Light-regulated single molecule crisper-cas detection method and kit

Abstract

The present application relates to a kind of light-regulated single molecule CRISPR-Cas detection method and kit.The detection method includes: S1, providing CRISPR reaction system and blocking sequence, configuration is premixed reaction liquid;S2, the target nucleic acid-containing sample to be measured is mixed with premixed reaction liquid to obtain mixed system, and the mixed system is dispersed to a large number of volume uniform microreaction unit;S3, UV light, and incubate microreaction unit;S4, read fluorescence signal, calculate the content of target nucleic acid.Blocking sequence can be combined with Cas effect protein in CRISPR reaction system to silence the activity of the protein, and the photofragmentation group in blocking sequence is broken after light, and is separated from Cas effect protein, so that its activity is unsilenced, this light-regulated mode can make the CRISPR catalytic system in each microreaction unit start at the same time, avoid the problem of background signal increase and inaccurate quantification caused by a small amount of Cas protein being activated to degrade the measured target and report probe before microreaction unit distribution is completed.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a light-controlled single-molecule CRISPR-Cas detection method and kit. Background Technology

[0002] Quantitative detection of nucleic acid molecules has significant applications in both scientific research and medical fields. For example, absolute quantification of microRNA expression levels, non-invasive prenatal screening, rare mutation frequency analysis, copy number variation (CNV) analysis, and gene editing efficiency assessment all require efficient and accurate quantitative detection using low-abundance, limited samples. Currently, quantitative PCR (qPCR), the gold standard for nucleic acid quantification, and third-generation droplet digital polymerase chain reaction (ddPCR) technologies both offer high detection sensitivity and quantification capabilities. However, the reverse transcription and nucleic acid amplification steps involved in these processes are susceptible to interference from various factors, affecting the accuracy of the detection. Methods that directly detect target DNA / RNA molecules without nucleic acid amplification not only fundamentally avoid the risk of aerosol contamination that may arise from conventional amplification methods but also avoid inaccuracies caused by amplification bias and incomplete transcription. In recent years, methods such as single-molecule fluorescence spectroscopy, nanopores, nano-atomic force microscopy, and DNA nanotechnology have been developed to detect nucleic acids at the single-molecule level. However, these sensing methods all rely on molecular diffusion, so they require high sample concentrations to be effective. They cannot detect low-abundance, limited samples, which to some extent restricts their widespread application.

[0003] The CRISPR-Cas system originates from the adaptive immune system of bacteria and archaea. Due to its programmability, high specificity, and isothermal reaction characteristics, numerous nucleic acid technologies based on CRISPR-Cas for pathogen detection, tumor diagnosis, and on-site detection have emerged in recent years. Because CRISPR-Cas proteins possess extremely efficient trans-cleavage activity—meaning they can non-specifically cleave thousands of reporter molecules after activation by target nucleic acid molecules—they achieve high signal amplification. Based on this highly efficient trans-cleavage signal amplification characteristic of the CRISPR-Cas system, a hyperlocal CRISPR-Cas reaction system has been developed, achieving single-molecule sensitivity for amplification-free absolute nucleic acid quantification. In particular, the sensitivity of this amplification-free direct detection method can be further improved by using multiple crRNAs combined to target different sites of the same target.

[0004] Because CRISPR catalysis retains residual activity at lower temperatures, the target and probe may be partially degraded by pre-activated Cas proteins during system preparation and microreaction unit distribution. This can lead to inconsistent reaction initiation points and increased background signals in different microreaction units, affecting the accuracy of subsequent quantification. Especially when using multiple crRNA combinations, while detection sensitivity can be improved, unblocked Cas proteins can cleave long-chain targets at different crRNA target sites. Target fragments can then be dispersed across different microreaction units, resulting in a higher-than-expected number of positive units and hindering accurate quantification. To achieve truly high-precision quantification, especially when requiring accuracy down to the single molecule level and the ability to distinguish subtle base differences and initial concentrations, precise control of the reaction is necessary. This involves temporarily "shutting down" Cas protein activity during system preparation and microreaction unit generation, and then "reviving" the protein using a simple, convenient, and universal method after microreaction unit distribution, allowing single-molecule reactions in each microreaction unit to "start" simultaneously. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a light-controlled single-molecule CRISPR-Cas detection method, which has the advantages of high accuracy, no amplification required, and high sensitivity.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A light-controlled single-molecule CRISPR-Cas detection method, comprising: S1. Provide the CRISPR reaction system and the blocking sequence, and configure them into a premixed reaction solution; The CRISPR reaction system described above includes Cas effector protein, crRNA or sgRNA, fluorescent reporter probe, blocking sequence and reaction buffer; The blocking sequence is a nucleotide sequence or nucleotide analogue modified with at least two modifications: a photocleavage group and thiophosphate. It is used to form a protein-nucleic acid complex with the Cas effector protein in the CRISPR reaction system to silence its enzyme activity. The photocleavage group has photo-cleavage properties and breaks after photoexcitation, so that the blocking sequence is separated from the Cas effector protein and the enzyme activity of the Cas effector protein is de-silenced. S2. The analyte containing the target nucleic acid is mixed with the above premixed reaction solution to obtain a mixed system, and then the mixed system is dispersed into a large number of uniformly sized microreaction units. S3. Start UV light irradiation and provide suitable temperature conditions to incubate the micro-reaction unit; The photo-induced cleavage group breaks after photoexcitation, thereby separating the blocked sequence from the Cas effector protein. The enzyme activity of the Cas effector protein is de-silenced, and the crRNA or sgRNA in all microreaction units starts recognizing the target nucleic acid sequence at the same time point and triggers the Cas effector protein to initiate a synergistic effect to continuously cleave the fluorescent reporter probe. S4. After the incubation reaction is completed, read the fluorescence signal in the microreaction unit, set the intensity threshold for judging negative / positive signals, and calculate the content of target nucleic acid in the sample to be tested.

[0007] The photo-regulated single-molecule CRISPR-Cas detection method described in this invention employs a designed blocking sequence, which is a nucleotide sequence or nucleotide analog modified with at least two modifiers: a photocleaving group and phosphate thiophosphate. Phosphate thiophosphate modification not only enables the blocking sequence to bind stably to the Cas effector protein, thereby silencing its activity, but also enhances the stability of the blocking sequence. Simultaneously, the photocleaving group in the blocking sequence exhibits photo-induced bond-breaking properties; upon exposure to UV light, it breaks down into small nucleotide fragments. The binding stability of these fragments or nucleotide analogs to the Cas effector protein decreases, allowing them to dissociate from the Cas effector protein surface into the reaction system, ultimately desilencing the Cas effector protease activity. Therefore, the blocking sequence... When added to the CRISPR reaction system, the activity of the Cas protein can be temporarily "silenced" during the preparation of the reaction system and the distribution of microreaction units. After the premixed reaction solution obtained by mixing the CRISPR reaction system to be analyzed with the blocking sequence is evenly dispersed into hundreds of thousands of independent, parallel cell-sized microreaction units through a specific microreaction unit distribution method, the CRISPR reaction system in each microreaction unit can be started simultaneously by simple light treatment. Due to the unique efficient trans-cutting signal amplification mechanism of the CRISPR-Cas system and the confinement effect generated by the small reaction volume, a microreaction unit containing a single analyte molecule can generate a detectable fluorescence signal for subsequent analysis and result interpretation without the need for nucleic acid amplification, thereby calculating the initial concentration of the analyte molecule. This light-controlled approach enables the CRISPR catalytic system in each microreaction unit to start simultaneously, avoiding the problem of background signal increase and inaccurate quantification caused by the degradation of the target and reporter probe due to the small amount of activated Cas protein before the microreaction unit is fully distributed. It achieves precise control of the CRISPR catalytic system in the microreaction unit, and activation can be achieved through simple light exposure, which helps to simplify experimental operations.

[0008] Compared with the prior art, the present invention has the following advantages: 1) It enables quantitative detection without the need for reverse transcription and nucleic acid amplification steps, allowing for direct detection with high specificity and sensitivity; 2) By using multiple crRNA combinations to target different regions of the same target to improve reaction efficiency and detection sensitivity, the integrity of the target to be tested in the microreaction unit before the reaction starts is ensured. This enables precise and rapid regulation of the CRISPR catalytic reaction in the microreaction unit, achieving single-molecule level nucleic acid detection and achieving truly high-precision absolute quantification without the need for internal reference calibration or reliance on standard curves. 3) The blocking sequence is applicable to various Cas effector proteins without the need for adjustment based on different Cas effector proteins. It is also applicable to various microunit distribution methods, which is universal and helps to save costs and simplify experimental operations. It is simple, convenient and fast to operate, and is a constant temperature reaction, so there is no need to use thermal cycling equipment. Furthermore, the closed sequence is a single-base repeat sequence or a random base sequence, with a length of 5~200 nt.

[0009] Furthermore, the photo-splitting group is embedded within the closed sequence, which facilitates the cleavage of the closed sequence into several small fragments after photolysis, thereby better detaching it from the Cas effector protein and restoring its activity.

[0010] Furthermore, the number of photo-splitting groups in the closed sequence is 3 to 199, wherein one photo-splitting group is inserted every 1 to 50 bases; the number of thiophosphate modification sites in the closed sequence is 4 to 199.

[0011] Furthermore, the photo-splitting group is PC-Linker, which, as a photosensitive group, can remain stable under natural light, thus not being limited by the sample preparation environment.

[0012] Furthermore, the closed sequence is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, a hybrid double strand formed by DNA and RNA, or a chimeric strand formed by DNA and RNA.

[0013] Further, in step S1, the molar ratio of the blocked sequence to the Cas effector protein in the CRISPR reaction system is 0.01~100:1.

[0014] Furthermore, the method of dispersing the mixed system into a large number of uniformly sized micro-reaction units in step S2 includes micro-chamber array and micro-droplet emulsification. Specifically, the means of forming micro-reaction units include, but are not limited to, passive micro-reaction unit formation methods such as cross-flow, co-flow, flow-focusing, stepemulsification, microchannel emulsification, and membrane emulsification, as well as active micro-reaction unit formation methods based on external forces (magnetic force, electric field force, centrifugal force, gravity, etc.) or fluid dynamic parameters (surface wetting, interfacial tension, fluid density, fluid velocity). Preferably, the effective volume of the microreaction unit does not exceed 20 pL, so that the number of targets to be detected in a single microreaction unit does not exceed 1 (0 or 1). For this method, while ensuring that a single microreaction unit can be effectively detected, reducing the volume of the reaction unit within a certain range is beneficial to shorten the detection time and improve the quantitative accuracy. The micro-reaction unit formed in step S3 can be incubated in situ in a static or continuous flow manner, with an incubation temperature in the range of 25~70 ℃ and an incubation time not exceeding 90 min; Step S4 includes counting the total number of positive microreaction units or the proportion of positive microreaction units to the total number of statistical microreaction units, counting the number of target nucleic acid molecules in the sample to be tested, or using the Poisson distribution principle to calculate the original concentration of target nucleic acid molecules in the sample to be tested.

[0015] Further, in step S2, the final concentration of the Cas effector protein in the mixing system is 10-200 nM, preferably 20 nM; the final concentration of crRNA / sgRNA in the mixing system is 20-400 nM, preferably 40 nM; the final concentration of the fluorescent reporter probe in the mixing system is 200-2000 nM, preferably 500-1000 nM; and the ratio of the final concentration of the blocking sequence to the final concentration of the Cas effector protein in the mixing system is 0.01-100:1, preferably 0.2-5:1. In step S3, the duration of UV irradiation is 5s to 30 min, the wavelength of the UV light used is 300 to 400 nm, and the power is 10 to 300 W.

[0016] In addition, embodiments of the present invention also provide a light-controlled single-molecule CRISPR-Cas detection kit, which includes the premixed reaction solution described above.

[0017] The analyte containing the target nucleic acid is directly added to the kit to form a mixed system. A large number of microreaction units are generated by a microreaction unit generator. The mixed system is dispersed into the microreaction units. Further light treatment of the microreaction units enables the CRISPR reaction system in each microreaction unit to start simultaneously, realizing single-molecule nucleic acid detection without amplification. It also achieves light regulation and avoids the problem of background signal increase and inaccurate quantification caused by the degradation of the target and reporter probe by a small amount of activated Cas protein before the microreaction units are fully distributed.

[0018] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the light-controlled single-molecule CRISPR-Cas detection method described in the embodiments of the present invention; Figure 2 This is a schematic diagram of the micro-reaction unit allocation according to an embodiment of the present invention; Figure 3 This is a graph showing the screening results of crRNA targeting different regions of long-chain targets in Example 1; Figure 4 This is a graph showing the effect of different crRNA combinations on detection efficiency in Example 1; Figure 5 This is a graph showing the effect of photomodulation of the confined CRISPR-Cas12a unit on the uniformity of fluorescence values ​​of the positive microreaction unit in Example 1. Figure 6 This is a graph showing the effect of photomodulation of the confined CRISPR-Cas12a unit on the uniformity of fluorescence values ​​of the positive microreaction unit in Example 2. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0022] This invention provides a light-controlled single-molecule CRISPR-Cas detection method, comprising: S1. Provide the CRISPR reaction system and the blocking sequence, and configure them into a premixed reaction solution; The CRISPR reaction system described above includes Cas effector protein, crRNA or sgRNA, fluorescent reporter probe, blocking sequence and reaction buffer; The blocking sequence is a nucleotide sequence or nucleotide analogue modified with at least two modifications: a photocleavage group and thiophosphate. It is used to form a protein-nucleic acid complex with the Cas effector protein in the CRISPR reaction system to silence its enzyme activity. The photocleavage group has photo-cleavage properties and breaks after photoexcitation, so that the blocking sequence is separated from the Cas effector protein and the enzyme activity of the Cas effector protein is de-silenced. The blocking sequence is a monobasic repeat sequence or a random base sequence, which can be single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, a hybrid double strand formed by DNA and RNA, or a chimeric strand formed by DNA and RNA, with a length of 5-200 nt; the photo-cleaving group is preferably PC-Linker, which, as a photosensitive group, can remain stable under natural light, so that it is not limited by the sample preparation environment. PC-Linker is embedded in the blocking sequence, which facilitates the cleavage of the blocking sequence into several small fragments after photolysis, thereby better detaching from the Cas effector protein and restoring its activity; preferably, the number of photo-cleaving groups in the blocking sequence is 3-199, wherein one photo-cleaving group is embedded every 1-50 bases; the number of phosphate thiocyanate modification sites in the blocking sequence is 4-199.

[0023] The reaction buffer contains 10 mM Tris-HCl, 3 mM MgCl2, 50 mM KCl, 0.005% (v / v) IGEPA-CA630, pH = 8.0; The following sample addition order is used for preparation: reaction buffer, Cas protein, crRNA combination, blocking sequence, reporter probe, and target to be tested. The molar ratio of the blocking sequence to the Cas effector protein in the resulting CRISPR reaction system is 0.01~100:1, preferably 0.2~5:1.

[0024] S2. The analyte containing the target nucleic acid is mixed with the above premixed reaction solution to obtain a mixed system, and then the mixed system is dispersed into a large number of uniformly sized microreaction units. After mixing, the final concentration of Cas effector protein in the mixing system is 10-200 nM, preferably 20 nM; the final concentration of crRNA / sgRNA in the mixing system is 20-400 nM, preferably 40 nM; the final concentration of fluorescent reporter probe in the mixing system is 200-2000 nM, preferably 500-1000 nM; and the ratio of the final concentration of the blocking sequence to the final concentration of Cas effector protein in the mixing system is 0.01-100:1, preferably 0.2-5:1. The resulting hybrid system can be achieved through either a microchamber array or a microdroplet emulsification method. The microreaction units can be formed in at least one of passive and active reaction unit formation methods. Passive reaction unit formation methods include cross-flow, co-flow, flow-focusing, step emulsification, microchannel emulsification, and membrane emulsification. Active reaction unit formation methods include those based on external forces (magnetic force, electric field force, centrifugal force, gravity, etc.) or fluid dynamic parameters (surface wetting, interfacial tension, fluid density, fluid velocity). Preferably, the effective volume of the microreaction unit does not exceed 20 pL, so that the number of targets to be detected in a single microreaction unit does not exceed 1 (0 or 1). For this method, while ensuring that a single microreaction unit can be effectively detected, reducing the volume of the reaction unit within a certain range is beneficial to shorten the detection time and improve the quantitative accuracy.

[0025] S3. Start UV light irradiation and provide suitable temperature conditions to incubate the micro-reaction unit; The duration of UV irradiation is 5s to 30min, the wavelength of the UV light used is 300 to 400 nm, and the power is 10 to 300W; The micro-reaction unit after light treatment can be incubated in situ in a static or continuous flow manner, with an incubation temperature in the range of 25~70 ℃ and an incubation time not exceeding 90 min. After UV irradiation, the photo-induced cleavage group is photoexcited and cleaved, thereby separating the blocked sequence from the Cas effector protein. The enzyme activity of the Cas effector protein is de-silenced, and the crRNA or sgRNA in all microreaction units is activated at the same time point to recognize the target nucleic acid sequence and trigger the Cas effector protein to initiate a co-operative effect to continuously cleave the fluorescent reporter probe.

[0026] S4. The signal intensity of the microreaction unit after the incubation reaction is measured by scanning imaging or flow cytometry. The intensity threshold for judging negative / positive signals is set, and the total number of positive units or the proportion of positive units to the total number of units counted is statistically analyzed. Alternatively, the original concentration of the target nucleic acid molecule in the sample to be tested can be calculated using the Poisson distribution principle.

[0027] Please refer to Figure 1-2 , Figure 1 This is a schematic diagram of the light-controlled single-molecule CRISPR-Cas detection method described in this embodiment of the invention. Figure 2 This is a schematic diagram of the microreaction unit allocation according to an embodiment of the present invention. As shown in the figure, the photo-regulated single-molecule CRISPR-Cas detection method of the present invention designs a blocking sequence, which is a nucleotide sequence or nucleotide analog modified with at least two modifications: a photocleaving group and phosphate thiophosphate. Phosphate thiophosphate modification not only enables the blocking sequence to bind stably to the Cas effector protein, thereby silencing the protein's activity, but also enhances the stability of the blocking sequence. Simultaneously, the photocleaving group in the blocking sequence has photo-induced bond-breaking properties; upon exposure to UV light, it breaks bonds, causing the blocking sequence to break into small nucleotide fragments. The binding stability of these small nucleotide fragments or nucleotide analog fragments to the Cas effector protein decreases, allowing them to be released from the Cas effector protein surface into the reaction system, ultimately de-silencing the Cas effector protease activity. Therefore, adding the blocking sequence to the CRISPR reaction system temporarily "silences" the Cas protein activity during the preparation of the reaction system and the allocation of the microreaction units. Figure 2 As shown, after the premixed reaction solution obtained by mixing the CRISPR reaction system to be analyzed and the blocking sequence is uniformly dispersed into hundreds of thousands of independent, parallel cell-sized microreaction units through a specific microreaction unit distribution method, the CRISPR reaction system in each microreaction unit can be simultaneously activated by simple light treatment. Due to the highly efficient trans-cleavage signal amplification mechanism unique to the CRISPR-Cas system and the confinement effect generated by the small reaction volume, a microreaction unit containing a single analyte molecule can generate a detectable fluorescence signal for subsequent analysis and result interpretation without nucleic acid amplification, thereby calculating the initial concentration of the analyte molecule. This light-controlled method enables the CRISPR catalytic system in each microreaction unit to be activated at the same time, avoiding the problem of background signal increase and inaccurate quantification caused by the degradation of the analyte target and reporter probe due to the small amount of activated Cas protein before the microreaction unit distribution is completed. This achieves precise control of the CRISPR catalytic system in the microreaction unit, and activation can be achieved through simple light treatment, which helps simplify experimental operations.

[0028] Based on this, embodiments of the present invention also provide a light-controlled single-molecule CRISPR-Cas detection kit, which includes the premixed reaction solution described above.

[0029] The analyte containing the target nucleic acid is directly added to the kit to form a mixed system. A large number of microreaction units are generated by a microreaction unit generator. The mixed system is dispersed into the microreaction units. Further light treatment of the microreaction units enables the CRISPR reaction system in each microreaction unit to start simultaneously, realizing single-molecule nucleic acid detection without amplification. It also achieves light regulation and avoids the problem of background signal increase and inaccurate quantification caused by the degradation of the target and reporter probe by a small amount of activated Cas protein before the microreaction units are fully distributed.

[0030] The following describes the solution of the present invention in conjunction with specific embodiments: Example 1 In this embodiment, the effect of crRNAs with different target locations on Cas12a detection is verified, as follows: 1. Screening crRNAs targeting different positions of long-chain test targets The target used in this embodiment is a synthetic plasmid containing a partial SARS-CoV-2 S-gene fragment. This gene fragment is 1000 bp in length, and its sequence information is as follows:

[0031] Based on the above sequence, 15 crRNAs were designed to target different positions of the sequence. The specific sequences of the crRNAs are shown in Table 1 below: Table 1

[0032]

[0033] Using the same target nucleic acid, the reaction efficiency of the above 15 crRNAs was verified respectively, and the test results are as follows: Figure 3 As shown; 2. Based on the above results, seven highly efficient crRNAs were selected. In this example, numbers 5, 6, 10, 11, 12, 13, and 15 were selected for combination to explore whether different crRNA combinations improved the efficiency of Cas12a in detecting target DNA. The test results are as follows: Figure 4 As shown; In this embodiment, a total of 7 combinations of crRNAs were verified, including: Combination 1: crRNA5; Combination 2: crRNA5 + crRNA6; Combination 3: crRNA5+ crRNA6+ crRNA10; Combination 4: crRNA5+ crRNA6+ crRNA10+ crRNA11; Combination 5: crRNA5+crRNA6+crRNA10+crRNA11+crRNA12; Combination 6: crRNA5+ crRNA6+ crRNA10+ crRNA11+ crRNA12+ crRNA13; Combination 7: crRNA5+ crRNA6+ crRNA10+ crRNA11+ crRNA12+ crRNA13+ crRNA15.

[0034] 3. The above seven crRNA combinations were used to conduct light-regulated microreaction unit experiments, as detailed below: The premixed reaction solution was prepared by adding the following samples in the following order: reaction buffer, Cas protein, crRNA combination, blocking sequence, and reporter probe. The target to be tested is added to the obtained premixed reaction solution to form a mixed system, which is then added to the chip injection port to generate micro-reaction units, thereby forming a large number of uniform water-in-oil discrete micro-reaction units with a diameter of 20-30 µm; the number of micro-reaction units is not less than 1×105; the number of micro-reaction units stacked due to vertical gravity does not exceed 2 layers to construct a micro-reaction unit array; The chip was then placed under a UV lamp for irradiation, and after irradiation, it was placed on a temperature-controlled heating device for incubation; the temperature of the chip in the microreactor was maintained at 45°C for 1 hour. After incubation, the chip was scanned under a fluorescence microscope to obtain fluorescence images, and the diameter D of the microreaction units was measured. A signal threshold for the microreaction units was set as S / N≥2, where S is the signal intensity of the microreaction unit and N is the average background signal. The microreaction unit signals were binarized based on the fluorescence intensity threshold; a fluorescence intensity higher than the threshold was considered positive ("1"), otherwise negative ("0"). The total number of microreaction units and the number of positive microreaction units were counted. The plasmid DNA content C was calculated based on the proportion p of positive microreaction units, using the following formula:

[0035] in, .

[0036] In addition, in this embodiment, an experimental group without the blocking sequence was set up as a control to explore the effect of light control on the fluorescence intensity of confined CRISPR-Cas12a reaction-positive microreaction units, as detailed below: Twenty positive microreaction units were randomly selected from the fluorescence images of both the uncontrolled and controlled-light experimental groups. The fluorescence intensity of these 40 positive microreaction units was statistically analyzed using ImageJ grayscale analysis. The results are as follows: Figure 5 As shown.

[0037] The statistical distribution of the results in the figure shows that the fluorescence intensity of different positive microreaction units in the uncontrolled group is relatively dispersed, while the fluorescence intensity of the positive microreaction units in the light-controlled experimental group is more uniform and concentrated. The uniformity of fluorescence intensity distribution is particularly important when resolving single-base mutations based on single-molecule fluorescence kinetics. More uniform fluorescence intensity allows even weak fluorescence intensity to serve as a standard for determining the single-molecule reaction kinetics in different microunits, eliminating errors caused by inconsistent reaction initiation and ensuring the accuracy of single-molecule catalytic reaction kinetics detection.

[0038] Example 2 In this embodiment, micro-17 is used as the detection target to verify the effect of illumination on Cas12a detection, as follows: 1. Prepare the premixed reaction solution by adding the following samples in the following order: reaction buffer, Cas protein, crRNA combination, blocking sequence, and reporter probe; The target to be tested is added to the obtained premixed reaction solution to form a mixed system, which is then added to the chip injection port to generate micro-reaction units, thereby forming a large number of uniform water-in-oil discrete micro-reaction units with a diameter of 20~30 µm; the number of micro-reaction units is not less than 1×105; the number of micro-reaction units formed is not more than 2 layers due to vertical gravity stacking, so as to construct a micro-reaction unit array. The chip was then placed under a UV lamp for irradiation, and after irradiation, it was placed on a temperature-controlled heating device for incubation; the temperature of the chip in the microreactor was maintained at 45°C for 1 hour. After incubation, the chip was scanned under a fluorescence microscope to obtain fluorescence images, and the diameter D of the microreaction units was measured. A signal threshold for the microreaction units was set as S / N≥2, where S is the signal intensity of the microreaction unit and N is the average background signal. The microreaction unit signals were binarized based on the fluorescence intensity threshold; a fluorescence intensity higher than the threshold was considered positive ("1"), otherwise negative ("0"). The total number of microreaction units and the number of positive microreaction units were counted. The plasmid DNA content C was calculated based on the proportion p of positive microreaction units, using the following formula:

[0039] in, .

[0040] In addition, in this embodiment, an experimental group without the blocking sequence was set up as a control to explore the effect of light control on the fluorescence intensity of confined CRISPR-Cas12a reaction-positive microreaction units, as detailed below: Twenty positive microreaction units were randomly selected from the fluorescence images of both the uncontrolled and controlled-light experimental groups. The fluorescence intensity of these 40 positive microreaction units was statistically analyzed using ImageJ grayscale analysis. The results are as follows: Figure 6 As shown.

[0041] The statistical results in the figure show that the fluorescence intensity of different positive microreaction units in the uncontrolled group is more dispersed, while the fluorescence intensity of the positive microreaction units in the controlled group is more uniform.

[0042] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A non-diagnostic, light-controlled single-molecule CRISPR-Cas detection method, comprising the following steps: S1. Provide the CRISPR reaction system and the blocking sequence, and configure them into a premixed reaction solution; The CRISPR reaction system includes a Cas effector protein, crRNA or sgRNA, a fluorescent reporter probe, a blocking sequence, and a reaction buffer; the Cas effector protein is Cas12a protein. The blocking sequence is a nucleotide sequence modified with both photolytic cleavage groups and phosphate thiophosphate. It is used to form a protein-nucleic acid complex with Cas effector proteins in the CRISPR reaction system, thereby silencing their enzyme activity. The photolytic cleavage groups have photo-cleaving properties, breaking upon photoexcitation to separate the blocking sequence from the Cas effector protein and de-silence its enzyme activity. The blocking sequence is single-stranded DNA or single-stranded RNA. The photolytic cleavage groups are embedded within the blocking sequence, with 3 to 199 photolytic cleavage groups, one photolytic cleavage group being embedded every 1 to 50 bases. The number of phosphate thiophosphate modification sites in the blocking sequence is 4 to 199. The closed sequence is a single-base repeat sequence or a random base sequence, with a length of 5~200 nt; S2. The analyte containing the target nucleic acid is mixed with the above premixed reaction solution to obtain a mixed system, and then the mixed system is dispersed into a large number of uniformly sized microreaction units. S3. Start UV light irradiation and provide suitable temperature conditions to incubate the micro-reaction unit; The photo-induced cleavage group breaks after photoexcitation, thereby separating the blocked sequence from the Cas effector protein. The enzyme activity of the Cas effector protein is de-silenced, and the crRNA or sgRNA in all microreaction units starts recognizing the target nucleic acid sequence at the same time point and triggers the Cas effector protein to initiate a synergistic effect to continuously cleave the fluorescent reporter probe. S4. After the incubation reaction is completed, read the fluorescence signal in the microreaction unit, set the intensity threshold for judging negative / positive signals, and calculate the content of target nucleic acid in the sample to be tested.

2. The light-controlled single-molecule CRISPR-Cas detection method according to claim 1, characterized in that: The photo-splitting group is PC-Linker.

3. The light-controlled single-molecule CRISPR-Cas detection method according to claim 1, characterized in that: The molar ratio of the blocked sequence to the Cas effector protein in the CRISPR reaction system is 0.01 to 100:

1.

4. The light-controlled single-molecule CRISPR-Cas detection method according to claim 1, characterized in that: In step S2, the method of dispersing the mixed system into a large number of uniformly sized microreaction units includes microchamber array and microdroplet emulsification. Step S4 includes counting the total number of positive microreaction units or the proportion of positive microreaction units to the total number of statistical microreaction units, counting the number of target nucleic acid molecules in the sample to be tested, or calculating the original concentration of target nucleic acid molecules in the sample to be tested using the Poisson distribution principle.

5. The light-controlled single-molecule CRISPR-Cas detection method according to claim 1, characterized in that: The final concentration of Cas effector protein in the mixed system was 10-200 nM, the final concentration of crRNA / sgRNA in the mixed system was 20-400 nM, the final concentration of fluorescent reporter probe in the mixed system was 200-2000 nM, and the ratio of the final concentration of the blocking sequence to the final concentration of Cas effector protein in the mixed system was 0.01-100:

1. In step S3, the duration of UV irradiation is 5s to 30 min, the wavelength of the UV light used is 300 to 400 nm, and the power is 10 to 300 W.

6. A light-controlled single-molecule CRISPR-Cas detection kit, characterized in that: Includes the premixed reaction solution according to any one of claims 1-5.

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