffer, etc.) to the bottom of a 96-well plate, and stand for 15 minutes.

[0176] (2) Add 10 pM of RNA target gene, and then add DEPC water to make the system volume 10 or 20 microliters and mix, and then transfer the obtained mixture to a PCR instrument for incubation at 37-42°C for 1-2 hours, and detect the fluorescence intensity (FAM: λex: 494 / 20 nm, λem: 522 / 30 nm) every minute.

[0177] After the reaction is completed, the fluorescence growth curves of the conventional CRISPR-Cas13 and the cascade amplification CRISPR-Cas13 are obtained. By analyzing the fluorescence curves obtained by different CRISPR-Cas13 reactions, the difference in trans-cleavage efficiency between the cascade amplification CRISPR-Cas13 detection and the conventional CRISPR-Cas13 detection is obtained.

[0178] By observing the fluorescence growth curves in Figure 9 , it can be seen that the cascade amplification CRISPR-Cas13 has a slightly higher blank background signal than the conventional CRISPR-Cas12 method, but has a higher trans-cleavage efficiency, a faster fluorescence amplification speed, and a higher final fluorescence intensity.

[0179] Test Example 7: Sensitivity test

[0180] 1. Sensitivity comparison experiment of cascade amplification CRISPR-Cas12 and conventional CRISPR-Cas12 detection based on polydisperse droplets, the system and steps refer to test example 4, different concentrations of target gene dsDNA1 are added to each tube for detection of sensitivity, and DEPC water is used as a blank control.

[0181] The results are shown in Figure 10 . The detection limit of droplet cascade amplification CRISPR-Cas12 can reach 100 aM, while the detection limit of droplet conventional CRISPR-Cas12 can only reach 100 fM. The detection limit of the droplet cascade amplification CRISPR-Cas12 method of the present application is at least three orders of magnitude higher than that of the droplet conventional CRISPR-Cas12 method.

[0182] 2. Sensitivity comparison experiment of cascade amplification CRISPR-Cas13 and conventional CRISPR-Cas13 detection based on polydisperse droplets, the system and steps refer to test example 5, different concentrations of target gene RNA are added to each tube for detection of sensitivity.

[0183] The results are shown in Figure 11The detection limit of the droplet cascade amplification CRISPR-Cas13 can reach 1aM, while the detection limit of the droplet conventional CRISPR-Cas13 can only reach about 1fM, and the detection limit of the droplet cascade amplification CRISPR-Cas13 method of the present application is at least two orders of magnitude higher than that of the droplet conventional CRISPR-Cas13 method.

[0184] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A CRISPR-Cas system-based cascade signal amplification detection system, characterized in that, The detection system comprises: (1) a target nucleic acid to be detected, a CRISPR effector protein and a crRNA thereof; (2) a cascade amplification probe, which is a complementary hybridization nucleic acid strand for blocking a next-stage reaction trigger element; the next-stage reaction trigger element comprises a split crRNA or a substrate DNA; the complementary hybridization nucleic acid strand is a complementary hybridization double strand or a self-complementary single strand comprising an uncomplementary single-stranded bubble region; the complementary hybridization nucleic acid strand comprises a chemical modification; the chemical modification comprises a locked nucleic acid modification, a 2'-fluoro nucleotide modification, and a 2'-methoxyethyl modification; (3) an auxiliary signal amplification element, which releases a trigger element of a next-stage reaction by cleavage of a previous-stage CRISPR effector protein, forming a self-catalytic cycle.

2. The detection system of claim 1, wherein, When the target nucleic acid to be detected is DNA, the detection system satisfies the following conditions: (a) the CRISPR effector protein is a Cas12a protein; the crRNA specifically recognizes the target nucleic acid DNA; the Cas12a protein and the crRNA form a Cas12a-crRNA complex; (b) the cascade amplification probe is a complementary hybridization double strand for blocking a split crRNA; the split crRNA and the chemically modified blocking strand hybridize to form a double-stranded structure comprising a 5-7 nt single-stranded bubble region; the split crRNA comprises a Split crRNA-handle and a Split crRNA-spacer; (c) the auxiliary signal amplification element is a pre-filled double-stranded DNA; when the target DNA exists, the Cas12a-crRNA complex recognizes the target DNA through the crRNA, activates the Cas12a enzyme activity, and cleaves the bubble region of the cascade amplification probe to release the Split crRNA-handle and the Split crRNA-spacer; the Split crRNA-spacer recognizes and binds to the auxiliary signal amplification element; the Split crRNA-handle binds to the Cas12a protein through a displacement mechanism, and, in combination with the Split crRNA-spacer and the pre-filled double-stranded DNA, further activates the Cas12a enzyme activity, forming a self-catalytic cycle.

3. The detection system of claim 2, wherein, the position of the chemical modification is 1-5 nucleotides at the 5' end and the 3' end of the blocking strand.

4. The detection system of claim 2, wherein, the nucleotide sequence of the bubble region is 5-7 consecutive C.

5. The detection system of claim 1, wherein, When the target nucleic acid to be detected is RNA, the detection system satisfies the following conditions: (d) the CRISPR effector protein and the crRNA thereof comprise a Cas12a protein and a crRNA thereof, and a Cas13a protein and a crRNA thereof; the Cas12a protein and the crRNA thereof form a Cas12a-crRNA complex; the Cas13a protein and the crRNA thereof form a Cas13a-crRNA complex; (e) the cascade amplification probe is a self-complementary single strand which seals the substrate DNA; the self-complementary single strand comprises a targeting DNA region and a sealing RNA region; the targeting DNA region is capable of activating Cas12a; the sealing RNA region seals the targeting DNA region by reverse complementation, and comprises a 5-7 nt non-complementary single-stranded bubble region; the self-complementary single strand contains chemical modification; (f) the auxiliary signal amplification element is substrate DNA released by cleavage; when the target RNA exists, the Cas13a-crRNA complex can recognize the target RNA by crRNA, activate the Cas13 enzyme activity and cleave the bubble region of the cascade amplification probe, release the substrate DNA in the probe, which is the trigger element for the next reaction, further activate the Cas12a-crRNA complex for secondary signal amplification.

6. The detection system of claim 5, wherein, The position of the chemical modification is 1-5 nucleotides from the 5' end and the 3' end of the self-complementary single strand.

7. A method for nucleic acid detection using the detection system according to any one of claims 1 to 6, characterized in that, It comprises the following steps: (1) mixing the nucleic acid sample with each component of the detection system; (2) adding a fluorescence detection probe, performing Cas enzyme cleavage reaction, detecting fluorescence signal, and determining the presence and concentration of the target nucleic acid by signal intensity.

8. The method of claim 7, wherein, The step (2) is combined with droplet digital detection technology for detection.

9. Use of the detection system according to any one of claims 1-6 in nucleic acid detection and / or preparation of nucleic acid detection products.

10. A nucleic acid detection product, characterized by It comprises the detection system according to any one of claims 1-6.

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