Method and application of single nucleotide polymorphism identification based on crRNA spacer splitting

Abstract

The invention discloses a method and application for single nucleotide polymorphism identification based on crRNA spacer splitting, by splitting the crRNA spacer complementary to the target base to obtain a recognition probe, when the recognition probe is fully complementary to the target sequence, Cas12a protein is activated to produce a significant fluorescent signal; when there is a base mismatched with the split spacer in the target sequence, the crRNA spacer of the split is difficult to assemble with the incomplete matching sequence, and the Cas12 system is not activated and does not produce a fluorescent signal. The method of the present invention enhances the high specificity recognition ability of CRISPR / Cas12a to single base mismatch, so that the detection of SNP is not limited by the position of the mismatched base, and still shows high resolution in low mutation abundance, for preparing a detection kit for single nucleotide mismatch, to meet the actual detection requirements for clinical samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of genome sequencing, and in particular relates to a method and application for single nucleotide polymorphism identification based on crRNA spacer splitting. Background Art

[0002] Advances in genome sequencing technologies have led to a growing interest in individual genetic variations, particularly single nucleotide polymorphisms (SNPs), which account for approximately 90% of genetic diversity. SNPs and insertion-deletion markers (InDels) are key genetic markers widely used in disease genetics, population genetics, pharmacogenomics, and biodiversity research. Notably, SNPs, such as the BRAF V600E mutation and EGFR L858R, have been implicated in cancer development and are widely used biomarkers for cancer diagnosis and treatment. Tumor cells gradually accumulate numerous mutations over time. Tumor progenitor cells rapidly proliferate and develop SNVs in multiple genes. Although only a few SNVs lead to tumor formation, as tumor clones expand, these SNVs are present in nearly every tumor cell. Therefore, detecting single nucleotide mismatches is crucial for understanding and addressing genetic predisposition to disease, particularly cancer.

[0003] In clinical practice, DNA hybridization probes, such as TaqMan probes, molecular beacons, toehold-exchange probes, and primers for Amplification Refractory Mutation System PCR (ARMS-PCR), are often used to identify disease-associated SNPs. The TaqMan SNP Genotyping Assay is a commonly used fluorescence-based method that exploits the properties of TaqMan probes, which selectively bind to different alleles of a target SNP site. During PCR amplification, when Taq DNA polymerase amplifies the target DNA sequence, the probes bind to it, and the SNP in the sample is determined by detecting the intensity of the fluorescent signal. Allele-specific PCR (AS-PCR) utilizes specific primers designed to bind only to one allele of the target SNP. The type of SNP in the sample can be determined by measuring the size of the amplified product or by other methods, such as gel electrophoresis or real-time fluorescence quantitative PCR. During the PCR reaction, only the target sequence that matches the primers is amplified, allowing the type of SNP in the sample to be determined. In the High-Resolution Melting Analysis (HRM) method, the target sequence amplified by PCR is heated through a specific temperature gradient, causing the DNA to unwind and form different melting curves. Different SNP types will result in different melting curve patterns, so the SNPs in the sample are determined by analyzing the shape and peak of the melting curve. Sanger sequencing, a classic DNA sequencing technology, uses sequence alignment software to convert fluorescent signals into DNA sequences. The SNPs in the sample are determined by comparing the sequencing results with the reference sequence. Compared with traditional PCR / genomic sequencing methods, molecular inversion probes (MIPs) are widely used as an alternative method for detecting SNPs. By designing two independent DNA chains to self-assemble into a dumbbell-shaped DNA probe, the dumbbell probe hybridizes with the target sequence in the presence of the target sequence, and then completes sequence-specific gap filling and ligation in a single step. Methods for detecting disease-associated SNPs using DNA hybridization probes and primers are usually designed to identify and label short 20-30 nucleotide sequences containing the target SNV, and show good performance in detecting high-abundance SNVs. However, the concentration pressure caused by the high abundance of WT may easily cover the subtle free energy differences (1.5 kcal / mol to 7 kcal / mol) introduced by single nucleotide mutations, and it is often difficult to detect SNVs with mutation abundances below 1%. In addition, exposed regions of the nucleic acid target or primer product may further reduce detection sensitivity and specificity through unwanted intra- or intermolecular hybridization.Low-throughput gel-based methods are limited by cumbersome operations and long analysis cycles, while high-throughput methods based on allele-specific primer extension, single-base extension, or target sequencing are extremely dependent on expensive instruments, complex primer design, and require precise temperature control to eliminate false-positive results. TapMan probes are specific to specific SNP sites, making some SNP detection difficult, especially in complex systems. The melting curves produced by HRM are complex and require experienced operators. In order to develop a low-cost, high-performance detection method that does not rely on instrumentation, the CRISPR / Cas system has become a useful tool in the detection field due to its inherent self-signal amplification and highly specific recognition capabilities, and has been widely used for SNP detection. Taking CRISPR / Cas12a detection of dsDNA targets as an example, when the mismatched base is located 5-10 bases proximal to the Protospacer Adjacent Motif (PAM), the incomplete match between crRNA and dsDNA causes Cas12a's cleavage ability to decrease, producing a low fluorescence signal, which is used to distinguish single-base mismatches from wild-type targets. Although the "seed region" proximal to the PAM site has strict base pairing requirements, there is a certain degree of tolerance for mismatches outside the seed region. How to achieve more accurate recognition has become a key development issue. The inherent performance of the CRISPR / Cas system is increasingly difficult to cope with different detection fields and clinical applications. To address the off-target effects in CRISPR / Cas12a and enhance the recognition of target DNA, one approach is to optimize the Cas protein through protein engineering to improve its specificity. Another approach is to modify crRNA through chemical changes and structural adjustments to improve the specificity of mismatched base recognition. Although chemical modification simplifies crRNA adjustment, it also increases the complexity of crRNA design for various sequences and poses a greater risk of biohazards. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method and application for single nucleotide polymorphism identification based on crRNA spacer splitting. We have developed a general method to enhance the high-specificity recognition ability of CRISPR / Cas12a for single base mismatches, making the detection of SNPs unrestricted by the position of the mismatched base and still showing high resolution even at low mutation abundance, thus meeting the actual detection requirements for clinical samples.

[0005] In order to solve the above technical problems, the present invention provides a method for single nucleotide polymorphism identification based on crRNA spacer splitting, wherein the crRNA spacer of the target is split to obtain a recognition probe, the recognition probe close to the 5' end is crRNA-Forward, and the recognition probe close to the 3' end is crRNA-Backward. When the crRNA-Forward and the crRNA-Backward are completely complementary to each other with a single-base mismatch sequence, the Cas12a protein is activated to generate a significant fluorescent signal; when there are bases mismatched with the spacer in the crRNA-Forward and the crRNA-Backward, the split crRNA spacer is difficult to assemble with the incomplete matching sequence, which hinders the activation of the Cas12a protein and does not generate a fluorescent signal.

[0006] The above method further comprises splitting the target crRNA spacer, specifically splitting the target crRNA spacer between the 6th base and the 14th base at the 5' end. These split positions cover the "seed region", "near the seed region" and "far from the seed region".

[0007] In the above method, further, the target is BRAF.

[0008] The above method further comprises splitting the crRNA spacer of BRAF from the 12th base of the 5' end to obtain a recognition probe, wherein the recognition probe close to the 5' end is 12F-BRAF, and the recognition probe close to the 3' end is 8H-BRAF. The DNA sequence of 12F-BRAF is shown in SEQ ID NO.1; the DNA sequence of 8H-BRAF is shown in SEQ ID NO.2.

[0009] In the above method, further, the target is EGFR.

[0010] The above method further splits the crRNA spacer region of the EGFR from the 6th base of the 5' end to obtain a recognition probe, the recognition probe close to the 5' end is 6F-EGFR, and the recognition probe close to the 3' end is 14H-EGFR. The DNA sequence of the 6F-EGFR is shown in SEQ ID NO.3; the DNA sequence of the 14H-EGFR is shown in SEQ ID NO.4.

[0011] Based on a general technical concept, the present invention provides an application of a recognition probe obtained by the method described above in preparing a detection kit for single nucleotide mismatch.

[0012] Based on a general technical concept, the present invention provides an application of a recognition probe obtained by the method described above in preparing a cancer detection kit.

[0013] Furthermore, the detection kit includes crRNA-Forward, crRNA-Backward and a signal reporter molecule. Furthermore, it also includes: DEPC-treated water, reaction buffer solution, 2μl 500nM LbCas12. The signal reporter molecule is fluorophore-quencher labeled DNA (Fluorophore-quencher labeled DNA (FQ)). The fluorophore-quencher labeled DNA sequence is: 5'-BHQ1-TCCCCCCT-3'-6-FAM).

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] (1) The present invention provides a single nucleotide polymorphism recognition probe based on crRNA spacer splitting. First, the 6th to 14th bases at the 5' end of the crRNA spacer are split for CRISPR / Cas12a to recognize the target. The splitting combination includes crRNA-Forward (6-14nt) and crRNA-Backward (6-14nt). After the split crRNA is paired with the target for recognition, the fluorescence value shows almost the same signal as when the complete crRNA is used for detection. Compared with the complete crRNA, the method of the present invention has three advantages. First, it eliminates the interference caused by the high abundance wild type and improves the superior specificity for low-frequency SNPs in various concentration ranges. Second, it has sequence universality and does not require complex chemical modification and protein editing of crRNA. Third, the high GC content of the sequence can be tolerated for high-specificity recognition of SNPs.

[0016] (2) The present invention provides an application of a single nucleotide polymorphism recognition probe in SNP detection. Based on the efficient trans-cleavage activity of the CRISPR / Cas12a system, we split multiple sites in the crRNA spacer. The results show that the split spacer does not affect the activation of Cas12a, and shows significant advantages over the intact crRNA spacer in the test of detecting SNPs. Based on the current experimental results, the advantages of the method of the present invention are as follows:

[0017] High Sensitivity: This method maintains the CRISPR / Cas12a system's high-speed flip-cleavage of ssDNA after target DNA recognition, demonstrating its inherent signal amplification capabilities in signal transmission. It can accurately detect even low levels of the target SNP.

[0018] High specificity: This method can effectively distinguish single-base differences between the target SNP and the wild-type sequence. Compared with the control group, this method is not restricted by the mismatch position and shows high specificity for SNPs that are difficult to distinguish at high GC content positions.

[0019] Fast response speed: This method has a simple operating procedure and a short experimental cycle, and can complete the detection and analysis of samples in a short time (within 1 hour).

[0020] Simple and easy-to-use workflow and high resistance to interference: This method does not require complex experimental conditions or specialized skills. It is also applicable to testing in serum samples, maintaining stable results even in the presence of high concentrations of wild-type gene sequences. This technology system produces remarkable results for SNP detection, with high sensitivity, good specificity, and strong resistance to interference, making it unparalleled.

[0021] (3) The present invention provides an application of a single nucleotide polymorphism recognition probe in SNP detection, using a split combination to test single base mismatches at different positions of the target, and using crRNA-Forwad-14nt and crRNA-Backward-6nt to test single base mismatches at the PAM-distal 17nt by mixing single base mutation sequences and wild-type sequences. The mutation abundance is tested from 0.05% to 50%, showing detection of mutation abundance as low as 0.05%. The results of detecting two mutant genes, EGFR L858R and BRAF V600E, in serum showed a good linear relationship of 100fM-100pM, R 2 =0.98. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0023] Figure 1 It is a schematic diagram of the crRNA spacer / Cas12a detection system for the splitting in Example 1 of the present invention.

[0024] Figure 2 Schematic diagram and heat map of the response of the crRNA spacer region after splitting to ssDNA and dsDNA in Experiment 1 of the present invention.

[0025] Figure 3 This is the real-time fluorescence curve of the crRNA spacer region in response to dsDNA and ssDNA after cleavage in Experiment 2 of the present invention.

[0026] Figure 4This is the real-time fluorescence curve of the crRNA spacer region in response to dsDNA and ssDNA after cleavage in Experiment 2 of the present invention.

[0027] Figure 5 This is the real-time fluorescence curve of the crRNA spacer region in response to dsDNA and ssDNA after cleavage in Experiment 2 of the present invention.

[0028] Figure 6 This is the real-time fluorescence curve of the crRNA spacer region in response to dsDNA and ssDNA after cleavage in Experiment 2 of the present invention.

[0029] Figure 7 This is the real-time fluorescence curve of the crRNA spacer region in response to dsDNA and ssDNA after cleavage in Experiment 2 of the present invention.

[0030] Figure 8 This is the electrophoresis analysis result of crRNA-Forward-14 and crRNA-Backward-6 in Experiment 3 of the present invention.

[0031] Figure 9 This is the test result of replacing the 3' end of the crRNA spacer region after cleavage with DNA in Experiment 4 of the present invention.

[0032] Figure 10 This is the result of investigating the specific recognition of single-base mismatches in dsDNA and ssDNA targets by intact crRNA and split crRNA spacer regions in Experiment 5 of the present invention.

[0033] Figure 11 This is the result of investigating the specific recognition of single-base mismatches in dsDNA and ssDNA targets by intact crRNA and split crRNA spacer regions in Experiment 5 of the present invention.

[0034] Figure 12 This is the result of investigating the specific recognition of single-base mismatches in dsDNA and ssDNA targets by intact crRNA and split crRNA spacer regions in Experiment 5 of the present invention.

[0035] Figure 13 This is a comparison of the specific recognition ability of intact crRNA and split crRNA spacer regions for single-base mismatches in dsDNA targets in Experiment 5 of the present invention.

[0036] Figure 14 This is the response of (14+6) / Cas12a to different concentrations of single-base mutant and wild-type sequences in Experiment 6 of the present invention.

[0037] Figure 15 Testing of (14+6) / Cas12a for different abundances of mutations in Experiment 7 of the present invention.

[0038] Figure 16 This is a simulated actual environment test of the split crRNA spacer / Cas12a detection system in Experiment 8 of the present invention on EGFRL858R and BRAF V600E in serum. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific preferred embodiments, but the scope of protection of the present invention is not limited thereby.

[0040] The materials, reagents, and instruments used in the following examples can all be purchased from commercial sources. The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0041] The probes involved in this method were purchased from Huzhou Hippo Biotechnology Co., Ltd. The specific probe sequences are shown in Table 1.

[0042] Table 1: Detailed list of probes and sequences used

[0043]

[0044]

[0045] The PAM sites in dsDNA are underlined and italicized in this sequence listing, and the mutation points are bolded. Target-1 and Target-2 are fully complementary single-stranded DNAs that were annealed to form a double-stranded DNA structure (dsDNA) for use in Experiments 1 to 4 of the present invention.

[0046] Example 1

[0047] A method for single nucleotide polymorphism identification based on crRNA spacer splitting, wherein the crRNA spacer that is complementary paired with the target base is split to obtain a recognition probe, the recognition probe close to the 5' end is crRNA-Forward, and the recognition probe close to the 3' end is crRNA-Backward. When crRNA-Forward and crRNA-Backward are completely complementary paired with the target sequence, the Cas12a protein is activated and a significant fluorescent signal is generated; when there are mismatched bases between the target sequence and crRNA-Forward and crRNA-Backward, the split crRNA spacer and the incompletely matched sequence are difficult to chain assemble, which hinders the activation of the Cas12a protein and does not generate a fluorescent signal.

[0048] The detection principle can be found in Figure 1:In order to meet the base sequence required by CRISPR / Cas12a in the split spacer, different positions of the crRNA spacer are split, specifically: from the 6th base to the 14th base of the 5' end of the spacer, the split is called crRNA-Forward near the 5' end, and crRNA-Backward near the 3' end, so it is combined to form split spacers of different lengths from the 5' end to the 3' end. Fluorophore-quencher labeled DNA (Fluorophore-quencher labeled DNA (FQ)) can be activated by the CRIPSR / Cas12a system to cut this as a signal reporter. The crRNA split spacer will only be successfully activated when it is fully complementary to the target sequence, generating a significant fluorescent signal. When the target has a base mismatch with the spacer, the split crRNA spacer and the incomplete matching sequence are difficult to chain assemble, hindering the activation of the Cas12a protein, and therefore cannot stimulate Cas12a to trans-cut the fluorescent signal reporter. Using this principle, mutant genes in samples can be accurately distinguished from a large number of wild-type sequences. In contrast, if intact but unsplit crRNA is used as a control, even though there is a sequence that does not completely match the crRNA, it cannot be distinguished, resulting in an erroneous activation output, making it impossible to distinguish between mutant genes and wild-type genes.

[0049] The present invention performs a split test on the spacer sequence of the complementary pairing of crRNA and target DNA bases, including a “seed region”, a close “seed region”, and a far-away “seed region” that has a great influence on the activity of the Cas12a protein. First, it is demonstrated that when the target is dsDNA and ssDNA, the split spacer successfully activates the CRISPR / Cas12a system, and then demonstrates the high specificity of the split spacer to SNP, which can achieve reliable detection of SNP at low allele frequencies. The test results show that compared with the traditional probe method for detecting SNPs that relies on mismatch positions, this method is not limited by mismatch points and can effectively distinguish single-base differences between target SNPs and wild-type sequences, and can maintain stable detection results even in the presence of high concentrations of wild-type gene sequences. In addition, compared with a class of CRISPR / Cas systems that perform complex protein editing and nucleic acid modification, this method has a fast reaction speed and a simple and easy operating procedure, making it an ideal choice for detecting samples such as circulating tumor DNA (ctDNA) directly from serum or plasma. Its outstanding performance provides a solid foundation for clinical diagnosis and personalized treatment, and provides important support for the further development of the field of molecular diagnosis.

[0050] In order to verify the feasibility of the method of this embodiment 1, a random sequence was designed: crRNA. The sequence of crRNA is: UAAUUUCUACUAAGUGUAGAUCUCAGGGCGGACUGGGUGCU.

[0051] The GC content of the spacer region in this sequence is 70%. Using this sequence as an example, a feasibility verification experiment for split crRNA was completed. When it came to actual samples, such as the mutant genes BRAF V600E and EGFR L858R, the split crRNA was designed based on the principle of base complementary pairing according to the target.

[0052] The crRNA was split from the sixth base of the 5' end of the spacer region to obtain: crRNA-Forward6 and crRNA-Backward14.

[0053] The crRNA was split at the 8th base from the 5' end of the spacer to obtain: crRNA-Forward8 and crRNA-Backward12.

[0054] The crRNA was split at the 10th base from the 5' end of the spacer to obtain: crRNA-Forward10 and crRNA-Backward10.

[0055] The crRNA was split at the 12th base from the 5' end of the spacer to obtain: crRNA-Forward12 and crRNA-Backward8.

[0056] The crRNA was split at the 14th base from the 5' end of the spacer to obtain: crRNA-Forward14 and crRNA-Backward6.

[0057] Experiment 1: Investigate the response of the crRNA spacer to dsDNA and ssDNA targets after cleavage at different positions.

[0058] The crRNA-Forward and crRNA-Backward were diluted in DEPC-treated water in an isocratic manner and mixed at a ratio of 1:1 to a final concentration of 500 nM. The crRNA was also diluted to a final concentration of 500 nM. The dsDNA was annealed and diluted to a concentration of 1 nM, and the ssDNA target was diluted to a concentration of 1 nM. The signal reporter (the signal reporter used in this experiment is a DNA fluorescent probe with a DNA sequence of 5'-BHQ1-TCCCCCCT-3'-6-FAM) was diluted to 2 μM, and the LbCas12a protein was diluted 10 times.

[0059] 10 μl DEPC-treated water, 2 μl 10x reaction buffer (reaction buffer: pH 9.0, 10 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl, 0.5% Tween-20, 1 mM DTT), 2 μl 500 nM (crRNA-Forward + crRNA-Backward), 2 μl 500 nM LbCas12a, 2 μl 2 μM signal reporter and 2 μl ssDNA or dsDNA target were mixed, and a total of 20 μl was detected in a Real Time PCR instrument at 37 ° C for 90 min, and fluorescence was measured every 60 seconds.

[0060] Results see Figure 2 :The 5' and 3' ends of the split spacer are combined for cleavage verification of the CRISPR / Cas12a system. We found that all the split patterns in the designed crRNA can achieve optimal activation of the Cas12a protein after meeting the 20nt recognition complementary pairing, even if the split point exists within the 5-10nt range close to the PAM site (i.e., the seed region of the crRNA). When the 5' and 3' ends of the split spacer meet the recognition of the 20nt target, the excess chain length exposed will inhibit the activity of Cas12a to a certain extent.

[0061] Experiment 2: Investigate the kinetic responses of the above spacer splitting combinations to dsDNA and ssDNA respectively.

[0062] Prepare the premixed solution: 10 μl DEPC-treated water, 2 μl 10x reaction buffer (reaction buffer: pH 9.0, 10 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl, 0.5% Tween-20, 1 mM DTT), 2 μl 500 nM LbCas12a, 2 μl 2 μM signal reporter, a total of 16 μl per tube.

[0063] Group 1: 2 μl of 500 nM crRNA, 2 μl of 500 nM crRNA-Forward-14 + crRNA-Backward-6 (shown as 14 + 6 in the figure), 2 μl of 500 nM 14F (14F represents the 14th base in the 5'-3' order of the spacer), and 2 μl of 500 nM 6H (6H represents the 6th base in the 3'-5' order of the spacer) were mixed with the above 16 μl premix solution, and finally 2 μl of 1 nM dsDNA / ssDNA target was added. The final volume was 20 μl and the detection was carried out in a Real Time PCR instrument at 37 ° C for 90 minutes, and the fluorescence was measured every 60 seconds. The results are shown in the table. Figure 3 .

[0064] Group 2: 2 μl of 500 nM crRNA, 2 μl of 500 nM crRNA-Forward-12 + crRNA-Backward-8 (shown as 12 + 8 in the figure), 2 μl of 500 nM 12F (12F represents the 12th base in the 5'-3' order of the spacer), and 2 μl of 500 nM 8H (8H represents the 8th base in the 3'-5' order of the spacer) were mixed with the above 16 μl premix solution, and finally 2 μl of 1 nM dsDNA / ssDNA target was added. The final volume was 20 μl and the detection was carried out in a Real Time PCR instrument at 37 ° C for 90 minutes, and the fluorescence was measured every 60 seconds. The results are shown in the table. Figure 4 .

[0065] Group 3: 2 μl of 500 nM crRNA, 2 μl of 500 nM crRNA-Forward-10 + crRNA-Backward-10 (shown as 10 + 10 in the figure), 2 μl of 500 nM 10F (10F represents the 10th base in the 5'-3' direction of the spacer), and 2 μl of 500 nM 10H (10H represents the 10th base in the 3'-5' direction of the spacer) were mixed with the above 16 μl premix solution, and finally 2 μl of 1 nM dsDNA / ssDNA target was added. The final volume was 20 μl and the detection was carried out in a Real Time PCR instrument at 37 ° C for 90 minutes, and the fluorescence was measured every 60 seconds. The results are shown in the table. Figure 5 .

[0066] Group 4: 2 μl of 500 nM crRNA, 2 μl of 500 nM crRNA-Forward-8 + crRNA-Backward-12 (shown as 8 + 12 in the figure), 2 μl of 500 nM 8F (8F represents the 8th base in the 5'-3' order of the spacer), and 2 μl of 500 nM 12H (12H represents the 12th base in the 3'-5' order of the spacer) were mixed with the above 16 μl premix solution, and finally 2 μl of 1 nM dsDNA / ssDNA target was added. The final volume was 20 μl and the detection was carried out in a Real Time PCR instrument at 37 ° C for 90 minutes, and the fluorescence was measured every 60 seconds. The results are shown in the table. Figure 6 .

[0067] Group 5: 2 μl of 500 nM crRNA, 2 μl of 500 nM crRNA-Forward-6 + crRNA-Backward-14 (shown as 6 + 14 in the figure), 2 μl of 500 nM 6F (8F represents the 6th base in the 5'-3' order of the spacer), and 2 μl of 500 nM 14H (14H represents the 14th base in the 3'-5' order of the spacer) were mixed with the above 16 μl premix solution, and finally 2 μl of 1 nM dsDNA / ssDNA target was added. The final volume was 20 μl and the detection was carried out in a Real Time PCR instrument at 37 ° C for 90 minutes, and the fluorescence was measured every 60 seconds. The results are shown in the table. Figure 7 .

[0068] from Figures 3 to 7 The results show that: a single cleavage 5' end or 3' end maintains a low activation state for Cas12a protease, that is, produces a lower fluorescence intensity; in addition, the above cleavage spacer combinations are used to identify ssDNA and dsDNA. The results show that the cleavage kinetic rate of the fluorescent probe by the cleavage spacer when the target is ssDNA is lower than that of dsDNA, which is consistent with the kinetic difference results of intact crRNA recognizing ssDNA and dsDNA reported in existing literature.

[0069] Experiment 3: Polyacrylamide (PAGE) gel electrophoresis analysis.

[0070] In the model where all split combinations satisfy the complementary pairing number of target bases of 20 nt, the 5' part of the split spacer is 14 nt (crRNA-Foarward-14) and the 3' end is divided into 6 nt (crRNA-Backward-6), showing a lower fluorescence kinetic rate. We selected this split combination for electrophoresis analysis.

[0071] The cleavage activity of the split spacer (crRNA-Forward-14 + crRNA-Backward-6) / Cas12a system was evaluated using a 20% PAGE assay. The gel was prepared from a solution containing 30% acrylamide, 10% ammonium persulfate, tetramethylenediamine, and 5xTAE buffer (TAE buffer: 200 mM Tris-Acetate, 5 mM EDTA, pH 8.0-8.6).

[0072] Results see Figure 8 : When the CRISPR / Cas12a cis-cleavage reaction was reacted at 37°C for 10 minutes, the cleavage bands of the split combination 14+6 pairs of dsDNA were brighter than the complete crRNA cleavage substrates in the control group, indicating that the former had more substrates remaining in the 10-minute cleavage reaction. After electrophoresis at 37°C for 30 minutes, the brightness of the two groups of substrate bands was almost the same, which was consistent with the fluorescence curve results.

[0073] Experiment 4: Replace crRNA-Backward with a DNA sequence to test the response of the CRISPR / Cas12a system.

[0074] Sequentially dilute DNA-6H, DNA-8H, DNA-10H, DNA-12H, and DNA-14H and mix them with crRNA-Forward-14, crRNA-Forward-12, crRNA-Forward-10, crRNA-Forward-8, and crRNA-Forward-6 at a 1:1 ratio until the final probe concentration is 500nM. Because this system can recognize both dsDNA and ssDNA targets, we considered the effect of replacing the 3' end of the cleavage spacer with DNA on the recognition of dsDNA and ssDNA targets. The specific experimental method is as follows:

[0075] 14F+DNA6H group: DNA-6H and crRNA-Forward-14 were mixed in a 1:1 ratio to a final concentration of 500 nM to obtain a crRNA-Forward+DNA mixture; 10 μl of DEPC-treated water, 2 μl of 10x reaction buffer, 2 μl of 500 nM crRNA-Forward+DNA mixture, 2 μl of 500 nM LbCas12a, 2 μl of 2 μM signal reporter, and 2 μl of 1 nM target DNA were mixed, and a total of 20 μl was mixed and tested in a Real Time PCR instrument at 37°C for 90 min, with fluorescence measured every 60 seconds.

[0076] 12F+DNA8H group: DNA-8H and crRNA-Forward-12 were mixed in a 1:1 ratio to a final concentration of 500 nM to obtain a crRNA-Forward+DNA mixture; 10 μl of DEPC-treated water, 2 μl of 10x reaction buffer, 2 μl of 500 nM crRNA-Forward+DNA mixture, 2 μl of 500 nM LbCas12a, 2 μl of 2 μM signal reporter, and 2 μl of 1 nM target DNA were mixed, and a total of 20 μl was mixed and tested in a Real Time PCR instrument at 37°C for 90 min, with fluorescence measured every 60 seconds.

[0077] 10F+DNA10H group: DNA-10H and crRNA-Forward-10 were mixed in a 1:1 ratio to a final concentration of 500 nM to obtain a crRNA-Forward+DNA mixture; 10 μl of DEPC-treated water, 2 μl of 10x reaction buffer, 2 μl of 500 nM crRNA-Forward+DNA mixture, 2 μl of 500 nM LbCas12a, 2 μl of 2 μM signal reporter, and 2 μl of 1 nM target DNA were mixed, and a total of 20 μl was mixed and tested in a Real Time PCR instrument at 37°C for 90 min, with fluorescence measured every 60 seconds.

[0078] 8F+DNA12H group: DNA-12H and crRNA-Forward-8 were mixed in a 1:1 ratio to a final concentration of 500 nM to obtain a crRNA-Forward+DNA mixture; 10 μl of DEPC-treated water, 2 μl of 10x reaction buffer, 2 μl of 500 nM crRNA-Forward+DNA mixture, 2 μl of 500 nM LbCas12a, 2 μl of 2 μM signal reporter, and 2 μl of 1 nM target DNA were mixed, and a total of 20 μl was mixed and tested in a Real Time PCR instrument at 37°C for 90 min, with fluorescence measured every 60 seconds.

[0079] 6F+DNA14H group: DNA-14H and crRNA-Forward-6 were mixed in a 1:1 ratio to a final concentration of 500 nM to obtain a crRNA-Forward+DNA mixture; 10 μl of DEPC-treated water, 2 μl of 10x reaction buffer, 2 μl of 500 nM crRNA-Forward+DNA mixture, 2 μl of 500 nM LbCas12a, 2 μl of 2 μM signal reporter, and 2 μl of 1 nM target DNA were mixed, and a total of 20 μl was mixed and tested in a Real Time PCR instrument at 37°C for 90 min, with fluorescence measured every 60 seconds.

[0080] Results see Figure 9 : When the RNA at the 3' end of the cleavage spacer is replaced by DNA and the recognition target is ssDNA, the activity of Cas12a is inhibited; when the 6nt RNA at the 3' end of the cleavage spacer is replaced by DNA and the recognition target is dsDNA, only part of the Cas12a protein is activated, and the substrate reporter probe is partially cut to produce a fluorescent signal. However, when the 3' end of the cleavage spacer is replaced by a DNA base greater than 6nt, the fluorescent signal is further weakened. Compared with the reported literature that the 3' end of the complete crRNA is replaced by DNA to improve the specificity of the CRISPR / Cas12a system, our test results not only show less tolerance in the number of base substitutions, but also the tolerable 3' end continuous 6nt replacement of the split crRNA with DNA has a greatly reduced cutting efficiency. We speculate that the split spacer may further enhance the specific recognition of Cas12a.

[0081] Experiment 5: The split spacer / Cas12a system recognizes single-base mismatches at different positions of the target (dsDNA, ssDNA).

[0082] The present application sets the single-base mismatch in the "seed region", the dsDNA and ssDNA adjacent to the "seed region" and the distal end of the "seed region" as targets. Among them, mutation-17-dsDNA represents the 17th position after the PAM site that is complementary paired with the spacer region (i.e., the mutation at the distal end of the seed region); the ssDNA target has no PAM site, and mutation-17-ssDNA represents the 17th position that is complementary paired with the spacer region. Mutation-11-dsDNA represents the 11th position after the PAM site that is complementary paired with the spacer region (i.e., the mutation adjacent to the seed region), and mutation-11-ssDNA represents the 11th position that is complementary paired with the spacer region. Mutation-5-dsDNA represents the 5th position after the PAM site that is complementary paired with the spacer region (i.e., the mutation in the seed region), and mutation-5-ssDNA represents the 5th position that is complementary paired with the spacer region.

[0083] The above dsDNA and ssDNA were diluted to a concentration of 1 nM respectively. Prepare the premix solution: 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM crRNA-Forward + crRNA-Backward, 2 μl 500 nM LbCas12, 2 μl 2 μM signal reporter and 2 μl 1 nM target DNA (dM-17-1 + dM-17-2, M-17, dM-11-1 + dM-11-2, M-11, dM-5-1 + dM-5-2, M5). Mix a total of 20 μl and test in a Real Time PCR instrument at 37 ° C for 90 min, measuring fluorescence every 60 seconds.

[0084] Figure 10 The response results of different splitting models and intact crRNA to mismatched targets when mutation-17-dsDNA and mutation-17-ssDNA are used as targets.

[0085] Figure 11 The response results of different splitting models and intact crRNA to mismatched targets when mutation-11-dsDNA and mutation-11-ssDNA are used as targets.

[0086] Figure 12 The response results of different splitting models and intact crRNA to mismatched targets when mutation-5-dsDNA and mutation-5-ssDNA are used as targets.

[0087] The fluorescence values ​​after 10 minutes and 45 minutes of reaction at 37℃ were compared. Figure 10 、 11The results of Figure 12 show that, regardless of whether single-base mismatches are recognized in dsDNA or ssDNA, at 45 minutes, the split point with a nearby single-base mismatch in the split model shows the lowest fluorescence measurement value. In contrast, the intact crRNA / Cas12a still shows high cleavage of the mutant sequence. Although studies have shown that Cas12a recognizes single-base mismatches in ssDNA poorly, in our system, appropriate split spacers can still be distinguished for accurate recognition of single-base mismatches at different positions.

[0088] Figure 13 For mutation-17-dsDNA, mutation-11-dsDNA, and mutation-5-dsDNA, the fluorescence value of the response of different splitting models and complete crRNA to mismatch targets was analyzed for 60 minutes. As can be seen from the figure: at the 60th minute, when the mismatch position is at the 5th position, crRNA-Forward-6 and crRNA-Backward-14 (6+14) significantly distinguish MT. For the 11th single base mismatch, crRNA-Forward-12 and crRNA-Backward-8 (12+8) show the lowest fluorescence value after recognizing the mutant sequence. crRNA-Forward-14+crRNA-Backward-6 (14+6) shows high resolution for the mismatch recognition at the 17th position, but there is no significant difference in the complete crRNA / Cas12a in distinguishing the wild type from the single base mismatch mutations at the above three different positions.

[0089] Experiment 6: Response of the (crRNA-Forward-14+crRNA-Backward-6) / Cas12a system to different concentrations of dsDNA targets.

[0090] Dilute the dsDNA target sequences (Target-1 and Target-2) to 10nM, 1nM, 100pM, and 1pM, respectively. Prepare a premix solution: 10μl DEPC-treated water, 2μl 10x reaction buffer, 2μl 500nM crRNA-Forward-14+crRNA-Backward-6, 2μl 500nM LbCas12a, and 2μl 2μM signal reporter; mix with 2μl 1nM target dsDNA, and test a total of 20μl in a Real Time PCR instrument at 37°C for 90 minutes, measuring fluorescence every 60 seconds.

[0091] Figure 14It is the response result of crRNA-Forward-14+crRNA-Backward-6 / Cas12a system to different concentrations of dsDNA target.Left figure: the response of the target of different concentrations of the crRNA spacer region of splitting to the complete complementary pairing of sequence, along with the rising of target (dsDNA) concentration, active Cas12a enzyme also increases accordingly, causing the fluorescence measurement value to increase proportionally. Investigating the significant mismatch base distinction specificity shown by this method, the spacer sequence of splitting is designed to be fully matched with the SNP sequence (MT), thus there is a single base mismatch with the wild type (WT), and further development is made for the detection application of SNP. In the right figure, the fluorescence response value of MT and WT for testing different concentrations when reacting for 30min is shown. In the right figure, MT is a single base mutation sequence, and WT is the wild-type sequence, i.e., there is a single base mismatch with the split spacer, because the wild-type has an incomplete complementary pairing with the split spacer, which hinders the activation of Cas12a protein, and the fluorescence value is relatively low. From the above, it can be seen that as the concentration of the target with a perfect base match increases, the active Cas12a enzyme also increases accordingly, resulting in a proportional increase in the fluorescence measurement value. At the corresponding concentration, we also tested a dsDNA target containing only one mismatched base at position 17 of the target. As the concentration of the single-base mismatched sequence (WT) increased, the fluorescence value hardly increased. The binary complex formed by the split crRNA spacer and the Cas12a protein effectively distinguished MT from WT.

[0092] Experiment 7: Testing of mutation abundance by the split spacer / Cas12a system.

[0093] Higher wild-type gene concentrations may introduce additional complexity because the abundant presence of wild-type genes may mask mutation signals, which places higher demands on mutation detection in such complex samples. To further verify the high selectivity of the split-spacer / Cas12a system for detecting low mutation abundance in the mixture, we fixed the mutant sequence concentration at 100pM and achieved fluorescence analysis of different mutation abundances (0% to 50%) by changing the wild-type sequence concentration (10nM to 20μM). 0% mutation abundance represents 20μM WT, while the MT concentration is 0. We have examined the performance of the split-spacer / Cas12a in single-base mismatch recognition. Given the low mutation abundance (0.01%-10%) in tumor samples and early-stage tumor biological tissue samples, we aim to evaluate the system's ability to detect low mutation abundance. Mutations of different abundances were configured as follows:

[0094] 0% MT (Mutation Type): 2μl DEPC-treated water+18μl 20μM WT (Wild Type).

[0095] 0.05% MT: 2 μl 100 pM MT + 18 μl 20 μM WT.

[0096] 0.1% MT: 2 μl 100 pM MT + 18 μl 10 μM WT.

[0097] 0.2% MT: 2 μl 100 pM MT + 18 μl 5 μM WT.

[0098] 0.4% MT: 2μl 100pM MT+18μl 2.5μM WT.

[0099] 0.5% MT: 2 μl 100 pM MT + 18 μl 2 μM WT.

[0100] 1% MT: 2μl 100pM MT+18μl 1μM WT.

[0101] 10% MT: 2μl 100pM MT+18μl 100nM WT.

[0102] 50% MT: 2μl 100pM MT+18μl 10nM WT.

[0103] MT (mutant type) indicates a single-base mutation sequence, and WT (wild type) indicates the wild-type sequence. 2 μl of each of the above mutant target solutions containing varying abundances was added to a premixed solution consisting of 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM crRNA-Forward-14 + crRNA-Backward-6, 2 μl 500 nM LbCas12a, and 2 μl 2 μM reporter. The total of 20 μl was assayed in a real-time PCR instrument at 37°C for 90 minutes, with fluorescence measured every 60 seconds.

[0104] Results see Figure 15 : Within the 10th and 20th minutes, the fluorescence response of crRNA-Forward-14+crRNA-Backward-6 / Cas12a to different MT mutation abundances at position 17 showed different results, and its detection limit reached 0.05%. As the mutation abundance increased, the fluorescence signal gradually increased.

[0105] Example 2

[0106] Use of the recognition probe obtained by the method of Example 1 in preparing a kit for identifying single nucleotide polymorphisms. The kit comprises: 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM 12F-BRAF + 8H-BRAF, 2 μl 500 nM LbCas12a, and 2 μl 2 μM signal reporter.

[0107] The sequence of 12F-BRAF is shown in SEQ ID NO. 1, specifically: UAAUUUCUACUAAGUGUAGAUGUCUAGCUACAG.

[0108] The sequence of 8H-BRAF is shown in SEQ ID NO. 2, specifically: AGAAAUCU.

[0109] Example 3

[0110] Use of the recognition probe obtained by the method of Example 1 in preparing a kit for identifying single nucleotide polymorphisms. The kit comprises: 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM 6F-EGFR + 14H-EGFR, 2 μl 500 nM LbCas12a, and 2 μl 2 μM signal reporter.

[0111] The sequence of 6F-EGFR is shown in SEQ ID NO. 3, specifically: UAAUUUCUACUAAGUGUAGAUGGCGGG.

[0112] The sequence of 14H-EGFR is shown in SEQ ID NO. 4, specifically: CCAAACUGCUGGGU.

[0113] Experiment 8: Anti-interference test in serum simulating actual environment.

[0114] In cancer patients, circulating DNA carries tumor-associated genetic and epigenetic changes associated with cancer development, progression, and drug resistance. We investigated BRAF V600E and EGFR L858R in serum, simulating a realistic environment. Split-12F-BRAF + 8H-BRAF / Cas12a and split-6F-EGFR + 14H-EGFR / Cas12a were used for anti-interference experiments, respectively. Targets (mutants) fully complementary to the split-spacer region were serially diluted, and the mutant targets were mixed with 200 nM WT and fetal bovine serum, each containing a single-base mismatch, to simulate a realistic environment.

[0115] BRAF V600E group: Anti-interference testing was performed in serum using BRAF V600E mutation targets (BRAF-1 and BRAF-2 in Table 1) to simulate the actual sample environment. Samples were processed as follows:

[0116] 10 nM: 10 μl 20 nM BRAF-1+BRAF-2, 5 μl 200 nM WT, 2 μl fetal bovine serum, 3 μl DEPC-treated water.

[0117] 1 nM: 10 μl 2 nM BRAF-1+BRAF-2, 5 μl 200 nM WT, 2 μl fetal bovine serum, 3 μl DEPC-treated water.

[0118] 500pM: 10μl 1nM BRAF-1+BRAF-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0119] 100pM: 10μl 200pM BRAF-1+BRAF-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0120] 10pM: 10μl 20pM BRAF-1+BRAF-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0121] 1pM: 10μl 2pM BRAF-1+BRAF-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0122] The target solution containing the mutant target in the simulated complex environment was used as the target, and 2 μl of the solution was added to a premixed solution consisting of 10 μl DEPC-treated water, 2 μl 10× reaction buffer, 2 μl 500 nM 12F-BRAF + 8H-BRAF, 2 μl 500 nM LbCas12a, and 2 μl 2 μM reporter. The total of 20 μl was assayed in a real-time PCR instrument at 37°C for 90 minutes, with fluorescence measured every 60 seconds.

[0123] EGFR L858R group: BRAF V600E mutation targets (EGFR-1 and EGFR-2 in Table 1) were used to conduct anti-interference testing in serum to simulate the actual sample environment. Samples were processed as follows:

[0124] 10nM: 10μl 20nM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0125] 1nM: 10μl 2nM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0126] 500pM: 10μl 1nM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0127] 100pM: 10μl 200pM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0128] 10pM: 10μl 20pM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0129] 1pM: 10μl 2pM EGFR-1+EGFR-2, 5μl 200nM WT, 2μl fetal bovine serum, 3μl DEPC-treated water.

[0130] 2 μl of the target solution containing the mutants in the simulated complex environment was added to a premixed solution consisting of 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM 6F-EGFR+14H-EGFR, 2 μl 500 nM LbCas12a, and 2 μl 2 μM reporter. The total of 20 μl was assayed in a real-time PCR instrument at 37°C for 90 minutes, with fluorescence measured every 60 seconds.

[0131] Results see Figure 16 :The assay showed good linearity within the target concentration range of 100 fM-100 pM, indicating that the assay has the potential to directly detect mutations in free DNA in serum or plasma without the need for DNA separation.

[0132] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for identifying single nucleotide polymorphisms based on crRNA spacer splitting, characterized in that The crRNA sequence is: 5'-UAAUUUCUACUAAGUGUAGAU-spacer-3'; the spacer is a sequence that is complementary to the target base, and the 5' end of the spacer is split between the 6th base and the 14th base to obtain a recognition probe, the recognition probe close to the 5' end is crRNA-Forward, and the recognition probe close to the 3' end is crRNA-Backward. The method comprises: (1) mixing the crRNA-Forward, the crRNA-Backward, the Cas12a protein and the signal reporter molecule; (2) Fluorescence detection is performed after adding the target; when the crRNA-Forward and the crRNA-Backward are completely complementary to the target sequence, the Cas12a protein is activated and a significant fluorescence signal is generated; when the crRNA-Forward and the crRNA-Backward have mismatched bases with the target sequence, the split crRNA spacer and the incompletely matched sequence are difficult to assemble, the Cas12a system will not be activated, and no fluorescence signal will be generated; The signal reporter molecule is: 5'-BHQ1-TCCCCCCT-3'-6-FAM; The methods are not for diagnostic or therapeutic purposes.

2. A kit for identifying BRAF V600E single nucleotide polymorphism using the method for identifying single nucleotide polymorphisms based on crRNA spacer splitting according to claim 1, characterized in that The kit comprises: 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM 12F-BRAF+8H-BRAF, 2 μl 500 nM LbCas12a, 2 μl 2 μM signal reporter; The sequence of the 12F-BRAF is shown in SEQ ID NO. 1; The sequence of the 8H-BRAF is shown in SEQ ID NO. 2; The signal reporter molecule is: 5'-BHQ1-TCCCCCCT-3'-6-FAM.

3. A kit for identifying EGFR L858R single nucleotide polymorphism using the method for identifying single nucleotide polymorphisms based on crRNA spacer splitting according to claim 1, characterized in that The kit comprises: 10 μl DEPC-treated water, 2 μl 10x reaction buffer, 2 μl 500 nM 6F-EGFR+14H-EGFR, 2 μl 500 nM LbCas12a, 2 μl 2 μM signal reporter; The sequence of the 6F-EGFR is shown in SEQ ID NO. 3; The sequence of 14H-EGFR is shown in SEQ ID NO. 4; The signal reporter molecule is: 5'-BHQ1-TCCCCCCT-3'-6-FAM.

4. Use of the kit according to claim 2 in preparing a cancer detection kit, characterized in that: The cancer detection kit is a detection kit based on cancer caused by BRAF V600E mutation.

5. Use of the kit according to claim 3 in preparing a cancer detection kit, characterized in that: The cancer detection kit is a detection kit based on cancer caused by EGFR L858R mutation.

Images