Method and application of a Cas12a sensing system based on sgRNA blocking strategy for single nucleotide polymorphism typing

Abstract

The application discloses a method and application of a Cas12a sensing system based on an sgRNA blocking strategy for single nucleotide polymorphism typing. The method comprises the following steps: pre-annealing sgRNA and blocking chains to form a complex; obtaining a single-stranded DNA target by using an asymmetric recombinase polymerase amplification; constructing a detection system comprising a Cas12a protein, the complex, a target and a fluorescent substrate; activating the trans-cleavage activity of Cas12a through a strand displacement reaction to generate a fluorescent signal, so as to realize SNP typing. The method combines the sgRNA blocking strategy with the Cas12a system for the first time, does not need to depend on a PAM sequence, has the advantages of high universality, high specificity, simple operation, rapidness, low cost and the like, and is suitable for ApoE gene typing and other SNP related detection.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a method and application of a Cas12a sensing system based on an sgRNA blocking strategy for single nucleotide polymorphism typing. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. As the most common form of heritable variation in humans, SNPs are crucial in areas such as susceptibility analysis for hereditary diseases, personalized medicine guidance, pathogen typing, and molecular breeding. Taking the apolipoprotein E (ApoE) gene located on human chromosome 19 as an example, SNPs such as rs429358 and rs7412 on its exons directly determine the composition of the ApoE ε2 / ε3 / ε4 alleles. The ε4 allele is the strongest genetic risk factor for Alzheimer's disease, while the ε2 allele is closely associated with type III hyperlipoproteinemia. Therefore, developing a rapid, accurate, and convenient SNP typing technique is essential for disease risk assessment and precision medicine practices.

[0003] Traditional SNP detection methods, such as Sanger sequencing, allele-specific PCR (AS-PCR), and high-throughput sequencing, while highly accurate, generally suffer from problems such as reliance on sophisticated instruments, cumbersome operation, long processing time, or high cost, making it difficult to meet the application needs of point-of-care testing (POCT) or resource-limited scenarios.

[0004] In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated proteins (CRISPR-Cas) system, especially Cas12a, has become the core of next-generation molecular diagnostic tools due to its highly specific target recognition ability and efficient trans-cleavage activity guided by guide RNA (sgRNA). It integrates recognition and signal amplification, bringing hope for the development of low-cost, highly sensitive SNP detection platforms.

[0005] However, existing SNP detection strategies based on CRISPR-Cas12a still face a series of technical bottlenecks in the pursuit of high specificity and universality: (1) PAM sequence dependence and site restriction: The natural activation of Cas12a is strictly dependent on the prototype spacer adjacent motif (PAM). This makes it impossible to effectively detect a large number of SNP sites not located near the PAM, which greatly limits the universality of the method. Although some studies have tried to circumvent the PAM restriction by introducing additional probes or chain substitution reactions, this often brings new complexity. (2) Trade-off between specificity and universality: In order to improve the ability to distinguish single base mismatches, existing technologies often adopt complex molecular designs. For example, Chinese patent application CN120272574A discloses an electrochemical sensor based on a dumbbell-shaped LSL probe, which requires careful design of a self-folding probe structure and detection through an electrochemical platform. The operation is complex and not easy to popularize. Chinese patent application CN113774165A improves the specificity of HBV genotyping by chemically modifying sgRNA with methoxy groups, but chemical modification increases cost and design complexity, and the universality of the strategy has not been verified. Chinese patent application CN117965698A proposes to use RPA-UDG (uracil-DNA-glycosylation enzyme) to generate specific double-stranded DNA targets and relies on an additional "regulatory strand" to trigger a toehold-mediated DNA strand displacement reaction (TSDR) to activate Cas12a. Although this method gets rid of the PAM limitation, it introduces multiple steps such as UDG enzyme digestion and regulatory strand pre-assembly, making the system more complex, and the multiple steps may affect the robustness and reproducibility of the reaction. (3) Challenges of operation simplification and cost control: The ideal POCT technology should pursue the fewest operation steps, the simplest reagent composition, and the fastest reaction time. Most of the strategies mentioned above, while introducing additional elements (such as special probes, modified RNA, additional enzymes or regulatory chains) to achieve the function, also increase the complexity of the system, detection costs and operational difficulties, which is not conducive to the promotion and commercialization of the technology.

[0006] Therefore, there is an urgent need in this field for a novel CRISPR-Cas12a detection strategy that can overcome the limitations of PAM, achieve highly versatile SNP detection, maintain system simplicity and ease of operation, and not rely on complex chemical modifications or additional regulatory elements. This is the key to driving this technology from the laboratory to widespread application. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method and application for single nucleotide polymorphism (SNP) typing using a Cas12a sensing system based on an sgRNA blocking strategy. It is the first invention to combine the sgRNA blocking strategy with the Cas12a system, achieving highly sensitive, highly specific, and convenient detection of SNPs.

[0008] This invention is achieved through the following technical solution:

[0009] A method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy includes the following steps:

[0010] Step 1) Pre-anneal the sgRNA with the blocking strand to form an sgRNA-blocking strand complex, wherein the blocking strand is partially complementary to the 3' end region of the sgRNA;

[0011] Step 2) Asymmetric recombinase polymerase amplification system with forward primer concentration of 5~20 nM and reverse primer concentration of 200~600 nM is used to amplify the target nucleic acid to obtain single-stranded DNA target;

[0012] Step 3) Mix the sgRNA-closed strand complex obtained in Step 1) with Cas12a protein, the single-stranded DNA target obtained in Step 2) and the single-stranded DNA fluorescent substrate to construct a Cas12a detection system. React at 37~42℃ for 30~90 min. Activate the trans-cleavage activity of Cas12a through strand displacement, and cleave the fluorescent substrate to generate a fluorescent signal.

[0013] Step 4) Detect the fluorescence signal intensity and perform SNP typing on the target nucleic acid.

[0014] Preferably, the sgRNA in step 1) is sgRNA-WT or sgRNA-Mutant, the nucleotide sequence of sgRNA-WT is shown in SEQ ID NO.1, the nucleotide sequence of sgRNA-Mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the closed strand is shown in SEQ ID NO.3.

[0015] Preferably, the target nucleic acid in step 2) is the rs429358 site of the ApoE gene, and its wild-type nucleotide sequence is shown in SEQ ID NO.4-5, and its mutant nucleotide sequence is shown in SEQ ID NO.6-7; the nucleotide sequence of the forward primer is shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.9.

[0016] Preferably, the Cas12a detection system in step 3) comprises: an sgRNA-blocking strand complex at a concentration of 30-50 nM, a Cas12a protein at a concentration of 15-25 nM, a single-stranded DNA target to be detected, and a single-stranded DNA fluorescent substrate at a concentration of 0.5-2 µM; the nucleotide sequence of the single-stranded DNA fluorescent substrate is 6-FAM-AATAA-BHQ1.

[0017] The above methods are applied to ApoE genotyping for non-diagnostic and non-therapeutic purposes.

[0018] An sgRNA-closed strand complex for SNP genotyping, the complex being formed by annealing sgRNA and a partially complementary closed strand thereto, the closed strand binding to the 3' end region of the sgRNA, for introducing a kinetic barrier in Cas12a detection to enable strand substitution-based SNP recognition.

[0019] Preferably, the sgRNA is sgRNA-WT or sgRNA-Mutant, the nucleotide sequence of sgRNA-WT is shown in SEQ ID NO.1, the nucleotide sequence of sgRNA-Mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the closed strand is shown in SEQ ID NO.3.

[0020] The above-mentioned sgRNA-closed strand complex is used in the construction of a Cas12a detection system for SNP genotyping.

[0021] A reagent kit for implementing the above method, characterized in that it comprises:

[0022] (a) sgRNA-closed strand complex;

[0023] (b) Cas12a protein;

[0024] (c) Asymmetric amplification primer pairs, wherein the concentration of the forward primer is 5~20 nM and the concentration of the reverse primer is 200~600 nM;

[0025] (d) Fluorescent substrates for single-stranded DNA;

[0026] (e) Reaction buffer.

[0027] The above-mentioned kit is used in the preparation of products for ApoE genotyping.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) This invention creatively uses the “sgRNA-closed strand” complex as the recognition core, and its detection mechanism does not depend on the PAM inherent in the Cas12a protein. This allows the invention to freely design sgRNAs for SNP sites at any location in the genome, greatly expanding the range of detectable sites and solving the site coverage limitations of existing Cas12a detection technologies due to PAM dependence, providing a universal platform for extensive genetic marker analysis and pathogen mutation detection.

[0030] (2) This invention employs a complex formed by a closed strand and sgRNA to construct a tunable "kinetic barrier" along the target binding pathway. A perfectly matched target can efficiently undergo strand substitution, rapidly activating Cas12a; while targets with single-base mismatches are significantly inhibited in their activation process because they cannot overcome this barrier. This discrimination mechanism based on differences in branch migration kinetics is highly sensitive to single-base mismatches, thus achieving highly specific SNP genotyping without the need for complex probe design or chemical modification, effectively distinguishing between wild-type, heterozygous, and homozygous mutants.

[0031] (3) The entire detection system of this invention integrates asymmetric recombinase polymerase amplification (As-RPA) and CRISPR-Cas12a detection. As-RPA amplification is performed under isothermal conditions, eliminating the need for sophisticated thermal cycling instruments; the subsequent Cas12a detection is also completed at a single temperature, simplifying the operation steps. This method reduces dependence on expensive equipment and professional operating environments, significantly simplifies the detection process, and shortens the detection time, making it particularly suitable for scenarios such as primary healthcare institutions, rapid on-site testing, and home health monitoring.

[0032] (4) The core detection system of this invention contains only Cas12a protein, sgRNA-closed strand complex, single-stranded DNA target and fluorescent reporter substrate, without the need to introduce additional regulatory proteins (such as UDG), complex hairpin probes or specially chemically modified nucleic acid components. The simple reagent composition reduces the preparation cost and the complexity of long-term storage, while also helping to ensure the reproducibility and stability of the detection system.

[0033] (5) The method of the present invention effectively amplifies low-abundance targets through As-RPA, and combines this with the amplification effect of the trans-cleavage signal of Cas12a to achieve extremely high detection sensitivity. Experiments show that it can stably detect target nucleic acids down to a single copy and can produce good linear response across a concentration range of several orders of magnitude, meeting the requirements for accurate quantitative analysis of samples of different concentrations. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the principle of the method of the present invention in Example 1 (using a human oral swab sample as an example).

[0035] Figure 2 This is a schematic diagram of the Cas12a activation kinetics mediated by sgRNA blockade in Example 1 (comparing perfectly matched targets with single-base mismatch targets).

[0036] Figure 3 This is a graph showing the comparison results of discriminant factors in Example 2 when the closed strand is located at the 3' end, 5' end, internal region, or is absent from the sgRNA.

[0037] Figure 4 This is a graph showing the results of detecting mutant target sequences covering 20 mutation sites to evaluate the system's universality in Example 3, with and without a 5' end closure strand.

[0038] Figure 5 This is a fluorescence result diagram of the sgRNA-blocked Cas12a system constructed in Example 4, used to detect wild-type targets with different copy numbers.

[0039] Figure 6 This is a fluorescence result diagram of the sgRNA-blocked Cas12a system constructed in Example 5 when detecting targets with different copy number mutations;

[0040] Figure 7 This is a graph showing the sgRNA ratio analysis results at the rs429358 site of the ApoE gene in Example 6;

[0041] Figure 8 This is a fluorescence result image of the ApoE gene rs429358 site in human oral swab samples from Example 7;

[0042] Figure 9 This is a graph showing the sgRNA ratio analysis results of the ApoE gene rs429358 site in human oral swab samples from Example 7. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0044] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] All nucleotide sequences involved in the following examples are shown in Tables 1-1, 1-2, and 1-3.

[0047] Table 1-1 Relevant nucleotide sequences

[0048]

[0049] Table 1-2 Related nucleotide sequences

[0050]

[0051] Table 1-3 Related nucleotide sequences

[0052]

[0053] Example 1

[0054] A method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy, such as... Figure 1 As shown, single-stranded DNA (ssDNA) was obtained through asymmetric recombinase polymerase amplification (As-RPA) to adapt to the sgRNA blocking-Cas12a detection system; the mutation site was set at position 19 of the protospacer to obtain the maximum discriminant factor. Subsequently, wild-type (WT) and mutant (Mutant) targets synthesized using As-RPA were amplified and Cas12a detected to achieve highly specific SNP genotyping. The proposed method was used for genotyping and analysis of the rs429358 locus in the human ApoE gene.

[0055] This invention proposes a novel detection strategy—"sgRNA occlusion," such as... Figure 2 As shown. Unlike existing methods that identify mutations by blocking ssDNA targets, this invention achieves mutation discrimination by blocking sgRNA, thus possessing universality independent of the target sequence and avoiding complex sample processing steps before detection. The blocking strand introduces a kinetic barrier on the sgRNA, making it easier for perfectly matched targets to undergo strand substitution and activate Cas12a, while mismatched targets are inhibited due to difficulty in crossing this barrier. This achieves high-specificity SNP discrimination based on differences in branch migration kinetics. Subsequent experimental results show that this sgRNA-blocked Cas12a system is a position-independent and mutation-type-independent universal mutation detection platform.

[0056] The specific steps of this method are as follows:

[0057] (1) sgRNA blocking strategy

[0058] In this design, the sgRNA and the blocking strand are pre-annealed to form a complex before Cas12a detection, which then hybridizes with the ssDNA target. The target binds to the toehold region of the sgRNA-blocking strand complex, forming a triple-stranded flap structure, which then completely hybridizes with the sgRNA and replaces the blocking strand through a branching migration process. The blocking strand is partially complementary to the 3' end region of the sgRNA.

[0059] (2) As-RPA

[0060] In the amplification system, the concentration of the forward primer of the target nucleic acid is reduced to 10 nM, and the target DNA sequence is amplified by combining the 400 nM reverse primer of the target nucleic acid to obtain the ssDNA target. The ssDNA target can be adapted to the sgRNA blocking-Cas12a detection system.

[0061] The amplification system includes: target DNA sequence, recombinase polymerase dry powder, forward primer, and reverse primer.

[0062] (3) Cas12a detection

[0063] A Cas12a detection system was constructed and reacted at 39°C for 1 h. The Cas12a detection system comprised: a 40 nM sgRNA-blocking strand complex, a 20 nM Cas12a protein, the aforementioned ssDNA target, and a 1 µM ssDNA fluorescent substrate. In this Cas12a detection system, the ssDNA target completely hybridized with the sgRNA, thereby activating the trans-cleavage activity of Cas12a, degrading the ssDNA fluorescent substrate, and generating a fluorescence signal. Specifically, the reacted solution was added to a cuvette, and the fluorescence signal was collected using a fluorescence spectrophotometer.

[0064] (4) The fluorescence signal is measured to achieve SNP typing of the target nucleic acid.

[0065] Example 2: Comparison of the effectiveness of sgRNA blocking strategies at different blocking sites

[0066] To verify the feasibility of the sgRNA blocking strategy and determine the optimal blocking site, sgRNA-action (SEQ ID NO. 10) targeting the model sequence and three blocking strands with different binding sites were designed and synthesized: CD44-5' at the 5' end of the sgRNA (SEQ ID NO. 12), CD44-3' at the 3' end (SEQ ID NO. 13), and CD44-internal in the internal region (SEQ ID NO. 14). An additional group of sgRNA without any blocking strand was included as a control (non-blocking control group).

[0067] 1. Experimental Procedure

[0068] 4 µM sgRNA-action was annealed with 4 µM of each blocking strand in 1×TAMg buffer (45 mM Tris, 7.6 mM MgCl2, pH 8.0) from 95 °C to 4 °C to form sgRNA-blocking strand complexes.

[0069] A Cas12a detection system was constructed in 1×NEBuffer 2.1, comprising 40 nM of one of the above-mentioned sgRNA-closed strand complexes, 20 nM LbaCas12a protein, 1 µM ssDNA fluorescent substrate (i.e., Cas12a-substrate in Tables 1-3, hereinafter the same), and 10 nM of a fully matched ssDNA target Action-target (SEQ ID NO.11). Simultaneously, single-base mismatched ssDNA targets (A1-20, SEQ ID NO.15-34) covering 20 different positions in the sgRNA complementary region were set up to evaluate the discriminative performance of the sgRNA-closed strand complex. All reactions were performed at 39 °C, and fluorescence signals (excitation / emission: 485 / 519 nm, hereinafter the same) were monitored in real time using a fluorescence spectrophotometer for 1 h.

[0070] The ratio of fluorescence signal of perfectly matched targets to that of each mismatched target at the reaction endpoint (1 h) (F) match / F mismatch It is evaluated as a discriminant factor (DF).

[0071] 2. Experimental Results and Analysis

[0072] The results are as follows Figure 3 As shown, the baseline discriminant factor in the control group without occluder was the lowest, indicating that the conventional Cas12a / sgRNA system has limited ability to resolve single-base mismatches. Introducing a blocking strand at the 5' end (CD44-5') or the internal region (CD44-internal) improved the average discriminant factor, but only to a limited extent. When the blocking strand was located at the 3' end of the sgRNA (CD44-3'), the average discriminant factor showed a highly significant increase, far exceeding the other two blocking configurations and the control group without occluder. This indicates that the 3' end blocking strategy most effectively introduces a kinetic barrier in the target hybridization pathway, thereby maximally suppressing the activation of Cas12a by single-base mismatch targets and achieving optimal single-base resolution performance.

[0073] The experimental results of this embodiment demonstrate that the sgRNA blocking strategy effectively improves the SNP resolution capability of the Cas12a system, with the most significant effect observed when the blocking strand is located at the 3' end of the sgRNA. Therefore, this optimized 3' end blocking configuration was adopted in all subsequent embodiments.

[0074] Example 3: Verification of the universality of the sgRNA blocking strategy

[0075] To demonstrate that the sgRNA blocking strategy is a universal method, it was applied to another CRISPR-Cas12a system designed for the ApoE gene rs429358 site. Wild-type sgRNA-WT (SEQ ID NO.1) and the corresponding 3' blocking strand Block-13 (SEQ ID NO.3) targeting this site were designed and synthesized.

[0076] The sgRNA-WT / Block-13 complex was prepared according to the method in Example 2. The Cas12a detection system was the same as in Example 1, and the targets were 20 single-base mutant ssDNA targets (M1-20, 10 nM, SEQ ID NO. 35-54) designed for this sgRNA at different positions.

[0077] The results are as follows Figure 4 As shown, in the presence of the 3' end occluder Block-13, the activation of all mutant targets by wild-type sgRNA-WT was effectively inhibited, while the perfectly matched wild-type target produced a strong fluorescent signal, further demonstrating the universality and stability of this occlusion strategy in different sgRNA-target systems.

[0078] Example 4: Sensitivity of wild-type target detection based on the As-RPA and sgRNA-blocked Cas12a system

[0079] Using the wild-type (T / T) target at the rs429358 locus of the ApoE gene as a model, the performance of the detection channel designed for the wild-type allele was validated.

[0080] 1. Experimental Procedure

[0081] (1) Asymmetric RPA (As-RPA) amplification

[0082] Forward primer WT-FP (SEQ ID NO. 8) and reverse primer WT-RP (SEQ ID NO. 9) were designed and synthesized targeting the rs429358 site. A 50 µL amplification system was constructed in RPA dry powder tubes containing: 29.4 µL resuspension buffer, 0.5 µL forward primer (10 nM), 2 µL reverse primer (400 nM), 2 µL wild-type (WT, SEQ ID NO. 4-5) template containing different copy numbers (1, 5, 10, 100, 1000 copies), 13.6 µL ddH2O, and 2.5 µL MgOAc (280 mM). The mixture was incubated at 39 °C for 20 min. Using wild-type double-stranded DNA (SEQ ID NO. 4-5) as a template, ssDNA products mainly derived from SEQ ID NO. 4 were obtained after As-RPA amplification and used as the target.

[0083] (2) Purification of amplification products

[0084] A DNA extraction buffer was prepared by mixing phenol, chloroform, and isoamyl alcohol in a volume ratio of 25:24:1. The amplification product was then mixed with the DNA extraction buffer in a 1:1 ratio. The mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected to obtain the ssDNA target product.

[0085] (3) Cas12a detection

[0086] A complex was prepared using sgRNA-WT (SEQ ID NO.1) designed for the wild-type sequence and the closed-chain Block-13 (SEQ ID NO.3) according to the method in Example 2. A 20 µL detection system was constructed in 1×NEBuffer 2.1, containing 40 nM sgRNA-WT / Block-13 complex, 20 nM LbaCas12a protein, 1 µM ssDNA fluorescent substrate, and 5 µL of the above ssDNA target product. After reacting at 39 °C for 1 h, the mixture was transferred to a cuvette and detected using a fluorescence spectrophotometer, and the fluorescence signal was recorded.

[0087] 2. Experimental Results and Analysis

[0088] The results are as follows Figure 5 As shown, when using sgRNA-WT to detect wild-type templates, the fluorescence signal intensity showed a good gradient response with the initial copy number of the template in the range of 1 to 1000 copies, demonstrating that the detection channel has high sensitivity to the target allele.

[0089] Example 5: Sensitivity of detecting mutant targets based on the As-RPA and sgRNA-blocked Cas12a system.

[0090] Using the ApoE gene rs429358 site mutant (C / C) target as a model, we validated the performance of an independent detection channel designed for mutant alleles.

[0091] 1. Experimental Procedure

[0092] (1) As-RPA amplification

[0093] The steps are the same as in Example 4, except that the amplification template is replaced with a mutant template (SEQ ID NO. 6-7) containing the same gradient copy numbers (1, 5, 10, 100, 1000 copies). Specifically, using the mutant double-stranded DNA (SEQ ID NO. 6-7) as a template, after As-RPA amplification, the ssDNA product mainly derived from SEQ ID NO. 6 is obtained as the target.

[0094] (2) Purification of amplification products

[0095] The steps are the same as in Example 4.

[0096] (3) Cas12a detection

[0097] A complex was prepared using sgRNA-Mutant (SEQ ID NO. 2) designed for the mutant sequence and the closed-chain Block-13 (SEQ ID NO. 3) according to the method in Example 2. The detection system was constructed in the same way as in Example 4, except that the sgRNA-WT / Block-13 complex was replaced with the sgRNA-Mutant / Block-13 complex.

[0098] 2. Experimental Results and Analysis

[0099] The results are as follows Figure 6 As shown, when using sgRNA-Mutant to detect mutant templates, the fluorescence signal intensity also showed a good gradient response with the initial copy number of the template in the range of 1 to 1000 copies, demonstrating that the detection channel for mutant alleles also has high sensitivity comparable to the wild-type channel.

[0100] Example 6: SNP genotyping and ratio analysis at the rs429358 locus of the ApoE gene

[0101] Using simulated clinical samples, the three genotypes (wild-type homozygous T / T, heterozygous T / C, and mutant homozygous C / C) at the rs429358 locus of the ApoE gene were classified.

[0102] 1. Experimental Procedure

[0103] (1) Sample simulation

[0104] Wild-type homozygote (T / T): 20 nM wild-type ssDNA target (WT, SEQ ID NO.4-5).

[0105] Mutant homozygote (C / C): 20 nM mutant ssDNA target (Mutant, SEQ ID NO.6-7).

[0106] Heterozygotes (T / C): equal amounts of 10 nM wild-type targets and 10 nM mutant targets.

[0107] (2) Dual-channel detection

[0108] Cas12a detection was performed in parallel using the sgRNA-WT / Block-13 complex designed for the wild-type sequence in Example 4 and the sgRNA-Mutant / Block-13 complex designed for the mutant sequence in Example 5, respectively. The detection system was constructed in the same way as in Example 4.

[0109] (3) Data analysis and classification

[0110] An sgRNA ratio model was constructed for the rs429358 site of ApoE by calculating fluorescence intensity. The calculation formula is as follows:

[0111]

[0112] Where Mi and Ni refer to the fluorescence intensity of the repeat number i of allele M or allele N detected by sgRNA, respectively. Each sgRNA replicates 3 times, therefore a and b equal 3. Each SNP site has two sgRNAs, so theoretically, the sgRNA ratio for homozygous wild-type (T / T) is 2, the sgRNA ratio for homozygous mutant (C / C) is 0, and in the ideal heterozygous (T / C) case, the sgRNA ratio should be 1. The synthetic DNA standards and their corresponding sgRNAs at the rs429358 site (including wild-type and mutant) were analyzed by calculating the fluorescence intensity.

[0113] 2. Experimental Results and Analysis

[0114] The results are as follows Figure 7 As shown, the measured sgRNA ratios of the three simulated samples are in high agreement with the theoretical values, clearly distinguishing the three genotypes. This indicates that the Cas12a detection system based on the sgRNA blocking strategy established in this invention, combined with ratio analysis, can achieve accurate and reliable SNP genotyping.

[0115] Example 7: Application of ApoE genotyping in human oral swab samples

[0116] This embodiment aims to verify the feasibility and accuracy of the Cas12a sensing system based on the sgRNA blocking strategy established in this invention for SNP genotyping in actual human biological samples. Using non-invasively obtained oral swab samples as the object, genotyping was performed on the ApoE gene rs429358 locus.

[0117] 1. Sample collection and genomic DNA extraction

[0118] Six volunteers were recruited, and epithelial cell samples were obtained from the buccal mucosa inside each volunteer's mouth using sterile oral swabs. A commercially available oral swab genomic DNA extraction kit was used, and the procedure was strictly followed according to the instructions. The simplified procedure included: placing the swab tip in lysis buffer to digest cells and denature proteins, followed by DNA binding via centrifugation, protein removal washing, and final elution to obtain a purified human genomic DNA solution. The concentration and purity were determined using a micro-spectrophotometer.

[0119] 2. Experimental Procedure

[0120] (1) As-RPA amplification

[0121] Using the extracted genomic DNA as a template, the amplification steps were exactly the same as in Example 4, that is, using the primer pair shown in SEQ ID NO.8 and SEQ ID NO.9, RPA amplification was performed under asymmetric conditions of 10 nM forward primer and 400 nM reverse primer, followed by purification to obtain ssDNA target product.

[0122] (2) Cas12a dual-channel detection

[0123] The purified product was divided into two portions, and each portion was analyzed in parallel.

[0124] ① Wild-type channel: The detection system was constructed in the same way as in Example 4, using an sgRNA-WT (SEQ ID NO.1) / Block-13 (SEQ ID NO.3) complex designed for wild-type sequences.

[0125] ② Mutant channel: The detection system was constructed in the same way as in Example 5 using an sgRNA-Mutant (SEQ ID NO.2) / Block-13 (SEQ ID NO.3) complex designed for mutant sequences.

[0126] The fluorescence intensity at the reaction endpoint (39℃, 1 h) of the two channels was recorded separately.

[0127] (3) Data analysis and classification

[0128] The calculation method is exactly the same as in Example 6. For each volunteer's sample, the sgRNA ratio is calculated based on the fluorescence intensity values ​​obtained in the two detection channels, and the genotype of the rs429358 locus is determined according to the preset ratio threshold range.

[0129] 2. Experimental Results and Analysis

[0130] (1) Fluorescence signal results

[0131] The samples from all six volunteers produced clear and distinguishable fluorescent signals in both testing channels, such as... Figure 8 As shown, the signal strength patterns differ among different samples, suggesting that they may have different genotypes.

[0132] (2) Genotyping results

[0133] By calculating the sgRNA ratio, clear genotyping results were successfully obtained for 6 samples, such as... Figure 9 As shown in the figure, the data points cluster in two different intervals based on their ratios.

[0134] The experimental results of this embodiment demonstrate that the detection method provided by this invention can be directly applied to real human oral swab samples, achieving rapid, accurate, and instrument-free SNP genotyping of the ApoE gene rs429358 locus. These results fully demonstrate the effectiveness and reliability of this invention in practical applications, providing a feasible technical solution for point-of-care testing (POCT) or large-scale population screening.

[0135] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy, characterized in that, Includes the following steps: Step 1) Pre-anneal the sgRNA with the blocking strand to form an sgRNA-blocking strand complex, wherein the blocking strand is partially complementary to the 3' end region of the sgRNA; Step 2) Asymmetric recombinase polymerase amplification system with forward primer concentration of 5~20 nM and reverse primer concentration of 200~600 nM is used to amplify the target nucleic acid to obtain single-stranded DNA target; Step 3) Mix the sgRNA-closed strand complex obtained in Step 1) with Cas12a protein, the single-stranded DNA target obtained in Step 2) and the single-stranded DNA fluorescent substrate to construct a Cas12a detection system. React at 37~42℃ for 30~90 min. Activate the trans-cleavage activity of Cas12a through strand displacement, and cleave the fluorescent substrate to generate a fluorescent signal. Step 4) Detect the fluorescence signal intensity and perform SNP typing on the target nucleic acid.

2. The method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy according to claim 1, characterized in that, Step 1) The sgRNA is sgRNA-WT or sgRNA-Mutant, the nucleotide sequence of sgRNA-WT is shown in SEQ ID NO.1, the nucleotide sequence of sgRNA-Mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the closed strand is shown in SEQ ID NO.

3.

3. The method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy according to claim 1, characterized in that, Step 2) The target nucleic acid is the rs429358 site of the ApoE gene, and its wild-type nucleotide sequence is shown in SEQ ID NO.4-5, and its mutant nucleotide sequence is shown in SEQ ID NO.6-7; the nucleotide sequence of the forward primer is shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

9.

4. The method for single nucleotide polymorphism typing using a Cas12a sensing system based on an sgRNA blocking strategy according to claim 1, characterized in that, Step 3) The Cas12a detection system includes: sgRNA-blocking strand complex at a concentration of 30-50 nM, Cas12a protein at a concentration of 15-25 nM, single-stranded DNA target to be detected, and single-stranded DNA fluorescent substrate at a concentration of 0.5-2 µM; the nucleotide sequence of the single-stranded DNA fluorescent substrate is 6-FAM-AATAA-BHQ1.

5. The application of the method as described in any one of claims 1-4 in ApoE genotyping for non-diagnostic and therapeutic purposes.

6. An sgRNA-closed strand complex for SNP genotyping, characterized in that, The complex is formed by annealing sgRNA and a partially complementary closed strand, which binds to the 3' end region of the sgRNA to introduce a kinetic barrier in Cas12a detection to enable strand substitution SNP recognition.

7. The sgRNA-closed strand complex for SNP genotyping according to claim 6, characterized in that, The sgRNA is sgRNA-WT or sgRNA-Mutant, the nucleotide sequence of sgRNA-WT is shown in SEQ ID NO.1, the nucleotide sequence of sgRNA-Mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the closed strand is shown in SEQ ID NO.

3.

8. The use of the sgRNA-closed strand complex as described in claim 6 or 7 in constructing a Cas12a detection system for SNP genotyping.

9. A kit for carrying out the method according to any one of claims 1-4, characterized in that, include: (a) sgRNA-closed strand complex; (b) Cas12a protein; (c) Asymmetric amplification primer pairs, wherein the concentration of the forward primer is 5~20 nM and the concentration of the reverse primer is 200~600 nM; (d) Fluorescent substrates for single-stranded DNA; (e) Reaction buffer.

10. The use of the kit as described in claim 9 in the preparation of a product for ApoE genotyping.

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