A detection system, detection method and application for single nucleotide polymorphism genotyping

Abstract

This invention discloses a detection system for single nucleotide polymorphisms (SNPs) and its applications. The detection system includes a nuclease and its variants, a splitting crRNA, a reporter molecule, and nucleic acid amplification products. The splitting crRNA consists of a first fragment containing a scaffold region and part of a spacer region, and a second fragment containing the remaining spacer region. This detection system is PAM-independent and highly specific, overcoming the limitations of traditional CRISPR nucleic acid detection systems. It expands the detection range to any target sequence of any species, solving the detection blind spots caused by PAM sequence limitations and the problem of insufficient specificity for SNP recognition in current technologies, making SNP detection more accurate and widespread. This system is applicable, but not limited to, non-invasive sample testing, point-of-care testing (POCT), and SNP genotyping scenarios for complex polyploid genomes. It can be applied to viral typing and variant identification, microbial detection, functional SNP detection in plant and animal genetics and mutations, crop germplasm phenotypic prediction and identification, and other fields.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a detection system for single nucleotide polymorphism (SNP) genotyping, its detection method, and its applications. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are the most common forms of genetic variation in the human genome, playing a significant role in disease susceptibility, drug response, and personalized medicine. With the development of precision medicine, higher demands are being placed on the accuracy, specificity, and convenience of SNP detection technologies.

[0003] In recent years, nucleic acid detection technology based on the CRISPR-Cas system has attracted much attention due to its high specificity and programmability. Traditional CRISPR / Cas12a detection systems achieve signal detection by designing specific crRNAs to guide the Cas12a protein to recognize target sequences and activate its trans-cleavage activity. For example, Chinese patent CN120005973A discloses a single nucleotide polymorphism (SNP) typing method based on CRISPR / Cas12a logic gates. This method introduces the PAM recognition sequence TTTN at positions -15 to -11 of the target site and uses two crRNAs to distinguish between wild-type and mutant SNPs. To further improve the specificity of SNP detection, Chinese patent CN118389648B discloses a method for SNP recognition based on crRNA spacer splitting. This method obtains recognition probes by splitting the crRNA spacer region. When the recognition probes are perfectly complementary to the target sequence, the Cas12a protein is activated, enhancing the ability to recognize single base mismatches. However, the detection system provided by this patent still requires the presence of PAM to recognize the target dsDNA.

[0004] Based on the above, the existing CRISPR-Cas12a SNP detection technologies are strictly dependent on PAM motifs, which limits the detection range of SNPs without nearby PAM sites, making it impossible to effectively detect many important genetic variation sites.

[0005] In addition, existing systems have high tolerance for mismatches. Traditional crRNAs have a strong tolerance for mismatched bases, making it difficult to accurately distinguish between perfectly matched SNP sites and mismatched targets, resulting in insufficient detection specificity. In particular, when dealing with key variants (such as the accurate identification of viral mutants), tolerance becomes a bottleneck for detection accuracy. Furthermore, high-fidelity Cas12a variants have difficulty achieving accurate SNP identification at multiple human DNA target sites.

[0006] Therefore, there is an urgent need to develop a universal SNP detection and typing strategy that is PAM-free, highly specific, highly sensitive, and applicable to both DNA and RNA targets. Summary of the Invention

[0007] To address the technical challenges of existing CRISPR-Cas SNP detection technologies, such as PAM motif dependence, high mismatch tolerance, limited applicability, and lack of a universal SNP genotyping strategy, and to achieve breakthroughs in PAM limitations, improved detection specificity, ultrasensitive detection, expanded applicability, and simplified and rapid operation, this invention initially uses ssDNA as the system detection sample (the sample can be generated through PCR or high-temperature denaturation pretreatment to form the system detection sample). This attempt aims to overcome the PAM dependence limitation of the endonuclease system. Research has shown that although this technique can detect some SNPs without PAM sites, its high tolerance for mismatched bases makes it unable to accurately distinguish between perfectly matched and mismatched targets. Therefore, based on the aforementioned scheme, the present invention provides an ultrasensitive single nucleotide polymorphism (SNP) genotyping detection system that does not require PAM motif sequences. This system is designed with splitting crRNA, which not only breaks through the PAM dependence of existing endonuclease systems and expands the detection range of SNPs, but also significantly improves the specificity of SNP identification, reduces the tolerance to mismatched bases, and can accurately distinguish between perfectly matched and mismatched targets.

[0008] The first aspect of the present invention provides a nuclease-based single nucleotide polymorphism (SNP) genotyping detection system.

[0009] Furthermore, the detection system includes: (1) Endonucleases and their variants; Furthermore, the nuclease is selected from members of the Cas family that have or can be modified to have auxiliary cleavage activity; wherein, members of the Cas family include, but are not limited to, Cas9, Cas12a, Cas12b, Cas12i, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12j, Cas13a, Cas13b, Cas13d, Cas13x, Cas13y or functional variants thereof; In some embodiments, the Cas9 nuclease is selected from SpCas9.

[0010] In some embodiments, the Cas12a nuclease is selected from AsCas12a, LbCas12a, seCas12a, FnCas12a, or functional variants thereof.

[0011] In some embodiments, the Cas13 nuclease is selected from LwaCas13a.

[0012] (2) Splitting crRNA; Furthermore, the splitting crRNA consists of a first segment containing a scaffold region and a spacer region X nucleotides from the 5' end, and a second segment containing the remaining nucleotides. Furthermore, X is an integer between 0 and 20; Preferably, X is an integer between 8 and 16; Preferably, X=13, meaning the splitting site of the splitting crRNA is located at nucleotide number 13; Furthermore, a single mismatched base is introduced at positions +1 to +23 in the original spacer region.

[0013] Preferably, a single mismatched base is introduced at the +4 to +20 sites in the original spacer region.

[0014] More preferably, a single mismatched base is introduced at the original spacer region +17 site.

[0015] Furthermore, the length of the spacer region can be 20 to 30 nucleotides.

[0016] (3) Reporting molecule.

[0017] Furthermore, the reporter molecule is a substrate for the trans-cleavage activity of endonucleases.

[0018] Furthermore, the reporter molecule is selected from fluorescent reporter probes, visual colorimetric probes, label probes recognizable by lateral flow chromatography test strips, or any other substrate molecule that can be trans-cleaved by endonucleases and can generate a corresponding detection signal.

[0019] In some embodiments, the reporter molecule is a fluorescence quenching probe.

[0020] Furthermore, the fluorescent quenching probe is specifically a hairpin-structured single-stranded DNA molecule with a fluorescent group labeled at the 5' end and a quenching group labeled at the 3' end, used for the visual detection of the Cas12a trans-cleavage effect.

[0021] In some embodiments, the reporter molecule is a labeled probe recognizable by a sideflow chromatography test strip.

[0022] Furthermore, the label probe that the lateral flow chromatography test strip can recognize is a single-stranded DNA molecule with a first label modified at the 5' end and a second label modified at the 3' end.

[0023] (4) Nucleic acid amplification system.

[0024] In some embodiments, the nucleic acid amplification system employs PCR or isothermal amplification (such as RPA) against the DNA target; In some embodiments, the nucleic acid amplification system first reverse transcribes RNA to generate cDNA before amplification, and after amplification, denaturation treatment is performed to enhance the generation of single-stranded DNA products.

[0025] Optionally, the detection system also includes a sample preprocessing module, which can lyse and purify different types of samples such as oral mucosa and rice leaves.

[0026] Optionally, the detection system also includes a freeze-drying module to prepare Cas12a and splitting crRNA into freeze-dried ribonucleoprotein complexes for sample transport and long-term preservation.

[0027] Furthermore, the detection system described in the aforementioned scheme does not rely on PAM for identifying and detecting SNPs, and can identify and detect SNPs without PAM motifs.

[0028] A second aspect of the present invention provides a method for SNP genotyping using the above-described detection system.

[0029] Furthermore, the method is a detection method that does not rely on PAM motifs.

[0030] Furthermore, the method includes the following steps: (1) Obtain the nucleic acid amplification of the sample to be tested.

[0031] Furthermore, the sample to be tested is one or more of the following: RNA target, genomic DNA target, non-invasive oral mucosal sample DNA, rice leaf or sugarcane tissue DNA.

[0032] Furthermore, when the sample to be tested is an RNA target, it needs to be converted into cDNA through reverse transcription, followed by isothermal amplification or PCR amplification.

[0033] In some embodiments, the sample to be tested is genomic DNA from rice leaves or sugarcane tissue, which is extracted using the CTAB method or a specific lysis buffer and then amplified at the target site.

[0034] (2) The nucleic acid amplified product obtained in the pretreatment step (1) is a single-stranded sample to be tested.

[0035] Furthermore, the specific pretreatment operation includes: denaturing the test sample in annealing buffer at 95°C for 2 minutes to accelerate the formation of single-stranded DNA.

[0036] (3) The endonuclease or its variant is pre-assembled in a buffer to form an endonuclease-crRNA complex with the splitting crRNA.

[0037] Furthermore, the buffer solution contains 1 mM-100 mM Mg²⁺.+ The cutting buffer.

[0038] Preferably, the buffer solution contains 1 mM to 12.5 mM Mg²⁺. + The cutting buffer.

[0039] Preferably, the buffer solution contains 2.5 mM-7.5 mM Mg²⁺. + The cutting buffer.

[0040] In some embodiments, the complex is pre-assembled in a cleavage buffer containing 2.5 mM MgCl2 to enhance the sensitivity of the endonuclease or its variants to distinguish between perfect match (PM) and mismatch (MM) targets.

[0041] (4) Incubation and signal detection: Further, the single-stranded sample to be tested, the endonuclease-mitotic crRNA complex, and the reporter molecule are mixed and incubated. If the target and the mitotic crRNA are a perfect match, the trans-cleavage activity of the endonuclease or its variant is activated, cleaving the reporter molecule to generate a detectable signal. If the target has an SNP mismatch, no signal is generated or a significantly reduced signal is generated, thereby achieving genotyping.

[0042] In some embodiments, the detection method distinguishes different subtypes of hepatitis B virus by identifying core SNPs (such as at HBV-1 and HBV-2 sites).

[0043] In some embodiments, the detection method is applied to ALDH2 *2、 PNPLA3 Allele detection was performed, and the high sensitivity and accuracy of the detection system were verified by comparing the results with Sanger sequencing.

[0044] In some embodiments, the detection method achieves an ultrasensitive detection limit for SARS-CoV-2 variants by designing specific splitting crRNAs (sequences selected from SEQ ID NO.296-SEQ ID NO.297) (e.g. for the P681R variant) and a fluorescent signal reporter system, enabling the detection of samples with concentrations as low as 1.8 copies / mL.

[0045] In some embodiments, the detection method further involves phenotypic analysis of the sample, such as detecting rice. OsUGT75A The association between gene SNP sites and coleoptile length is used for crop germplasm identification and phenotype prediction.

[0046] A third aspect of the invention provides a kit for SNP genotyping, the kit comprising the detection system described above.

[0047] A fourth aspect of the present invention provides the application of the above-described detection system, detection method and reagent kit in the field of genotyping detection.

[0048] In some embodiments, the application is microbial SNP detection.

[0049] In some embodiments, the microbial SNP detection can be used for strain typing and tracing to track the transmission routes of pathogenic microorganisms.

[0050] In some embodiments, the microbial SNP detection is used to detect SNP mutations related to functions such as microbial drug resistance, to quickly determine the drug resistance spectrum of strains, and to guide precise clinical medication.

[0051] In some embodiments, the microbial SNP detection is used to analyze the genetic diversity and evolutionary history of microbial populations and to clarify the phylogenetic relationships between different strains.

[0052] In some embodiments, the microbial SNP detection is used to achieve rapid identification and diagnosis of pathogenic microorganisms in samples, distinguish between pathogenic and non-pathogenic strains, and improve diagnostic efficiency.

[0053] In some embodiments, the microbial SNP detection is applied to detect SNP sites of industrial microorganisms and screen strains with excellent traits such as high yield and high stability.

[0054] In some embodiments, the microbial SNP detection is applicable to the detection of functional microbial SNPs. Specifically, taking Salmonella and Escherichia coli as examples, it involves synthesizing variant DNA fragments of key functional SNPs in Salmonella (S80I mutation, associated with multidrug resistance in Salmonella) and key functional SNPs in Escherichia coli (such as G80A, S119A, E84P), and simultaneously synthesizing corresponding fission-type crRNAs for detection. After reverse transcription and isothermal amplification, it achieves highly specific recognition of SNP mutations in the target microbial strain. In some embodiments, the application is for the identification of viral variants.

[0055] Furthermore, the application of the detection system of the present invention in the viral SNP detection scenario was verified, with a focus on functional viral SNP detection evaluation for RNA virus SARS-CoV-2 and hepatitis B virus (HBV).

[0056] Furthermore, for SARS-CoV-2, variant RNA fragments containing multiple key functional SNPs such as L452R, T478K, N501Y, D614G, and P681R, along with their corresponding mitotic crRNAs, were synthesized. After reverse transcription and isothermal amplification, the SNIPER technology, using a fully automated medical PCR analysis system and ultraviolet light transmission detection, identified all target variants with high specificity. Simultaneously, the S235F mutation was used to distinguish wild-type SARS-CoV-2 from the more transmissible B.1.1.7 (Alpha) variant, achieving high detection rates using commercially available lateral flow test strips. The detection limit for the P681R variant was as low as 1.8 copies / mL. Comparative experiments showed that the SHERLOCK and Cas13a-occluder methods could not distinguish between wild-type and the three SARS-CoV-2 variants, while the SNIPER technology performed superiorly.

[0057] Furthermore, DNA sequence alignment revealed three core SNP sites that can be used for HBV subtype identification, confirming that the detection system of this invention can efficiently, sensitively, and accurately detect SNPs in both DNA and RNA virus systems without the need for laboratory equipment support, and has significant application potential in the field of disease prevention and control.

[0058] In some embodiments, the application is for human functional SNP detection.

[0059] Furthermore, the detection system of this invention identified functional SNPs in several known human populations, with results completely consistent with those obtained from commercial standard assays. Simultaneously, this detection system was used to detect human functional variants in non-invasive oral mucosal samples. Results showed that for the PNPLA3 mutation, SNIPER and Sanger sequencing results were completely consistent.

[0060] Furthermore, the detection system of this invention achieved a detection sensitivity of 0.05% for the EGFR T790M mutation and 0.5% for the NPM1 gene TCTG insertion mutation (c.863_864insTCTG). In addition, detection was performed using a lyophilized ribonucleoprotein complex containing Cas12a and fission-type crRNA. It was found that the complex remained detectable after being stored at 45°C for 72 hours, and the PNPLA3 mutation allele was successfully detected. This indicates that the detection system is suitable for non-invasive samples and has high stability, making it particularly suitable for home testing, preclinical applications, and testing in remote areas.

[0061] In some embodiments, the application is crop germplasm identification and phenotypic prediction.

[0062] Furthermore, the detection system of this invention was used to detect 13 core SNP sites annotated by genome-wide association studies (GWAS). The results showed that the variation patterns of these SNPs among germplasms enabled SNIPER to clearly distinguish 25 rice germplasms in the rice mini core germplasm bank.

[0063] Furthermore, the above-mentioned detection system was used to test rice. OsUGT75A The SNP loci of the gene (the gene regulating coleoptile length) were analyzed, and the results were consistent with the corresponding phenotypes. This example also found that SNIPER can be used to distinguish different alleles, and this technology can be applied to complex polyploid genomes such as sugarcane. The results indicate that SNIPER has great potential in agricultural applications, including phenotypic prediction, variety identification, and marker-assisted selection.

[0064] Furthermore, some embodiments of the present invention also provide specific sequences of synthesized splitting crRNAs applicable to various scenarios, the specific sequence information of which is as follows: Table 1. Sequence information of fission-type crRNA

[0065] The present invention has the following beneficial effects: 1. Overcoming PAM-dependent limitations and achieving high identification specificity: The dependence of existing endonuclease systems on PAM sequences limits their ability to detect SNPs across the entire genome, especially SNPs without PAM sites, affecting the comprehensiveness and flexibility of detection. This invention provides a detection system containing splitting crRNA, which not only overcomes the PAM dependence of existing endonuclease systems and expands the detection range of SNPs, but also significantly improves the identification specificity of SNPs, reduces the tolerance for mismatched bases, and can accurately distinguish between perfectly matched and mismatched targets.

[0066] 2. Strong technical adaptability: This invention can simultaneously cover DNA and RNA targets, and can be used in multiple fields such as viral mutation monitoring, detection of clinically relevant functional SNPs in humans, crop germplasm identification and phenotypic prediction.

[0067] 3. Meets the needs of non-invasive sample and complex genome detection: This invention has fast detection speed, simple operation, and high detection sensitivity, which can meet the needs of non-invasive sample detection; at the same time, it can be used with a variety of reporter molecules to meet the detection needs of different scenarios, including point-of-care testing (POCT).

[0068] 4. Overcoming the limitations of high-throughput detection: This invention can meet the needs of large-scale sample detection, improve the detection efficiency and stability of existing technologies, and especially meet the needs of clinical testing centers or agricultural breeding experiments that need to process large numbers of samples. Attached Figure Description

[0069] Figure 1 : A schematic diagram of the detection principle of the SNIPER platform of the present invention; Figure 2 : Fluorescence detection experiment of Cas12a in a standard system for PCR products; Figure 3 : Fluorescence detection experiments of Cas12a standard systems containing PAM and without PAM for dsDNA and ssDNA; Figure 4 Fluorescence detection experiments on non-PAM motif sites in the Cas12a conventional system after denaturation treatment; Figure 5 :by OsDEP1 Design principles for different lengths of splitting crRNAs, for example; Figure 6: Detection of different mitotic crRNAs and full-length crRNAs using the Cas12a system. OsDEP1 Fluorescence detection experiment of gene target sequence SNP; among which, Figure 6A The results of fluorescence detection experiments for mitotic crRNAs of different lengths; Figure 6B The results of fluorescence detection experiments for mitotic crRNAs with mitotic sites X located at different mitotic sites from 0 to 20; Figure 6C To design spacer regions of different lengths, the corresponding splitting crRNAs were synthesized, and the fluorescence detection results were obtained. Figure 7 A fluorescence detection experiment comparing the resolution of SNPs in different target gene sequences using the Cas12a system combined with full-length crRNA and splitting crRNA-13; Figure 8: Different Mg² + SNIPER detection system under concentration conditions OsEPSPS ( Figure 8A )and OsDEP1 ( Figure 8B A fluorescence detection experiment comparing the detection efficiency of gene SNPs; Figure 9Fluorescence detection experiments comparing the detection effects of different Cas12a enzyme variant detection systems and the SNIPER system on SNPs; Figure 10: Appendix Figure 10A and 10B yes Figure 9 Raw fluorescence readings from the fluorescence detection experiment; Figure 11 The SNIPER system was used in fluorescence detection experiments to detect 18 functional variations among multiple species, including plants, animals, microorganisms, and viruses. Figure 12 : A schematic diagram of the detection process of SNIPER for detecting viral single-base variations; Figure 13 : Fluorescence detection experiment of SARS-CoV with 5 functional SNP variants using the SNIPER system; Figure 14 Using an LED ultraviolet transilluminator to... Figure 13 Fluorescence visualization detection experiments were conducted on the five samples involved. Figure 15 Fluorescence detection experiment using the SNIPER system to distinguish SARS-CoV-2 from the alpha variant (B.1.1.7) through the S235F mutation; Figure 16 Commercial test strips are used for reading. Figure 15 SNP identification results in the data; Figure 17 Comparison of the detection efficacy of three platforms—SNIPER, SHERLOCK, and Cas13a-occluder—in detecting SARS-CoV-2 variants using fluorescence detection experiments; Figure 18 Fluorescent detection experiment to validate the sensitivity of SNIPER detection, taking the P681R mutation of SARS-CoV-2 as an example; Figure 19 SNIPER was used to design experiments for HBV genotyping. Figure 20 : Verification experiments using different crRNA-mediated SNIPER systems to distinguish HBV genotypes (B, C, and D). Figure 21 : Schematic diagram of the SNIPER detection process for functional SNPs in the human genome; Figure 22 : Fluorescence detection assays using the SNIPER system for commercial standards with human functional variations; Figure 23 SNIPER and Sanger sequencing technologies were used to analyze oral mucosal samples. HsPNPLA3(c.444 C>G & c.447 C>T) and HsALDH2*2 Gene mutation detection was performed at the (c.1510 G>A) locus; Figure 24 : HsEGFR -T790M and HsNPM1 Experimental analysis of SNIPER fluorescence detection sensitivity and signal difference of -insTCTG variant at different allele frequencies; Figure 25: Freeze-drying design experiment and the effects of using different treatments on freeze-dried mixtures HsPNPLA3 Fluorescence detection experiment, Figure 25A For the experimental procedure, Figure 25B For the test results; Figure 26 SNIPER detection process for plant genotyping; Figure 27 Genotyping and germplasm differentiation experiments were conducted on 25 germplasm resources from the rice mini core germplasm bank using SNIPER based on 13 SNP loci validated by Sanger sequencing. Figure 28 SNIPER was used to analyze 13 germplasm accessions. OsUGT75A Fluorescence detection experiments were conducted to verify the site-specific SNPs. Figure 29 Results of experiments using SNIPER to distinguish different alleles in sugarcane. Detailed Implementation

[0070] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] Terminology Explanation The Single Nucleotide Identification and Precise Evaluation Reporting (SNIPER) technology described in this invention is an integrated technology system for specific identification, high-sensitivity detection, and accurate result evaluation of single nucleotide polymorphisms (SNPs).

[0073] The detection system, detection method, and related reagent kits provided by this invention are all based on the SNIPER technology and its derivative application scenarios.

[0074] Nucleotide endonuclease Nucleotide endonucleases are a class of hydrolytic enzymes that break down the phosphodiester bonds within nucleic acid molecules, thus cleaving the DNA / RNA chain. The nucleotide endonucleases described in this invention can be natural or artificially synthesized.

[0075] variants The variants described in this invention are functional variants of nucleases. The variants described in this invention refer to mutant proteins that, through site-directed mutagenesis, protein modification, natural mutation, etc., alter the recognition specificity, chain cleavage ability, catalytic activity, PAM, modified base preference, and thermostability of wild-type endonucleases, retaining nucleic acid binding but with a directional change in enzymatic cleavage function.

[0076] fission-type crRNA Split-type crRNA is a modularly modified crRNA that is artificially split into two or more independent RNA fragments at the scaffold and spacer junction sites of the naturally integrated crRNA molecule. Each fragment cannot assemble into a functional CRISPR-RNP complex when it exists alone. Only when the target nucleic acid is present and the complete crRNA spatial structure is restored by tandem splicing based on the complementary bases of the target, can it bind to Cas protein and activate endonuclease activity.

[0077] Preferably, the splitting crRNA of the present invention comprises a first segment including a scaffold region and a spacer region X nucleotides from the 5' end, and a second segment including the remaining nucleotides in the spacer region; more preferably, X is any integer between 0 and 20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, X is any integer between 8 and 16; more preferably, X is = 13, that is, the splitting site of the splitting crRNA is located at the 13th nucleotide.

[0078] Reporting molecules The reporter molecule described in this invention is a substrate with trans-cleavage activity of a nuclease. Preferably, the reporter molecule is selected from fluorescent reporter probes, visual chromogenic probes, labeled probes recognizable by lateral flow chromatography test strips, or any other substrate molecule that can be trans-cleaved by a nuclease and can generate a corresponding detection signal.

[0079] Transformation The denaturation treatment described in this invention involves subjecting the sample (including but not limited to ssDNA and cDNA) to high-temperature annealing (around 95°C) to alter the properties of the sample.

[0080] Materials and Methods 1. Procurement and synthesis of general reagents The specially modified nucleic acid probes were synthesized by GenScript in Nanjing; the certified standards were purchased from the National Institute of Metrology of China and COBIOER in Nanjing; the Twist Amp Basic kit and the RT-basic Nucleic Acid Amplification Kit were purchased from TwistDX in the UK and GenDx in Suzhou, respectively; the CRISPR single-target lateral flow detection test strip was from ToloBiotech in Shanghai; and the PCR amplification reagent 2×KeyPo Master Mix (Dye Plus) was purchased from Novizan in Nanjing.

[0081] 2. DNA Materials and Plasmid Construction DNA sequences were obtained from GenBank. Target fragments could be cloned from HEK293T cells, the genomic DNA of japonica rice 'Zhonghua 11', or the sugarcane hybrid 'Xintaitang 22', or they could be synthesized by GenScript in Suzhou. The fragments were then constructed into the pUC57 plasmid. Single-base mismatches were introduced into the plasmid via PCR amplification.

[0082] 3. RNA materials Full-length crRNA or split crRNA was designed based on the target sequence and experimental requirements and synthesized by Nanjing GenScript; the SARS-CoV-2 RNA target sequence was obtained from GenBank and synthesized by Suzhou Cwbio.

[0083] 4. Expression and purification of LbCas12a protein (1) The LbCas12a-6xHis prokaryotic expression vector was transformed into Escherichia coli BL21(DE3) competent cells. Positive clones were picked and cultured in Luria-Bertani (LB) medium (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride) at 37°C with shaking at 200 rpm. (2) Measure the absorbance of the bacterial culture at OD600nm. When the absorbance reaches about 0.6, add isopropylthio-β-galactopyranoside (IPTG) to a final concentration of 0.5 mM and culture at 21°C and 200 rpm for 16 h with shaking. (3) Collect the bacterial cells by centrifugation at 4℃, 4000 rpm for 20 min, add 100 ml of lysis buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM mercaptoethanol, 10% glycerol, 10 mM imidazole) and 1 ml of protease inhibitor, and resuspend the bacterial cell pellet. (4) The suspended bacterial cells were lysed by ultrasound in an ice bath. The ultrasound conditions were: 500W, ultrasound on for 1s, off for 1.5s, for 30min. (5) After the bacterial cells become transparent, dispense them into 50 ml centrifuge tubes, centrifuge at 15000g for 20 min, and remove the precipitate (the precipitate and supernatant should be sampled into 1.5 ml centrifuge tubes respectively). (6) Add 1 ml of Ni-NTA-tagged protein purification medium to the supernatant and incubate at 4°C for 1-2 h; (7) After incubation, collect the medium into a gravity empty column, and add 1 empty column volume of lysis buffer, 3 empty column volumes of high-salt buffer (20 mM Tris (pH 7.5), 1 M NaCl, 10 mM mercaptoethanol, 10% glycerol, 10 mM imidazole), 1 empty column volume of lysis buffer, and flow through the washing medium. (8) Add an appropriate amount of elution buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM mercaptoethanol, 10% glycerol, 500 mM imidazole), collect the effluent, determine the protein concentration, and detect the bands by SDS-PAGE electrophoresis. (9) Add the effluent obtained in step 8 to the dialysis membrane (RC membrane, molecular weight cutoff of 3 kDa), seal the openings at both ends, and place it in the storage solution overnight at 4°C to replace the buffer solution.

[0084] (10) SDS-PAGE gel electrophoresis identification and storage at -80 ℃ 5. Oral sample preparation Volunteers collected oral mucosal samples, centrifuged them at 12000g for 10 min, collected the precipitate, and used the Novizan room temperature sample lysis kit to lyse the sample at room temperature for 3 min to obtain a crude lysis buffer. 1 µL of the buffer was used as an amplification template for SNIPER detection and Sanger sequencing.

[0085] 6. Conventional PCR preparation of DNA targets This invention amplifies a fragment approximately 500 bp flanking the target site using 2× KeyPo Master Mix (Dye Plus) targeting plasmid vectors, purified human cell line genomic DNA, or rice genomic DNA. The amplified fragment can be diluted 10-fold or purified before being used as a target template. The purified product is quantified using a spectrophotometer.

[0086] 7. Preparation of DNA targets via recombinase polymerase amplification (RPA) Primer design was performed using the EZassay online tool (Shenzhen, China; https: / / www.ezassay.com), following the specifications in the TwistAmp Basic kit instruction manual. Each 50 µL reaction volume was prepared according to the instructions, with 1 µL of DNA target added, and reacted at 37°C for 10–30 minutes using the TwistAmp Basic kit. The DNA template concentration was adjusted according to specific experimental requirements.

[0087] 8. Preparation of RNA targets through reverse transcription and amplification To detect functional SARS-CoV-2 single nucleotide polymorphisms, reverse transcription was performed using a 10 nM RNA target and SuperScriptIV reverse transcriptase. Each 20 µL reaction volume, containing 0.5 nM RNA target, was prepared according to instructions, incubated at 55 °C for 10 minutes, and then treated at 80 °C for 10 minutes. The resulting cDNA was used as a template for subsequent target amplification.

[0088] For one-step reverse transcription-enzymatic recombinase amplification (RT-ERA), the reaction was performed using the RT-basic Nucleic Acid Amplification Kit. The master mixture was prepared by reconstituted a single lyophilized reaction precipitate by mixing 20 µL of rehydration buffer, 2.5 µL of forward primer (10 µM), 2.5 µL of reverse primer (10 µM), and 8 µL of nuclease-free water. The master mixture was then aliquoted into five centrifuge tubes. 3 µL of target RNA (at the specified concentration) was added to each aliquot. 0.4 µL of activator was then coated onto the inside of each tube cap, and the mixture was briefly centrifuged to mix the reagents. The reaction mixture was incubated at 42 °C for 20 minutes.

[0089] 9. Preparation of Spiked-in DNA Targets Will HsEGFR T790M, HsNPM1 The c.863_864insTCTG mutant DNA fragment was serially diluted to a wild-type DNA background to construct a mutant allele frequency gradient sample (total concentration maintained at 10 nM). PCR amplification of the target mixture was performed using primers with unique barcodes, and the purified products were validated for mutation ratios using NGS on the NovaSeq platform (analysis workflow available on GitHub open-source code). For each target site, amplicon sequencing was performed three times using genomic DNA from three independent samples. Allele frequency analysis for each sample was performed according to the previously described method.

[0090] 10. DNA samples used for rice germplasm identification and phenotypic analysis Thirteen SNP loci in the rice genome were selected from the organized rice GWAS database, based on publicly available data from the GWAS Atlas (https: / / ngdc.cncb.ac.cn / gwas). All genomic locations were reported according to the IRGSP-1.0 reference genome assembly standard. The p-value reflects the association strength between each SNP and its corresponding phenotype.

[0091] For the 25 rice germplasm resources collected, seeds were cultured for 7 days to promote germination and seedling growth. Leaf fragments of 1 cm × 1 cm were collected, and genomic DNA was extracted using the CTAB method. DNA extraction was performed in a CTAB buffer containing 2% (w / v) CTAB, 200 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), and 1.4 M NaCl, and incubated at 65 °C for 30 minutes. Crude genomic DNA was then obtained by chloroform extraction and isopropanol purification. This genomic DNA served as a template for subsequent amplification of target sites. The resulting PCR products were diluted 10-fold, denatured, and then used for SNIPER detection.

[0092] For rice OsUGT75A For SNP phenotypic testing and detection of the gene, 13 dried seeds were placed in test tubes and cultured in water for 7 days under a 12-hour light / 12-hour dark cycle at 25℃, with three independent biological replicates. Phenotypic characteristics of each rice germplasm were recorded on day 7, and coleoptile tissue was collected for subsequent DNA extraction using the method described above.

[0093] The genotype of each rice germplasm resource was confirmed by Sanger sequencing. When the allele frequency determined by Sanger sequencing was approximately 50% (heterozygous) or 100% (homozygous), the variant was considered to have been confirmed.

[0094] 11. Hepatitis B virus (HBV) genotyping DNA sample Genomic DNA sequences of various HBV subtypes were obtained from the NCBI database. Selected genomic fragments were synthesized and cloned into the pUC57 vector as amplification templates. Through sequence alignment analysis, this invention identified fourteen target sites and designed corresponding crRNAs for subsequent detection.

[0095] During SNIPER assay, the HBV PCR amplification products were annealed with annealing buffer (RTE3105; Real). The sample was diluted tenfold and annealed to generate a detection template. (Times [Beijing] Biotechnology, China)

[0096] 12. Sugarcane genotyping DNA samples Retrieve from NCBI database ShSPS , ShGA1 and ShSUT The gene sequence was obtained and cloned from the genomic DNA of the sugarcane hybrid 'Xintai Sugar 22' into the pUC57 vector. The gene fragment was amplified by PCR, and the PCR product was diluted 10-fold and annealed in annealing buffer to generate a template for SNIPER detection.

[0097] 13. Traditional Cas12a-full-length crRNA detection method For PCR product detection experiments, LbCas12a and full-length crRNA were prepared using a pre-assembly method: 200 nM LbCas12a and 250 nM crRNA were incubated in a cleavage buffer (20 mM HEPES pH 7.5, 250 mM KCl, 2.5 mM MgCl2, 0.5 mM DTT, 1% glycerol) at 37 °C for 30 min. The pre-assembled complex was diluted 5-fold and mixed with 500 nM 5'-FAM-labeled hairpin DNA-BHQ reporter gene and 3.3 nM target or non-target DNA to a 10 µL reaction volume to initiate the cleavage reaction. The reaction was performed in a fully automated medical PCR analysis system (LEPGEN-96; Lepu Diagnostics; Beijing, China) at 37 °C for 1 hour, simultaneously exposed to 485 nm blue light. Fluorescence intensity was measured after 1 hour of incubation.

[0098] To achieve denaturation-assisted DNA target detection, the sample was first denatured in annealing buffer at a concentration of 10 nM, incubated at 95 °C for 2 minutes, and then immediately placed on ice for rapid cooling before the above method was used.

[0099] 14. SNIPER detection method 10 nM purified DNA template or 1 µL of target DNA amplification fragment was denatured at 95 °C for 2 minutes in 10 µL annealing buffer (as described above), followed by rapid cooling in an ice bath. Simultaneously, 200 nM LbCas12a and 250 nM splitting crRNA were pre-assembled at 37 °C for 30 minutes in cutting buffer (as described above). A 10 µL SNIPER reaction mixture contained 50 nM Cas12a, 62.5 nM splitting crRNA, 3 µL denatured single-stranded DNA, and 500 nM fluorescent reporter probe (5'-FAM-labeled hairpin DNA-BHQ probe). The reaction was performed in a fully automated medical PCR analysis system (LEPGEN-96) at 37 °C under 485 nm blue light irradiation.

[0100] (1) Endpoint reading method: Measure fluorescence intensity after 1 hour of incubation. Curve determination method: Incubate the reaction at 37℃ for 1 hour, and record the fluorescence value every 30 seconds.

[0101] (2) Lateral flow assay (LFA): The 5'-FAM-labeled hairpin DNA-BHQ reporter gene was replaced with a 5'-FAM-labeled single-stranded DNA-biotin reporter gene (total input: 20 pmol). The SNIPER reaction product was diluted to 50 µL and loaded onto a commercial CRISPR single-target nucleic acid test strip (Tolo Biotech) to read the results. The result interpretation criteria are as follows: positive (both the control line [C] and the test line [T] show double red bands), negative (only the control line [C] shows a single red band), and invalid (no band on the control line [C]).

[0102] (3) Using an LED ultraviolet transilluminator (Shenhua), after incubation at 37℃ for 20-60 minutes, the fluorescence intensity was evaluated by direct observation and the data was recorded.

[0103] 15. RNA detection using LwaCas13a The LwaCas13a-crRNA complex was pre-assembled using the following steps: 180 nM LwaCas13a and 90 nM crRNA were incubated in 1× NEB 3.1 buffer at 37 °C for 30 minutes. For initial detection, the complex was diluted with nuclease-free water to a final concentration of 45 nM LwaCas13a and 22.5 nM crRNA, and mixed with 500 nM RNA FQ reporter molecule to prepare a 10 µL reaction system. The reaction was performed at 37 °C for 1 to 3 hours using an automated medical PCR analysis system (LEPGEN-96), with fluorescence signals recorded every 30 seconds.

[0104] 16. Preparation of freeze-dried mixtures Prepare a premixed stock solution containing 12.50% sucrose, 8.33% mannitol, 10.00% polyethylene glycol 8000, 0.83% Tween-20, and 1.66% bovine serum albumin. Vortex mix and centrifuge to remove surface air bubbles. Add an equal volume of 50 mM Tris-HCl buffer (pH=7.4) and homogenize completely by inverting or vortexing.

[0105] Prepare 1 mL of lyophilization mixture by adding 2.5% amino acids (glycine / monosodium glutamate / histidine), 10 µM LbCas12a, and 12 µM crRNA sequentially to the premixed stock solution. After thorough mixing and centrifugation, aliquot the mixture into PCR tubes. Immerse the tubes in liquid nitrogen until the contents form white frozen microspheres, then transfer to -80 °C and freeze for 4–16 hours, followed by lyophilization for 4 hours. Store and process the resulting powder according to the requirements of subsequent experiments.

[0106] Buffer A, Buffer B, and Buffer C were respectively prepared with glycine, without glycine, and with water treatment without nuclease.

[0107] The crRNA and splitting crRNA sequence information used in the following specific embodiments of the present invention are selected from SEQ ID NO.187-SEQ ID NO.378 / SEQ ID NO.396-SEQ ID NO.411.

[0108] The sequence information of LbCas12a, seCas12a, and LbCas12a-HF used in the following specific embodiments is selected from SEQ ID NO.390-SEQ ID NO.392.

[0109] Example 1 This embodiment provides a detection system for single nucleotide polymorphism (SNP) genotyping, which includes four core components: a nuclease, a fissile crRNA, a reporter molecule, and a nucleic acid amplification system.

[0110] The endonuclease used is Cas12a nuclease, specifically LbCas12a nuclease (SEQ ID NO. 390). LbCas12a nuclease possesses strong trans-cleavage activity, enabling non-specific cleavage of single-stranded DNA substrates upon activation. After binding to fissile crRNA and recognizing the target sequence, this enzyme is activated and exhibits trans-cleavage activity, thereby cleaving reporter molecules to generate a detection signal.

[0111] The split crRNA is a key innovative component of this detection system, consisting of two separate fragments. The first fragment contains the complete scaffold region and 13 nucleotides from the 5' end of the spacer region, while the second fragment contains the remaining nucleotides of the spacer region.

[0112] In this embodiment, the splitting site of the splitting crRNA is precisely located at nucleotide 13 of the spacer region. This splitting design makes the detection system more sensitive to SNPs; simultaneously, since the detection substrate is ssDNA, the crRNA does not rely on the PAM motif to guide the endonuclease to recognize the target sequence, significantly expanding the range of target sequence selection, and is particularly suitable for the precise recognition of SNP sites. When two crRNA fragments bind to the target sequence simultaneously, a functional crRNA-Cas12a complex is formed, activating the trans-cleavage activity of the nuclease.

[0113] In a preferred embodiment, when the SNP is located at the original spacer region +17 site, the detection system significantly enhances its ability to distinguish between perfectly matched and mismatched targets. This design overcomes the dependence of the traditional CRISPR-Cas12a system on PAM motifs, improves the sensitivity to detect mismatched bases, and thus makes SNP genotyping detection more accurate and comprehensive, extending the detection range to DNA and RNA targets without PAM sites.

[0114] The reporter molecule employs a fluorescent quenching probe, which is a single-stranded DNA molecule capable of forming a hairpin structure. The 5' end is modified with a fluorescent group, and the 3' end with a quenching group. Under normal conditions, the fluorescent signal is quenched because the hairpin structure brings the fluorescent group and the quenching group close together. When the Cas12a nuclease is activated, its trans-cleavage activity cuts the fluorescent quenching probe, disrupting the hairpin structure and separating the fluorescent group from the quenching group, thus generating a detectable fluorescent signal.

[0115] In a preferred embodiment, the reporter molecule may also be a labeled probe recognizable by a lateral flow chromatography strip. This labeled probe is a single-stranded DNA molecule with a first label modified at its 5' end and a second label modified at its 3' end. Upon cleavage by the LbCas12a nuclease, the labels are separated and can be visually detected using a lateral flow chromatography strip, eliminating the need for complex fluorescence detection equipment.

[0116] The nucleic acid amplification system provides the reaction environment for nucleic acid amplification, including an isothermal amplification system. This isothermal amplification system utilizes recombinase polymerase amplification technology, enabling rapid amplification of target DNA at a constant temperature of 37°C without the need for thermal cycling equipment. The amplification products directly serve as recognition targets for fissile crRNA, achieving target sequence enrichment and detection signal amplification.

[0117] The working principle of this detection system is as follows: First, the isothermal amplification system amplifies the target DNA containing the SNP site; then, the splitting crRNA binds to LbCas12a to form a complex, which recognizes and binds to the target, thereby activating Cas12a and exerting cleavage activity; finally, the activated nuclease cleaves the reporter molecule, producing a detectable signal change. The entire detection process is completed in a single tube reaction system, which is simple to operate, has a short detection time, high specificity, and can accurately distinguish single nucleotide differences, achieving rapid detection of SNP genotyping.

[0118] According to the technical solution provided in this embodiment, the design of splitting crRNA reduces the tolerance of Cas12a to mismatched bases. In particular, the reporter molecule, as a substrate for the trans-cleavage activity of Cas12a, generates a signal after cleavage that can directly reflect the presence or absence of the target, thereby achieving accurate SNP identification. Therefore, the technical solution of this embodiment effectively solves the problems of limited detection range, insufficient detection specificity, and poor adaptability in specific application scenarios (such as rapid point-of-care testing, non-invasive sample testing, and SNP genotyping of complex polyploid genomes) caused by the dependence on PAM sequences in existing SNP detection methods. This enables SNP detection technology to be more widely promoted and applied in fields such as genetic locus identification, medical diagnosis, infectious disease control, and agricultural germplasm identification.

[0119] This technical solution not only greatly improves the specificity of SNP detection, but also broadens the detection range, enabling it to be more effectively applied to the detection of DNA or RNA without PAM sequence restrictions, as well as rapid on-site detection in non-laboratory environments, meeting the needs of modern biotechnology for efficient, convenient and accurate detection.

[0120] Example 2 This embodiment provides a method for SNP genotyping detection using the detection system in Embodiment 1. This method is a detection method that does not rely on PAM motifs.

[0121] Step 1: Obtain the nucleic acid amplified product of the sample to be tested. When the sample is an RNA target, it is necessary to first reverse transcribe the sample to obtain cDNA before nucleic acid amplification. The reverse transcription process involves incubating the sample with reverse transcriptase at 55 °C for 10 minutes, followed by treatment at 80 °C for 10 minutes to convert the RNA template into stable cDNA. Subsequently, the target sequence is amplified by PCR or isothermal amplification techniques to obtain the nucleic acid amplified product containing the SNP site.

[0122] Step 2: Pre-process the nucleic acid amplifier to obtain the test sample in a single-stranded state. The test sample is denatured in annealing buffer at 95°C for 2 minutes, and then rapidly cooled in an ice bath to maintain the target strand as single-stranded DNA.

[0123] Step 3: Complex formation. Pre-assemble Cas12a nuclease with cleaving crRNA in a buffer solution to form the Cas12a-cleaving crRNA complex. A Mg²⁺-containing buffer solution is used. + The cleavage buffer was used as the reaction system, and Mg²⁺ was added. + The final concentration was 2.5 mM. The Cas12a nuclease from Example 1 was mixed with the fission-type crRNA at a molar ratio of 1:1.25 and incubated at 37°C for 15-30 min to form a stable complex.

[0124] Mg² + Ions, as cofactors, are crucial for the catalytic activity of Cas12a nuclease, and a concentration of 2.5 mM is sufficient to ensure optimal enzyme activity in most SNP assays.

[0125] Step 4: Incubation and Signal Detection. The single-stranded sample, the Cas12a-split crRNA complex, and the reporter molecule are mixed and incubated. The reaction system is incubated at 37°C for 60 minutes. If the target and split crRNA are perfectly matched, both fragments of the split crRNA can simultaneously and stably bind to the target sequence, forming a functional crRNA structure and activating the trans-cleavage activity of Cas12a. The activated Cas12a nuclease non-specifically cleaves the fluorescent quencher probe, disrupting the probe's hairpin structure and separating the fluorescent group from the quencher group, generating a detectable fluorescent signal. If there is an SNP mismatch in the target, even a difference of only one nucleotide will significantly reduce the binding stability of the split crRNA to the target sequence, resulting in the Cas12a nuclease not being effectively activated and producing no or significantly reduced fluorescent signal. By comparing the fluorescence signal intensity of different samples, the genotype of the SNP site can be accurately determined, achieving precise genotyping detection.

[0126] The core advantage of this method lies in its use of splitting crRNA design and high-temperature denaturation to make the detection substrate ssDNA. This enables target sequence recognition independent of the PAM motif, significantly expanding the range of detectable SNP sites. Simultaneously, the high sensitivity of splitting crRNA to single nucleotide mismatches ensures the accuracy and specificity of SNP genotyping. The entire detection process is simple to operate and quick, making it suitable for various applications such as viral variant identification, human functional SNP detection, and crop germplasm identification and phenotypic prediction.

[0127] Example 3 This embodiment provides a kit for SNP genotyping, which includes the detection system described in Example 1. The specific process for SNP genotyping using this kit is as follows: 1) Sample pretreatment: Obtain the nucleic acid amplified product of the sample to be tested. The DNA sequence containing the target SNP site is amplified by PCR amplification or isothermal amplification to obtain a sufficient concentration of amplified product.

[0128] 2) Complex pre-assembly: The Cas12a nuclease and the splitting crRNA are pre-assembled in the buffer to form the Cas12a-splitting crRNA complex. That is, the two fragments of Cas12a protein and splitting crRNA are incubated in 5*CB buffer at 37°C for 15-30 min to form a stable complex.

[0129] 3) Incubation and signal detection: The single-stranded sample to be tested, the Cas12a-split crRNA complex, and the reporter molecule are incubated at 37°C for 15 min to 2 h. If the target and the split crRNA are perfectly matched, the trans-cleavage activity of Cas12a is activated, cleaving the reporter molecule to generate a detectable signal; if there is an SNP mismatch in the target, no signal is generated or a significantly reduced signal is generated, thereby achieving genotyping.

[0130] In some embodiments, the following steps are also included: Single-strand conversion: The nucleic acid amplified product obtained in pretreatment step 1 is converted into a single-stranded sample for testing. The double-stranded DNA amplification product is then converted to a single-stranded state through heat denaturation or alkaline treatment, enabling it to bind to dividing crRNA.

[0131] The kit provided in this embodiment utilizes the synergistic effect of the Cas12a nuclease and a specifically designed split crRNA molecule. The design principle of the split crRNA is to enhance the specific recognition of target sequences by dividing it into two parts—one containing the first 13 nucleotides of the scaffold and spacer regions, and the other containing the remaining spacer region. This is particularly effective in increasing sensitivity to SNP sites and reducing tolerance to mismatched bases. This design allows Cas12a to more accurately distinguish between perfectly matched and mismatched targets, especially when SNPs are located at +17 sites, maximizing detection efficiency and discriminative power.

[0132] Meanwhile, the detection system in the kit does not rely on PAM sequences, which expands the application scope of SNP detection and covers more gene sequence regions that cannot be identified by traditional systems. The signal reporting system in the system, such as fluorescent reporter probes and lateral flow test strips, can generate easily detectable signals under the trans-cleavage action of Cas12a, enabling visual genotyping of SNPs.

[0133] Furthermore, the kit also considers the needs of point-of-care testing (POCT) by preparing lyophilized Cas12a-split crRNA ribonucleoprotein complexes, ensuring their stability at room temperature and long-term storage, facilitating transportation and use. This combination of technical features not only improves the speed and sensitivity of SNP detection but also greatly simplifies the detection process, enabling it to provide efficient detection capabilities and practical operability in various applications such as medical diagnosis, infectious disease control, and agricultural germplasm identification, overcoming the limitations of traditional techniques in terms of sensitivity, specificity, and sample type adaptability.

[0134] Example 4 The purpose of this embodiment is to verify that the detection system and detection method provided in Embodiment 1 do not depend on the PAM sequence.

[0135] Specifically, to investigate whether the PAM sequence is necessary for the combined use of CRISPR-Cas12a nucleic acid detection and PCR, this embodiment performed detections at 12 gene loci (see Table 2 for gene loci information) with and without PAM.

[0136] Table 2 Information on 12 sites

[0137] The results are as follows Figure 2 As shown, even in the absence of PAM, most gene loci in the PCR product group can still be directly detected, and the fluorescence intensity is greater than 50 a.u. The negative control group (a non-target DNA segment that does not match the crRNA sequence) has no obvious fluorescence intensity and can not be directly detected. The results indicate that the PAM motif is not necessary for the detection of PCR products.

[0138] Based on this result, this embodiment initially assumes that single-stranded DNA (ssDNA) generated during PCR can be detected via PAM-independent methods. To verify whether Cas12a can utilize ssDNA lacking the PAM sequence, this embodiment synthesized ssDNA containing and without the PAM motif, as well as double-stranded DNA (dsDNA), and evaluated the detection performance of CRISPR-LbCas12a. The amino acid sequence of LbCas12a used in this embodiment is SEQ ID NO. 390. Consistent with previous studies, ssDNA, rather than dsDNA, can serve as a PAM-independent substrate for CRISPR-Cas12a. Figure 3 ).

[0139] This embodiment further introduces a denaturation (high-temperature denaturation) treatment, which enhances the generation of ssDNA products and increases the fluorescence signal intensity. Figure 4 ).

[0140] The results of this study demonstrate that the ssDNA fragments generated during PCR can be directly and efficiently recognized by Cas12a. Therefore, PCR-based detection can use PAM-free ssDNA as a Cas12a target.

[0141] Example 5 This embodiment further optimizes the design of splitting crRNA in the detection system provided in Embodiment 1, thereby improving the specificity of SNP detection.

[0142] The splitting crRNA can maintain cis and trans cleavage activity of Cas12a in vitro. In this embodiment, the crRNA is separated into two fragments: (1) RNA containing the scaffold region and X nt from the 5' end of the spacer region; (2) the remaining RNA in the spacer region.

[0143] In this embodiment, the spacer region is 23 nt in length. For the remaining (23-X) nt RNA, due to its shorter length, the requirement for target DNA sequence complementarity is higher, thus reducing the tolerance for mismatches and improving the specificity and sensitivity of the detection. Therefore, this embodiment designs multiple splitting crRNAs with splitting sites located at nucleotides 10 to 16 (X=10-16). Figure 5 The study found that when crRNA was split into a scaffold region - 13 nt spacer region and a 10 nt spacer region (split crRNA-13), its ability to detect mismatched nucleotides was significantly improved compared to the traditional full-length crRNA design. Figure 6A Furthermore, under the aforementioned conditions, this invention also designed cleavage-type crRNAs with cleavage sites located at different sites between 0 and 20, where X is 0, 6, 13, or 20. The crRNA sequences are selected from sequences SEQ ID NO.396-SEQ ID NO.401. Results showed that mismatched nucleotides could still be detected under these conditions. Figure 6B ).

[0144] This embodiment further designed spacer regions of different lengths and synthesized corresponding splitting crRNAs. The sequences of the synthesized splitting crRNAs are SEQ ID NO.402-SEQ ID NO.411. The results showed that when the length of the spacer region was 20nt-30nt, the synthesized splitting crRNAs could clearly detect SNPs. Figure 6C ).

[0145] This embodiment also introduces a single mismatched base at positions +14 to +21 in the original spacer region. When the SNP is located at position +17, Cas12a exhibits the highest sensitivity for identifying SNPs among 11 plant and human gene loci. Figure 7Cas12a successfully detected all these SNPs; however, at some sites, the full-length crRNA showed a high background signal. Ultimately, this embodiment found that at a concentration of 2.5 mM MgCl2, the fluorescence value change between perfectly matched (PM) targets and mismatched (MM) targets was the most significant. Figure 8A Furthermore, this embodiment also designs different Mg... 2+ Buffer solutions with concentration gradients were found to be effective when Mg 2+ SNPs can be clearly detected at concentrations ranging from 1.5 mM to 100 mM. Figure 8B ).

[0146] Example 6 This embodiment verifies the application of the detection system provided in Embodiment 1 in the microbial SNP detection scenario.

[0147] The detection of microbial SNPs can be used for: 1. Strain typing and tracing, tracking the transmission routes of pathogenic microorganisms; 2. Detecting SNP mutations related to functional traits such as drug resistance, rapidly determining the drug resistance spectrum of strains, and guiding precise clinical medication; 3. Analyzing the genetic diversity and evolutionary history of microbial populations, clarifying the phylogenetic relationships between different strains; 4. Achieving rapid identification and diagnosis of pathogenic microorganisms in samples, distinguishing between pathogenic and non-pathogenic strains, and improving diagnostic efficiency; 5. Detecting SNP sites in industrial microorganisms, and screening strains with excellent traits such as high yield and high stability.

[0148] Specifically, this embodiment uses Salmonella and Escherichia coli as examples to evaluate whether the SNIPER technology is suitable for the detection of functional microbial SNPs. Figure 11 ).

[0149] SNIPER synthesized mutant DNA fragments (sequences selected from SEQ ID NO. 23, NO. 25, NO. 30, NO. 39) of key functional SNPs in Salmonella (S80I) (associated with multidrug resistance in Salmonella) and key functional SNPs in Escherichia coli (G80A, S119A, E84P) (functional mutations in the LexA protein, an important factor in E. coli's DNA damage response, affecting the bacteria's radiation resistance), and simultaneously synthesized corresponding fission-type crRNAs (sequences selected from SEQ ID NO. 240, NO. 241, NO. 244, NO. 245, NO. 256, NO. 257, NO. 274, NO. 275) for detection. After reverse transcription and isothermal amplification, SNIPER successfully identified SNP mutations in target microbial strains with high specificity. Table 3 Figure 11 Sequences and information of four microbial functional SNP sites involved

[0150] Example 7 This embodiment verifies the functionality of the detection system provided in Embodiment 1 for detecting SNPs.

[0151] The detection system provided in Example 1 utilizes the synergistic effect of Cas12a and splitting crRNA-13 to identify targets carrying SNPs at the +17 site in the original spacer region. Figure 1 Next, this embodiment compares the differences in SNP detection capabilities between LbCas12a, high-fidelity LbCas12a variants (seCas12a and HF high-fidelity versions), and SNIPER, wherein the LbCas12a, seCas12a, and LbCas12a-HF sequences are selected from SEQ ID NO.390-SEQ ID NO.392.

[0152] The results showed that LbCas12a, seCas12a, and LbCas12a-HF failed to recognize SNPs at any of the four human DNA target sites, while SNIPER exhibited significantly enhanced sensitivity, showing a significant improvement over wild-type LbCas12a. Figure 9 , Figure 10A -B). Of the 18 functional SNPs in different species, SNIPER can sensitively distinguish 17 of the variations from the wild type. Figure 11 The 18 functional SNPs shown are illustrated in Table 4, highlighting their robustness and sensitivity in SNP identification. Figure 11 ).

[0153] Table 4 Figure 11 Sequences and information of 18 functional SNP sites involved

[0154] Figure 11 The specific sequences corresponding to each figure are as follows: picture OsPi2 middle ， The horizontal axis T corresponds to the high signal mutation (SEQ ID NO.22), and the horizontal axis A corresponds to the low signal wild-type base in the figure (its sequence information is that the 51st position of SEQ ID NO.22 is replaced with A). picture CaTsw middle ， The horizontal axis G corresponds to the high signal mutation (SEQ ID NO.32) in the figure, and the horizontal axis A corresponds to the low signal wild-type base in the figure (its sequence information is that the 50th position of SEQ ID NO.32 is replaced with A). picture EclexIn G8OA, the horizontal axis G corresponds to the high signal mutation in the figure (SEQ ID NO.25), and the horizontal axis A corresponds to the low signal wild-type base in the figure (its sequence information is that the 50th position of SEQ ID NO.25 is replaced with A). picture AtGun4 middle ， The horizontal axis T corresponds to the high signal mutation (SEQ ID NO.24) in the figure, and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.24 is replaced with G). picture MmGrin1 In the diagram, the horizontal axis T corresponds to the high signal mutation (SEQ ID NO.26), and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.26 is replaced with C). picture MmTuba1 In the diagram, the horizontal axis G corresponds to the high signal mutation (SEQ ID NO.27), and the horizontal axis A corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.27 is replaced with A). picture HsCXCR2 In the diagram, the horizontal axis T corresponds to the high signal mutation (SEQ ID NO.28), and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.28 is replaced with C). picture HsCRC In the diagram, the horizontal axis T corresponds to the high signal mutation (SEQ ID NO.29), and the horizontal axis G corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.29 is replaced with G). picture SeparC middle, x-axis T corresponds to the high-signal mutation in the figure (SEQ ID NO.30), and G on the horizontal axis corresponds to the low-signal wild-type base in the figure (its sequence information is that the 50th position of SEQ ID NO.30 is replaced with G). picture HsTLR9 In the diagram, the horizontal axis T corresponds to the high signal mutation (SEQ ID NO.33), and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.33 is replaced with C). picture AaCDC25A In the diagram, the horizontal axis C corresponds to the high signal mutation (SEQ ID NO.31), and the horizontal axis A corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.31 is replaced with A). picture HsIRF5In the diagram, the horizontal axis A corresponds to the high signal mutation (SEQ ID NO.34), and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 50th position of SEQ ID NO.34 is replaced with C). picture SIBBX18 In the diagram, the horizontal axis C corresponds to the high signal mutation (SEQ ID NO.36), and the horizontal axis T corresponds to the low signal wild-type base (its sequence information is that the 51st position of SEQ ID NO.36 is replaced with T). picture ASFV- In B646L, the horizontal axis G corresponds to the high signal mutation in the figure (SEQ ID NO.35), and the horizontal axis A corresponds to the low signal wild-type base in the figure (its sequence information is that the 50th position of SEQ ID NO.35 is replaced with A). picture HsHBB In the diagram, the horizontal axis G corresponds to the high signal mutation (SEQ ID NO.38), and the horizontal axis C corresponds to the low signal wild-type base (its sequence information is that the 100th position of SEQ ID NO.38 is replaced with C). picture OsJNBa0083M16.2 In the diagram, the horizontal axis G corresponds to the high signal mutation (SEQ ID NO.37), and the horizontal axis T corresponds to the low signal wild-type base (its sequence information is that the 97th position of SEQ ID NO.37 is replaced with T). picture Eclex- In S119A, the horizontal axis AGC corresponds to the high signal mutation in the figure (SEQ ID NO.39), and the horizontal axis CGA corresponds to the low signal wild-type sequence in the figure (its sequence information is that the 50th-52nd positions of SEQ ID NO.39 are replaced with CGA). picture Eclex In E84P, the horizontal axis CCG corresponds to the high signal mutation (SEQ ID NO.23) in the figure, and the horizontal axis TTC corresponds to the low signal wild-type sequence in the figure (its sequence information is that the 30th-32nd positions of SEQ ID NO.23 are replaced with TTC).

[0155] Example 8 This embodiment verifies the application of the detection system provided in Embodiment 1 in the scenario of virus SNP detection.

[0156] Specifically, this embodiment uses the RNA virus SARS-CoV-2 as an example to evaluate whether the SNIPER technology is suitable for the detection of functional viral SNPs (SNPs). Figure 12 ).

[0157] RNA fragments of SARS-CoV-2 variants containing key functional SNPs (L452R, T478K, N501Y, D614G, and P681R) (SEQ ID NO. 49-SEQ ID NO. 53) were synthesized, and corresponding splitting crRNAs (SEQ ID NO. 292-301) were synthesized simultaneously for detection.

[0158] The results are as follows Figure 13 As shown in the figure, the corresponding sequence information is as follows: L452R corresponds to the high-signal mutation CGG (SEQ ID NO.49) in the figure, and 452L corresponds to the low-signal wild-type sequence in the figure (its sequence information is that the 46th-48th positions of SEQ ID NO.49 are replaced with CUG). T478K corresponds to the high-signal mutation AAA (SEQ ID NO. 52) in the figure, and 478T corresponds to the low-signal wild-type sequence in the figure (its sequence information is that the 56th to 58th positions of SEQ ID NO. 52 are replaced with ACA). N501Y corresponds to the high-signal mutation UAC (SEQ ID NO. 53) in the figure, and 501 N corresponds to the low-signal wild-type sequence in the figure (its sequence information is that the 51st to 53rd positions of SEQ ID NO. 53 are replaced with AAU). D614G corresponds to the high-signal mutation GGU (SEQ ID NO.50) in the figure, and 614D corresponds to the low-signal wild-type sequence in the figure (its sequence information is that the 54th-56th positions of SEQ ID NO.50 are replaced with GAU). P681R corresponds to the high-signal mutation CGU (SEQ ID NO.51) in the figure, and 681 P corresponds to the low-signal wild-type sequence in the figure (its sequence information is that the 60th-62nd positions of SEQ ID NO.51 are replaced with CCU).

[0159] The RNA fragment sequence information of the SARS-CoV-2 variant strains was selected from: SEQ ID NO.49 SARS-COV-2 (L452R) target gene sequence; SEQ ID NO.50 SARS-COV-2 (D614G) target gene sequence; SEQ ID NO.51 SARS-COV-2 (P681R) target gene sequence; SEQ ID NO.52 SARS-COV-2 (T478K) target gene sequence; SEQ ID NO.53 SARS-COV-2 (N501Y) target gene sequence.

[0160] After reverse transcription and isothermal amplification, SNIPER successfully identified all target variants with high specificity. This result was validated by both a fully automated medical PCR analysis system and ultraviolet light transmission detection. Figure 13 , Figure 14 ).

[0161] Furthermore, this embodiment also utilizes SNIPER technology to distinguish wild-type SARS-CoV-2 (SEQ ID NO.47) and its B.1.1.7 (Alpha) (SEQ ID NO.48 SARS-CoV-B1.1.7) variant (the latter has approximately 40-80% higher transmissibility) through the S235F mutation, and achieves a high detection rate using commercially available lateral flow test strips. Figure 15 , Figure 16 ).

[0162] This embodiment also compares the performance of SNIPER with other RNA detection methods. Specifically, for the SHERLOCK detection method, please refer to [link to relevant documentation]. Gootenberg, JS et al. Nucleic acid detection with CRISPR-Cas13a / C2c2. Science 356, 438–442 (2017); For the Cas13a-occluder detection method, please refer to [link / reference]. Larsen, BB et al. RNA structure modulates Cas13 activity and enables mismatch detection. Nat. Biotechnol. (2025). Preprint at https: / / www.nature.com / articles / s41587-025- 02868-6. The results showed that the SHERLOCK and Cas13a-occluder methods could not distinguish between wild-type and the three SARS-CoV-2 variants. Figure 17 This embodiment also found that the SNIPER technology can achieve ultrasensitive detection of the P681R mutant strain, with a validation detection limit as low as 1.8 copies / mL. Figure 18 ).

[0163] Furthermore, this embodiment performed DNA sequence alignment of hepatitis B virus (HBV) subtypes and found three core SNP sites that can be used for HBV subtype identification (SEQ ID NO.54-SEQ ID NO.73). Moreover, the SNIPER technology described in this invention can achieve sensitive detection of hepatitis B virus (HBV). Figure 19 , Figure 20The HBV sequence information used in this embodiment is selected from the following sequences: SEQ ID NO.54 HBV-B1 target gene sequence, SEQ ID NO.55 HBV-B2 target gene sequence, SEQ ID NO.56 HBV-B3 target gene sequence, SEQ ID NO.57 HBV-B4 target gene sequence, SEQ ID NO.58 HBV-B6 target gene sequence, SEQ ID NO.59 HBV-B7 target gene sequence, SEQ ID NO.60 HBV-C1 target gene sequence, SEQ ID NO.61 HBV-C2 target gene sequence, SEQ ID NO.62 HBV-C3 target gene sequence, SEQ ID NO.63 HBV-C4 target gene sequence, SEQ ID NO.64 HBV-C5 target gene sequence, SEQ ID NO.65 HBV-C6 target gene sequence, SEQ ID NO.66 HBV-C11 target gene sequence, SEQ ID NO.67 HBV-C12 target gene sequence, SEQ ID NO.68 HBV-D1 target gene sequence, SEQ ID NO.69; HBV-D2 target gene sequence, SEQ ID NO.70; HBV-D3 target gene sequence, SEQ ID NO.71; HBV-D4 target gene sequence, SEQ ID NO.72; HBV-D5 target gene sequence, SEQ ID NO.73; HBV-D7 target gene sequence.

[0164] The above results demonstrate that SNIPER technology has high sensitivity and accuracy in detecting SNPs in various viral systems, and has significant application potential in the field of disease prevention and control. Furthermore, it can efficiently detect SNPs in both DNA and RNA without the need for laboratory equipment.

[0165] Example 9 This embodiment utilizes the detection system provided in Embodiment 1 to detect functional SNPs in a human population.

[0166] This embodiment successfully identified functional SNPs in four known human populations using SNIPER technology. ALDH2 *2 (c.1510 G>A), PNPLA3 (c.444 C>G and c.447 C>T) CYP2C9 *3 (c.1075 A>C) and CYP2C19 *2 (c.681 G>A)], the results of which are completely consistent with those of commercial standard products. Figure 22 ).

[0167] Figure 22 The sequence information involved is as follows: picture ALDH2In *2, c.1510 A corresponds to the high-signal mutation in the figure (SEQ ID NO.41), and c.1510 G corresponds to the low-signal wild-type base in the figure (its sequence information is that the 143rd position of SEQ ID NO.41 is replaced with G). picture PNPLA3 In the figure, c.444 G / c.447 T corresponds to the high signal mutation (corresponding to SEQ ID NO.40), and c.444 C / c.447 C corresponds to the low signal wild-type bases in the figure (their sequence information is that the 148th position of SEQ ID NO.40 is replaced with C, and the 151st position is replaced with C). picture CYP2C9 In *3, c.1075 C corresponds to the high-signal mutation in the figure (SEQ ID NO.42), and c.1075 A corresponds to the low-signal wild-type base in the figure (its sequence information is that the 154th position of SEQ ID NO.42 is replaced with A). picture CYP2C19 In *2, c.681 A corresponds to the high-signal mutation in the figure (SEQ ID NO.43), and c.681 G corresponds to the low-signal wild-type base in the figure (its sequence information is that the 154th position of SEQ ID NO.43 is replaced with G).

[0168] This embodiment uses SNIPER technology to detect human functional variants from non-invasive oral mucosal samples. The empirical value of "mean signal of negative sample ± 6 standard deviations" commonly used in digital PCR experiments is used as the reference value. The results show that for PNPLA3 mutation, SNIPER and Sanger sequencing results are completely consistent (100%, n=51). ALDH2 *2 (c.1510G>A) Genotyping: Cohen's κ = 0.957 (n = 48) Figure 23 This embodiment uses SNIPER and synthesizes classifying crRNAs (SEQ ID NO.284-287) to achieve [the desired result]. EGFR The detection sensitivity for the T790M (SEQ ID NO.44-45) mutation is 0.05%, and NPM1 The detection sensitivity for the TCTG insertion mutation (c.863_864insTCTG) (SEQ ID NO.46) was 0.5%. Figure 24 ).

[0169] Furthermore, this embodiment also used a lyophilized ribonucleoprotein complex containing Cas12a and cleaving crRNA for detection, and found that it was still detectable after being placed at 45°C for 72 hours, and was successfully detected. PNPLA3 Mutant alleles ( Figure 25A-B). These results indicate that the SNIPER testing system is suitable for non-invasive samples and has high stability, making it particularly suitable for home testing, preclinical applications, and testing scenarios in remote areas.

[0170] Example 10 This embodiment applies the detection system provided in Example 1 to the agricultural field, specifically for crop germplasm identification and phenotypic prediction.

[0171] The above embodiments do not involve the need to use a small number of core SNPs to predict the phenotype of a large sample population. Figure 26 This example selects 13 core SNP sites annotated through genome-wide association studies (GWAS), as shown in Table 5 below.

[0172] Table 5 Information on 13 core SNP sites

[0173] The aforementioned loci may be associated with stress resistance, yield, and quality traits, and are used for germplasm identification. The results of this embodiment demonstrate the variation patterns of these SNPs among germplasms, enabling SNIPER to clearly distinguish 25 rice germplasms in the rice mini core germplasm bank (see Table 6). Figure 27 ).

[0174] Table 6 Information on 25 Rice Varieties

[0175] This embodiment also uses SNIPER to detect rice. OsUGT75A The SNP sites of the gene (the gene that regulates coleoptile length) were analyzed, and the results were consistent with the corresponding phenotype. Figure 28 ).in, OsUGT75A The horizontal axis A corresponds to the high signal wild-type base (SEQ ID NO.87) in the figure, and G corresponds to the low signal mutation in the figure (its sequence information is that the 158th position of SEQ ID NO.87 is replaced with G).

[0176] This embodiment Figure 29 SNIPER has been found to be effective in distinguishing different alleles, and this technology can be applied to complex polyploid genomes such as sugarcane (SEQ ID NO. 88-SEQ ID NO. 90). To further verify the accuracy and reliability of the results, it is evident that the SNIPER provided by this invention can be effectively used to distinguish different alleles.

[0177] The results indicate that SNIPER has great potential in agricultural applications, including phenotypic prediction, variety identification, and marker-assisted selection.

[0178] The above examples demonstrate that SNIPER exhibits high specificity for SNP detection in both DNA and RNA targets, combining broad applicability with high sensitivity. This technology requires only basic laboratory equipment and technical expertise, making it a powerful tool applicable to a variety of scenarios, including medical applications (especially clinical testing, POCT) and agriculture.

[0179] The specific sequence information used in the above embodiments is as follows: SEQ ID NO.1 HsDNMT1 target gene sequence TAAGACATGGGTCCAGCAAAGCATTCCTCATGCTGCTGCCTGCACTTACCCTCTAGGAAGGCCAGCCTGGAGGAGCCACTTCTGCCCTTCAGGCAAATGATAACTGAGCACCTGTCTGTGCGGGTGCTAGGTGGAAAGGTCCAGAGATGG CAGTGGCCTGGTCCCTGCCTGGGGCACCTTCTCTCCACCAACAGACACTTACCCAGCTGGTTAGGCCTGTTCTCACCCCGCAGTCTGTAGTTAGCACCCAGGCTGGGAACAGCCTGTGTTCCTCTCCCTTTCCTTGGTACCTTCTCT SEQ ID NO.2 HsVEGFA target gene sequence CTCCCCTGGGAAGCATCCCTGGACACTTCCCAAAGGACCCCAGTCACTCCAGCCTGTTGGCTGCCGCTCACTTTGATGTCTGCAGGCCAGATGAGGGCTCCAGATGGCACATTGTCAGAGGGACACACTGTGGCCCCTGTGCCCAGCCCT GGGCTCTCTGTACATGAAGCAACTCCAGTCCCAAATATGTAGCTGTTTGGGAGGTCAGAAATAGGGGGTCCAGGAGCAAACTCCCCCCACCCCCTTTCCAAAGCCCATTCCCTCTTTAGCCAGAGCCGGGGTGTGCAGACGGCAGTCACT SEQ ID NO.3 HsRNF2 target gene sequence TTTATTTTTTTTATTTTTTGAGATGGAGTCTTGCTGTGTCACCCAGGCCAGAGTGCAGTGGCACCATCTTGGCTCACTGCAACCTCTGCCTCCTGGGTTTCCAGCGATTCTCCTGCTTTAGCGTCTTGAGTAGCTGGGAGTACAGGTGCACGCCACCATGCCCAGCTAATTTTTGTATTTTTTAGTAGAGATGGGGTTTCACCATATTGGCGAGGCTGGTCTCAACTCCTGACCTTGTGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGAGATTACAGGTGTGATCCACCACACCTGGCC SEQ ID NO.4 HsHEK4 Target gene sequence CTACTGAGATCCTGTCCTTAGTTTACTGCGTGGAGACAGACCACAAGCAGGTAAACAAGCAAATATGTAAGTCCCAGGTCAGATAAATTTTAGGAAGTGCTGTTTTCCAGTGGTTCAATGGTCATCCCAGGGCAGAGGTGGGGAGACCTGCTGAGGGCGGCTTCTCCCTCAGTCAGTCCATGCCTGCAGGGTCTGGAACCCAGGTAGCCAGAGACCCGCTGGTCTTCTTTCCCCTCCCCTGCCCTCCCCTCCCTTCAAGATGGCTGACAAAGGCCGGGCTGGGTGGAAGGAAGGGAGGAA SEQ ID NO.5 HsHEK3 Target gene sequence CCACTAAAGTGCTCTGGTGAATTTCTTTTCCAACACCCTAATGCTGAGGCTTTCCAGCCATCCACCCATCCATCCATCACTCATTTCCTTGGCCATCCACCACCCATTAAATGTGAGGATACTATGGCCCAGGCACTGAGCCTAGTGCTGAGGATACAGGGCTGAATCAGACCCAGCCTTGATCCTCAGGGTGGTCAGGGCCAGTGCAAAAACAAGCAAGGAGATAAAATATGCTGGTACTAGGCTTTCCTGCTGCATTCTCATTGCAGACTTGCGTGAAAGGGAGCTTACAGCAGGAGT SEQ ID NO.6 HsHEK2 Target gene sequence GGCTTCTCACTTGGGAAGTTCAATCCTGTAATCACCTGCTTGAAAGACTAAACAAACCTTAACTGTCATGTGCTTGATTCAAGTACCACCAGTTTTATGAAGAAAAATGATATCCATTATTAGTGGCTGAATGCTGAGGGGAGCAGTGGGGGAGAGGGTGTTGCTGTGGATTAATGCATCATCTCTGCTTTCAGCTGTGGGCCAGGTTTTGAGTCTAGCTCTGACTGAATCCTGCCATACACTTTGAATAGCATAATGAAGTATTGTTATTGCCTCAGTCTAGTCATTACATCAGGGAAA SEQ ID NO.7 HsFANCF Target gene sequence TAAGTTGCCCAGAGTCAAGGAACACGGATAAAGACGCTGGGAGATTGACATGCATTTCGACCAATAGCATTGCAGAGAGGCGTATCATTTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTCAGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCATTCGCACGGCTCTGGAGCGGCGGCTGCACAACCAGTGGAGGCAAGAGGGCGGCTTTGGGCGGGGTCCAGTTCCGGGATTAGCGAACTTCCAGGCCCTCGGTCACTGTGACGTCCTGCTCTCTCTGCGCCTGCTGGAGAACCGGGCCCTCGGGGATGCAGCTCGTTACCACCTGGTGCAGC SEQ ID NO.8 OsGAPDH Target gene sequence AACTTGATATTGATATTACAGCTTAGATGAAAGTAACTTGTTTATGGTAGCGAGCGTGGAACTGATGTTTGATCCTGTGCAATTTGAAAGGAACCCTGACGAGATTCCGTGGGCTGAGGCTGGTGCTGAGTATGTCGTGGAGTCCACCGGTGTCTTCACTGACAAGGAGAAGGCTGCTGCTCACTTGAAGGTATTATCACTGTTGCTTTTCATTCCAACAAATTGGCATATTGTTACATATTGTTCCCATGGGATTGCATACAAGCAAGTAAATAATGCTATGGTTATTTTTAAGAATTA SEQ ID NO.9 OsAAT Target gene sequence CTGGATTTCCAAGGTACCAATTTCAATTTTCCATGGAGGAACTTGTTCTTATGAAGAAGATTAATTGCAAGATTGTTTGCACCTGTCTGACTTGATCAAGGATGTGTTTTCGTGTTACAGGGCTGTTAGAAGATCTCGGTTCAGCTCCTTCAGGTGCAATTGTACTGCTTCATGCTTGTGCCCACAACCCTACTGGAGTAGACCCAACTTTGGACCAGTGGGAACAGATCAGGCAGTTGATGAGATCAAAAGCATTGCTGCCATTCTTTGATAGCGCTTATCAGGTAAATTATTGGCATC SEQ ID NO.10 OsDEP1 Target gene sequence CTTGGCCCTCTTTGGGCGTGGCGCCATGGCTGTACTACCTTTGTCGTTGTTTGGTTGGGCTCCTCGTTGGAGAAAAGAAGAGCGTGGGCATGGACAACTGACCTGAGTGGCCTTGTCAGGGAGAGCCATAGCAGTGGACGTGTCTATCTCCGCCATTGCTTCGTCGACACTGGACGTGCAGACGGCATGGCCATGAGGGCTTTGCACGATGGGTGGTGCCGTGTTGGTGTTATGGGCTGCCACCATGGTTTGAGGCTTTTGATGTTGCTAGATTTTGTGTTTAACGAGGGAGGGAAGAAT SEQ ID NO.11 OsEPSPS Target gene sequence GGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACGTATGTTTTTTTTTTTAATGTTTATGAAAATATGTATGGAATTCATGGGGTATGTTTTATGACCTTTTTCTTTACCATCAGTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAG SEQ ID NO.12 OsACC1 Target gene sequence GGTGATCTTATAGCTAGGCTGGATCTTGATGACCCTTCTGCTGTTAAGAGAGCTGAGCCGTTCGAAGATACTTTTCCACAAATGGGTCTCCCTATTGCTGCTTCTGGCCAAGTTCACAAATTATGTGCTGCAAGTCTGAATGCTTGTCGAATGATCCTTGCGGGGTATGAGCATGATATTGACAAGGTAAACATCATGTCCTCTTGTTTTTTCTTTTGTTTATCATGCATTCTTATGTTCATCATGTCCTCTGGCAAATCTAGATTCCGCTGTCGTTTCACACAGATTTTTCTCATTCTC SEQ ID NO.13 OsCDC48 Target gene sequence GCTACAAACCGGCCAAACAGTATTGATCCTGCTCTCAGAAGGTTTGGTAGGTTCGATCGGGAGATTGACATTGGTGTTCCTGATGAAGTTGGGCGGCTTGAAGTTCTTCGGATTCACACCAAAAACATGAAGCTGGCTGAAGATGTGAGTTATCACCACTACAGTCTAGATGCAAAATATTCTTTATTGGTTGCATTTTTTTTTAATAGTTATTACATTGTTGTCGCAGGTTGACCTGGAACACATTGCCAAAGACACTCATGGGTATGTGGGTGCTGATCTTGCTGCTCTTTGCACTGA SEQ ID NO.14 OsALS Target gene sequence ACTGGTGTTGGGCAGCACCAGATGTGGGCGGCACAATATTACACCTACAAGCGGCCACGGCAGTGGCTGTCTTCGGCTGGTCTGGGCGCAATGGGATTTGGGCTGCCTGCTGCAGCTGGTGCTTCTGTGGCTAACCCAGGTGTCACAGTTGTTGATAaTGATGGGGATGGTAGCTTCCTCATGAACATTCAGGAGCTGGCATTGATCCGCATTGAGAACCTCCCTGTGAAGGTGATGGTGTTGAACAACCAACATTTGGGTATGGTGGTGCAATGGGAGGATAGGTTTTACAAGGCGAAT SEQ ID NO.15 OsBADH2 Target gene sequence AATGCAAATTTGGAGGGAAGAAGTTTTTGGTCCAGTGCTCTGTGTGAAAGAATTTAGCACTGAAGAAGAAGCCATTGAATTGGCCAACGATACTCAGTGAGTTTTTTTTTTAATACAGTTCATTGTCCTGTTCAATCTTGCAGCATATGTATATACTCTGTGGCATATGAACTTATTCTGCTACTACTACTTTTGATAGTTATGGTCTGGCTGGTGCTGTGCTTTCCGGTGACCGCGAGCGATGCCAGAGATTAACTGAGGTATATCCAAGTGAAGGGGGTTGGCATTGTTTGATTCATA SEQ ID NO.16 HPT Target gene sequence ATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTT SEQ ID NO.17 Kana Target gene sequence ATGGGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGACGAGGCAGCGCG SEQ ID NO.18 ASFV target gene sequence GAATTCTGTGAACAAAAGTTATGGGAAACCCGACCCCGAACCCACTTTGAGTCAAATCGAAGAAACACATTTGGTTCATTTTAATGCGCATTTTAAGCCTTATGTTCCAGTAGGGTTTGAATACAATAAAGTACGCCCGCATACGGGTACCCCCACCTTGGGAAACAAGCTTACCTTTGGTATTCCCCAGTACGGAGACT SEQ ID NO.19 OsGAPDH Target gene sequence with PAM TCAGGTAACTTGATATTGATATTACAGCTTAGATGAAAGTAACTTGTTTATGGTAGCGAGCGTGGAACTGATGTTTGATCCTGTGCAATTTGAAAGGAACCCTGACGAGATTCCGTGGGCTGAGGCTGGTGCTGAGTTTGTCGTGGAGTCCACCGGTGTCTTCACTGACAAGGAGAAGGCTGCTGCTCACTTGAAGGTATTATCACTGTTGCTTTTCATTCCAACAAATTGGCATATTGTTACATATTGTTCCCATGGGATTGCATACAAGCAAGTAAATAATGCTATGGTTATTTTTAA SEQ ID NO.20 OsCDC48 Target gene sequence with PAM GGTGCTACAAACCGGCCAAACAGTATTGATCCTGCTCTCAGAAGGTTTGGTAGGTTCGATCGGGAGATTGACATTGGTGTTCCTGATGAAGTTGGGCGGCTTGAAGTTCTTCGGATTCACACCAAAAACATGAAGtttCTGAAGATGTGAGTTATCACCACTACAGTCTAGATGCAAAATATTCTTTATTGGTTGCATTTTTTTTTAATAGTTATTACATTGTTGTCGCAGGTTGACCTGGAACACATTGCCAAAGACACTCATGGGTATGTGGGTGCTGATCTTGCTGCTCTTTGCACT SEQ ID NO.21 OsDEP1 Target gene sequence with PAM AGCTTGGCCCTCTTTGGGCGTGGCGCCATGGCTGTACTACCTTTGTCGTTGTTTGGTTGGGCTCCTCGTTGGAGAAAAGAAGAGCGTGGGCATGGACAACTGACCTGAGTGGCCTTGTCAGGGAGAGCCATAGCAGtttACGTGTCTATCTCCGCCATTGCTTCGTCGACACTGGACGTGCAGACGGCATGGCCATGAGGGCTTTGCACGATGGGTGGTGCCGTGTTGGTGTTATGGGCTGCCACCATGGTTTGAGGCTTTTGATGTTGCTAGATTTTGTGTTTAACGAGGGAGGGAAGA SEQ ID NO.22 OsPi2 Target gene sequence CAGGAAAACACTCGCCACATAGCATTCCATGGGAGTATGTCCTGCAAAACTGGATTGGATTGGAGCATTATTCGATCATTAGCTATTTTTGGTGACAGAC SEQ ID NO.23 EcLex E84P target gene sequence AGGAAGAGGAAGAAGGGTTGCCGCTGGTACCGCGTGTGGCTGCCGGTCCGCCACTTCTGGCGCAACAGCATATTGAAGGTCATTATCAGGTCGATCCTTC SEQ ID NO.24 AtGun4 Target gene sequence CGCCACAACTGCCGAAACCGCGACCATATTCGACGTTCTGGAGAACCATTTCGTCAATCAAAACTTCAGACAAGCCGACGAGGAGACACGGAGATTACTC SEQ ID NO.25 EcLex G80A target gene sequence ACGCGGGATTCGTCTGTTGCAGGAAGAGGAAGAAGGGTTGCCGCTGGTAGCCCGTGTGGCTGCCGGTGAACCACTTCTGGCGCAACAGCATATTGAAGGT SEQ ID NO.26 MmGrin1 Target gene sequence GATCTTCCTCATTTTCATCGAGATCGCCTACAAGCGACACAAGGATGCCTGTAGGAAGCAGATGCAGCTGGCTTTTGCAGCCGTGAACGTGTGGAGGAAG SEQ ID NO.27 MmTuba1 Target gene sequence CAGCCTGATGGCCAGATGCCAAGTGACAAGACCATTGGGGGAGGAGATGGCTCCTTCAACACCTTCTTCAGTGAGACAGGAGCTGGCAAGCATGTGCCCC SEQ ID NO.28 HsCXCR2 Target gene sequence TTCTTCTTGGTCTCAGTGTCAATGCAGCCCCCATTGTGGTCACAGGAAGTAGAGGAGGCCACGTTCTTACTAGTTTCCCTTGCATGGTTTAGAAAGCTTG SEQ ID NO.29 HsCRC Target gene sequence GTAGCCAGAGTTAATACCCTCATCGTCCTTTGAGCTCAGCAGATGAAAGTCACTGAGAAAAGTACAAAGAATTTTTATGTGCTATTGACTTTATTTTATT SEQ ID NO.30 SeparC Target gene sequence GCCCGTACCGTTGGTGACGTACTGGGTAAGTATCACCCGCATGGCGACATCGCCTGCTATGAAGCCATGGTGCTGATGGCGCAGCCGTTCTCTTACCGTT SEQ ID NO.31 AaCDC25A Target gene sequence CAGCAGCTCAAGGCCAGGACAGCCGGCTGGACAGGAGCCTCAGTGCTCCCGAATTGGGCCCTGTGTTTGTGAGGCAAGGCCCAGTCCTTGGGTCATCCAG SEQ ID NO.32 CaTsw Target gene sequence GATTAGTTGAAGATATGGAGGAATTTGAGAATCGCGAAAAACCCAATCTGGAAAAAACTAAAACAGTCAACTTGGGAGATCACGATAAAAATCAAAGAGA SEQ ID NO.33 HsTLR9 Target gene sequence GCATGCCAAAGGGCTGGCTGTTGTAGCTGAGGTCCAGGGCCTCCAGTCGTGGTAGCTCCGTGAATGAGTGCTCGTGGTAGAGGTCCAGCTTATTGTGGGA SEQ ID NO.34 HsIRF5 Target gene sequence TGCTATTTGATAAATGATCACAATGTGTAAAACACTGTAGTTACAAGATATCATTTAATCCGCCTAACAACCTTGCCAAGTATTAATAAAACCCGTTTTA SEQ ID NO.35 ASFV B646L target gene sequence CATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCGATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAA SEQ ID NO.36 SIBBX18 SNP-328 target gene sequence AGTTCTCTGTGTGTGCAATGTGACATGATGGTATACACGTTGGCGGTAAACGAACACACAGTAGATATCTCCTGTTGAGGCAGAAAGTTGAGTTTCCAGG SEQ ID NO.37 OsJNBa0083M16.2 Target gene sequence AAAATCTACTCTGCCGTAGACTGTATAATCTCCCGTGTTTGTTGAACTAACTACCATATCTCAAATCTGAATGTATAATCTGCCAGTGTCAACTGCGTGCTTCCATTTCAACCCGAGGTGTAACATGTGCACACAAAAGCTCTTCTCTGAATTCCGCAAATAAACTCTGCTTAATTTGCAGATGAGTTCGTAGTTGTCAC SEQ ID NO.38 HsHBB Target gene sequence GAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACGCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGA SEQ ID NO.39 EcLex -S119A target gene sequence TTCCTTATTCAAGCCGAATGCTGATTTCCTGCTGCGCGTCAGCGGGATGAGCATGAAAGATATCGGCATTATGGATGGTGACTTGCTGGCAGTGCATAAA SEQ ID NO.40 HsPNPLA3 Target gene sequence ACCTACTCTGTGCAAAGGGCATTTTCAAGTTTGTTGCCCTGCTCACTTGGAGAAAGCTTATGAAGGATCAGGAAAATTAAAAGGGTGCTCTCGCCTATAACTTCTCTCTCCTTTGCTTTCACAGGCCTTGGTATGTTCCTGCTTCATGCCTTTCTACAGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTGGTAAGTCGGCTTTCTCTGCTAGCGCTGAGTCCTGGGGGCCTCTGAAGTGTGCTCACACATCTCCTGCCTGCAGGGCACTGGTGTCGGGCACCTCAGGGTCTGTCCCATGG SEQ ID NO.41 HsALDH2*2 Target gene sequence CCCCAAGAGTGATTTCTGCAATCTCGTTTCAAATTACAGGGTCAACTGCTATGATGTGTTTGGAGCCCAGTCACCCTTTGGTGGCTACAAGATGTCGGGGAGTGGCCGGGAGTTGGGCGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTGGGACCTGCTGGGGGCTCAGGGCCTGTTGGGGCTTGAGGGTCTGCTGGTGGCTCGGAGCCTGCTGGGGGATTGGGGTCTGTTGGGGGCTCGGGGCCTGCCAGAGGTTCAGGACCTGCCGGGGACTCAGGGCCTGCTGG SEQ ID NO.42 HsCYP2C9*3 Target gene sequence TTTTCTCCTTTTCCATCAGTTTTTACTTGTGTCTTATCAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACCTTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCAACTCCATGTTTTCGAAGTCCCCAAATTCATAGTATCATTTTTAAACC SEQ ID NO.43 HsCYP2C19*2 Target gene sequence ATATACAATATATTTTATTTATATTTATAGTTTTAAATTACAACCAGAGCTTGGCATATTGTATCTATACCTTTATTAAATGCTTTTAATTTAATAAATTATTGTTTTCTCTTAGATATGCAATAATTTTCCCACTATCATTGATTATTTCCCAGGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCTCGGGACTTTATTGATTGCTTCCTGATCAAAATGGAGAAGGTAAAATG SEQ ID NO.44 HsEGFR Target gene sequence CTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAG SEQ ID NO.45 HsEGFR (T790M) Target gene sequence CTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAG SEQ ID NO.46 HsNPM1 Target gene sequence TTTTTTTTTTAAGATTTAACTTTCGGGGGGTAAAATGAAAAATAGATGTTGAATTTTGCAAAGAGCCTTTTAATTTTTTGATGTTTAAAAAGGGTTGGGGTCCCTTACCCCCTTTTTTTTTTTTTTTTCCAGGCTATTCAAGATCTCTGTCTGGCAGTGGAGGAAGTCTCTTTAAGAAAATAGTTTAAACAATTTGTTAAAAAATTTTCCGTCTTATTTCATTTCTGTAACAGTTGATATCTGGCTGTCCTTTTTATAATGCAGAGTGAGAACTTTCCCTACCGTGTTTGATAAATGTTGTCCA SEQ ID NO.47 SARS-COV-WT target gene sequence UUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGC SEQ ID NO.48 SARS-COV-B1.1.7 target gene sequence UUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUUUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGC SEQ ID NO.49 SARS-CoV-2 (L452R) target gene sequence AAUUCUAACAAUCUUGAUUCUAAGGUUGGUGGUAAUUAUAAUUACCGGUAUAGAUUGUUUAGGAAGUCUAAUCUCAAACCUUUUG SEQ ID NO.50 SARS-CoV-2 (D614G) target gene sequence GUGUUAUAACACCAGGAACAAAUACUUCUAACCAGGUUGCUGUUCUUUAUCAGGGUGUUAACUGCACAGAAGUCCCUGUUGCUAUUCAUGCAGAUCAACUUACUCCUACUUGGCGUGUUU SEQ ID NO.51 SARS-CoV-2 (P681R) target gene sequence AGUGUGACAUACCCAUUGGUGCAGGUAUAUGCGCUAGUUAUCAGACUCAGACUAAUUCUCGUCGGCGGGCACGUAGUGUAGCUAGUCAAUCCAUCAUUGCCUACACUAUGUCACUUGGG SEQ ID NO.52 SARS-CoV-2 (T478K) target gene sequence UAAUCUCAAACCUUUUGAGAGAGAUAUUUCAACUGAAAUCUAUCAGGCCGGUAGCAAACCUUGUAAUGGUUGAAGGUUUUAAUUGUUACUUUCCUUUA SEQ ID NO.53 SARS-CoV-2 (N501Y) target gene sequence AAGGUUUUAAUUGUUACUUUCCUUUACAAUCAUAUGGUUUCCAACCCACUUACGGUGUUGGUUACCAACCAUACAGAGUAGUAGUACUUUCUUUUGAACU SEQ ID NO.54 HBV-B1 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTGCCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGACCATGCAAGACCTGCACAACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.55 HBV - B2 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTGCCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGACCATGCAAAACCTGCACAACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.56 HBV - B3 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAACCCTGTTCAGAACACTGCCTCTCCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTGCCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGAAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACTTGTTGTCCTCCGATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACCGGACCCTGCAAAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.57 HBV-B4 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAGCCCTGCTCAGAATACTGCCTCTGCCATATCGTCAACCTTCTTGAAGACTGGGGACCCTGTACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACTTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACCGGACCATGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.58 HBV - B6 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAACCCTGCTCAGAGTACTGCCTCTGCCATATCGTCAACCTTATCGACGACTGGGGACCCTGCACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGAAATGCCCGGGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTGTCCTCCAATCTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTTTGCATCCTGCTGCTATGCCTCACCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACCGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.59 HBV-B7 target gene sequence TTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAACCCTGTTCAGACCACTGCCTCTTCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTGCCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACTTGTTGTCCTCCGATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACCGGACCATGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.60 HBV-C1 target gene sequence TTCCTGCTGGTGGCTCAAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAATATGGAGAGCACCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTTGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAATTGCACTTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.61 HBV-C2 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAACTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.62 HBV-C3 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCTCATTTCGTCAATCTTCTCGAGGATTGGGGACCCTGTAACGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCGTGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGTAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.63 HBV-C4 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGAATACTGTCTCTCACATCTCATCAATCTTCACGAAGACTGGGGACCCTGCATCGAACATGGAGAGCACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCTCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCTCCCGCGTGTCTTGGCCAAAATTCGCAGTCCCAAACCTCCAATCACTCACCAACCTCTTGTCCTCCAACCTGTCCTGGCTATCGTTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGATCAACGACCACCAGCACGGGACCTTGCAGAACCTGCACGATCACTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.64 HBV-C5 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGCAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAATATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGAACATCCACAACAAGCACGGGACCATGCAAGACCTGCACGACTCCTGCTCAAGGCACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAAATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCATGGCTCAGTTTA SEQ ID NO.65 HBV-C6 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACTATTGCCTCTCCCATCTCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAACATGGAGAGCACAACATCCGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGTGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGAAGCACCCATGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGATTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCTTGCAAGACCTGCACAATTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.66 HBV-C11 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGATTACTGCCTCTCCCATATCGTCAATCTTCTTGAGGACTGGGGACCCTGCACCGAACATGGACAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGTGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGAAGCTCCCATGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGATTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCACCACGGGACCATGCAGGACCTGCACAATTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.67 HBV-C12 target gene sequence TTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCTCGAGGATTGGGGACCTTGCACCGAACATGGAGAGCACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGTGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGAAGCACCCGCGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGATTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACAACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.68 HBV-D1 target gene sequence TCCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGTTCCGACTACTGTCTCTCACATATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCTTCAACCACCAGCACGGGACCATGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.69 HBV-D2 target gene sequence TCCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTCTGTCCTCTAATTCCAGGATCTTCAACCACCAGCGTGGGACCATGCAGAACCTGCACGACTACTGTTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.70 HBV-D3 target gene sequence TCCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCACTTATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAGCACGGGACCATGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.71 HBV-D4 target gene sequence TCCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCACATATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGACCCTGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCGTCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCAGTTTA SEQ ID NO.72 HBV-D5 target gene sequence TCCCTGCTGGTGGCTCAACTTCAGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATTGTCAATCTTCTCGAAGATTGGGGACCCTGCACCGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAGCTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCTTCGACCACCAGCACGGGACCATGCAGAACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.73 HBV-D7 target gene sequence TCCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCTTCAACCACCAGCACGGGACCATGCAGAACCTGCACGACTCCTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCATCATGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTA SEQ ID NO.74 Rice-chr1:2271427 target gene sequence CCCAGCTACGAACTGTGGGGGTGATGAGAGGAAATATCTTGGCCTTCTCGTACTTGTCAGGGAGCTCCTCGAGCTGATGCAGTCAGGGAAGCTGGAGCCGG SEQ ID NO.75 Rice-chr2:24573278 target gene sequence AATCATTACCGAAGCCAAGAGCGACGCCGCCACCACCGGCATCACTGATTAGCCGTTGGACACAATGGCCGCGTAAGCCATGCGAGCTATCAGAATGCCTT SEQ ID NO.76 Rice-chr3:17741591 target gene sequence GTATTTTGTTATAACATTCATGCTGAGGAAACAAAGCGTCTTCTATTACAGAGTTGCAGTTCGTCTGAATCGGTTAAATTAAAACCTGCCCTGAATTGGCG SEQ ID NO.77 Rice-chr3:29589636 target gene sequence AATGATTATGGATCCTTTTATCTAAGAGGGGATTGTTGAACACCAACCAAACATGTAAACTACCTACTTCTCAGTTTACTTATCCAAATTAGCAGCCCAAA SEQ ID NO.78 Rice-chr4:30726420 target gene sequence TAGTTTCTTCTGATTGGCCACATTCCGTAATTTTGTGATCCCCATTGTTTGCAGGACTGTTGAATTCAATGTCCAAGCTGATTGAAGGTAAACTTCTCTGG SEQ ID NO.79 Rice-chr4:31079938 target gene sequence TTGAGACCGGTGATAGTATCCAGCAGCCAATGAGTCCACCACCATTGTGGACACTGCAGCAGACATTGAAACAAGGCCCGCAAAGGGGAAGGGATTCCTAT SEQ ID NO.80 Rice-chr5:5361894 target gene sequence CAGTGGCCACGAGTCAAGATGCTGTATATATGTATGGATGAGTCATCAGTGTGATGCATGATCTCACATCTCGTCACTGATGATCTCCAGCTTGAGCTTGC SEQ ID NO.81 Rice-chr6:1768724 target gene sequence ACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGGATCCCAGCAAGGACAAGTACATCACCGCCAAGTACGACGCAACCACGGTAAGAAC SEQ ID NO.82 Rice-chr6:1768006 target gene sequence GGCTCCTAGGATCCTAAACCTCAACAACAACCCATACTTCAAAGGAACTTCTGGTGAGTTACAATTGATCTCAAGATCTTATAACTTTCTTCGAAGGAATC SEQ ID NO.83 Rice-chr7:28305040 target gene sequence GTTGTTGCTTAAGGAGAATGATGGTGACAAAAAATGTATGTTAGAGATCAAAGTCTTGGAGTTCTATTATAAAGCCCCGTTCGGTAGTGAGCTAGTAGTGG SEQ ID NO.84 Rice-chr8:20897662 target gene sequence ACATCCAGACCAGAAAGCTGTTGTCCCAACAACTGTCTGCAATATCACGTCATGCACAGTCATTATTTGAATGCTACTCCCTTCATCCTAAAATAAACCAA SEQ ID NO.85 Rice-chr8:26726297 target gene sequence AATTAACCAACTAATTAAAGTTCGGTAGTAATCCACACTGCTAAATTCTGGCCGTCATCGATCCGGCCATCGTCGTCGTCTTCTTCATCAGTAGCGAGAAT SEQ ID NO.86 Rice-chr12:13698063 target gene sequence TCTTGCTATTTCTTGAGTTCCCTTGCCCCCCAACTTGCCATGCTGAAACATCTGATCAGAGGCGTGGCAAGGCTGTTCTTATGGAAGGTGACAGTGAACAG SEQ ID NO.87 OsUGT75A target gene sequence CATTGCATGTAGCCTGCGTCGTCGTCTTGCTCGAAGAGGTCGCCGCCGCCGTCGGCCTCCGTGTCGGTGACCATGGGTCCAACCGGGATGAGGTCAATGCCGGGGACGGACGCGAGCGCCTTCGGCTCGAGCGCGTCGAAGGTGTTCACAAGCACCCACGGCTTCGCGCCACTCTGGCCATGGGCATGGCACGACGCGGCGGCGGTGGCCTCGACGACGTGGAACATCTCCCGGAAGG SEQ ID NO.88 ShSPS Target gene sequence SEQ ID NO.89 ShSUT Target gene sequence CCGGAACAATGCCACCACATCAATCTGCTACAACGATGCTGCACTACAGAAGTGTTCCTCAAACATCAGTTGCTCTCATGAATTCTTGGGAGTCTTCAATGAGATCATGGGAGGGAAGATGTGGCGGGTGAACATGCTGATAGTCATGGACTCGGTCTTGGCGGGAGTCATAGTTGTTATCGGAGCTTATGCTCGGCGGTATCGGTACCACTCCTTAACGCGCTTCATCTTCCTTGGAGCTACAACTTTGTTCCTCCCCATCATCTCCTATGTTGTCTCCACCATC SEQ ID NO.90 ShGA1 Target gene sequence GCACTCCCACTTCCCAATCCTGGATCCAAATCCCAAGCTATCCCAGAACCGAAACCGAGGCGCGCAAGCAATTATTATCTGGCTAGCTAGGCCTGTAGCTCGGAGATCATGAAGCGCGAGTACCAAGACGCCGGCGGGAGCGGCGGCGACATGGGCTCCTCCAAGGACAAGATGATGGTGGCGGCGGCGGGGGCAGGAGAGCAGGAGGAGGAGATGGACGAGATGCTGGCCGCGCTCGGGTACAAGGTGCGTTCGTCGGATATGGCGGACGTCGCACAGAAGCTGGAGCAGCTCGAGATGGCCATGGGGATGGGCGGCGTTGGCGGCGCCGGCGCTACCGCTGATGACGGTTTCATCTCCCACCTCGCCACGGACACCGTGCACTACAATCCCTCCGACCTGTCGTCCTGGGTCGAGAGCATGCTGTCCGAGCTCAACGCGCCCCCGCCGCCGCTCCCGCCGGCGACGCCGCCGGCCCCGAGGCTCGCGTCCACCTCGTCCACCGTCACAAGTGGCGCCGCCGCCGGTGCTGGCTACTTCGATCTCCCGCCCGCCGTCGACTCGTCCAGCAGCACCTACGCCCTGAAGCCGATCCCCTCGCCGGTGGTGGCGTCGGCCGACCCGTCGTCCACGGACTCGACGCGGGAGCCCAAGCGGATGCGGACTGGCGGCGGCAGCACGTCGTCTTCCTCTTCCTCGTCGTCATCCATGGATGGCGGCCGCACTAGGAGCTCCGTGGTCGAAGCTGCCCCGCCCGCGACGCAAGCGTCCGCGGCGGCCAACGGGCCCGCGGTGCCGGTGGTGGTGATGGACACGCAGGAGGCTGGGATCCGGCTCGTGCACGCGCTGCTGGCGTGCGCGGAGGCCGTGCAGCAGGAGAACTTCTCT SEQ ID NO.91 - 114 HsDNMT1 Single - base mismatch sequence

[0180] SEQ ID NO.115-138 HsVEGFA Single base mismatch sequence

[0181] SEQ ID NO.139-162 OsEPSPS Single base mismatch sequence

[0182] SEQ ID NO.163-186 OsDEP1 Single base mismatch sequence

[0183] SEQ ID NO.187-SEQ ID NO.378 / SEQ ID NO.396-SEQ ID NO.411 crRNA and splitting crRNA sequence information

[0184] SEQ ID NO.379-386 RPA primers

[0185] SEQ ID NO.387-389 Fluorescent reporter sequences

[0186] SEQ ID NO.390 LbCas12a amino acid sequence SEQ ID NO.391 seCas12a amino acid sequence SEQ ID NO.392 LbCas12a-HF amino acid sequence SEQ ID NO.393-395 Occluder sequence

[0187] The above description is merely a preferred embodiment of this embodiment and is not intended to limit this embodiment in any way. Although this embodiment has been disclosed above with reference to a preferred embodiment, it is not intended to limit this embodiment. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above to create equivalent embodiments without departing from the scope of the technical solution of this embodiment. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this embodiment without departing from the content of the technical solution of this embodiment shall still fall within the scope of the technical solution of this embodiment.

Claims

1. A detection system for single nucleotide polymorphism (SNP) genotyping, characterized in that, The detection system includes: (1) Endonucleases and their variants; (2) A splitting crRNA, wherein the splitting crRNA consists of a first fragment and a second fragment, the first fragment comprising a scaffold region and a spacer region X nucleotides from the 5' end, and the second fragment comprising the remaining nucleotide sequence of the spacer region, wherein X is an integer between 0 and 20; preferably, X is an integer between 8 and 16. (3) Reporter molecule, wherein the reporter molecule is a substrate for the trans-cleavage activity of the endonuclease.

2. The detection system according to claim 1, characterized in that, In step (1), the endonuclease is selected from members of the Cas family that have or can be modified to have auxiliary cleavage activity; wherein, members of the Cas family include, but are not limited to, Cas9, Cas12a, Cas12b, Cas12i, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12j, Cas13a, Cas13b, Cas13d, Cas13x, Cas13y or their functional variants.

3. The detection system according to claim 1, characterized in that, The first fragment includes a scaffold region and a spacer region 13 nucleotides from the 5' end, i.e., X=13.

4. The detection system according to claim 1, characterized in that, A single mismatched base is introduced at positions +1 to +23 in the original spacer region; preferably, a single mismatched base is introduced at positions +4 to +20 in the original spacer region; more preferably, a single mismatched base is introduced at position +17 in the original spacer region.

5. The detection system according to claim 1, characterized in that, The report molecules are selected from one of the following: (1) A fluorescent reporter probe, which is a single-stranded DNA molecule that can form a hairpin structure by having a fluorescent group modified at the 5' end and a quenching group modified at the 3' end; (2) Visualized colorimetric probes; (3) Identifiable labeled probes on the side-flow chromatography test strip; (4) Any substrate molecule that can be trans-cleaved by a nuclease and can generate a corresponding detection signal.

6. The detection system according to any one of claims 1 to 5, characterized in that, The detection system identifies and detects SNPs independently of PAM and can identify and detect SNPs without PAM motifs.

7. The detection system according to claim 1, characterized in that, The detection system also includes a nucleic acid amplification system.

8. The detection system according to claim 1, characterized in that, The spacer region can be 20 to 30 nucleotides in length.

9. A method for SNP genotyping detection using the system described in any one of claims 1-8, characterized in that, The method includes the following steps: (1) Obtain the nucleic acid amplification product of the sample to be tested; (2) The nucleic acid amplified product obtained in the pretreatment step (1) is used to obtain a single-stranded sample to be tested; (3) The Cas12a nuclease and the fission crRNA are pre-assembled in a buffer solution to form a Cas12a-fission crRNA complex; (4) Incubation and signal detection: The single-stranded sample to be tested, the Cas12a-split crRNA complex and the reporter molecule are mixed and incubated; if the target and the split crRNA are completely matched, the trans-cleavage activity of Cas12a is activated, and the reporter molecule is cleaved to generate a detectable signal; if there is an SNP mismatch in the target, no signal is generated or a significantly reduced signal is generated, thereby achieving genotyping.

10. The method according to claim 9, characterized in that, In step (1), if the sample to be tested is an RNA target, the sample needs to be reverse transcribed to obtain cDNA before nucleic acid amplification.

11. The method according to claim 9, characterized in that, The buffer solution mentioned in step (3) contains 1 mM to 100 mM g². + The cleavage buffer preferably contains 1 mM-12.5 mM Mg²⁺. + The cleavage buffer preferably contains 2.5 mM-7.5 mM Mg²⁺. + The cutting buffer.

12. A kit for SNP genotyping, characterized in that, The kit comprises the detection system according to any one of claims 1-8.

13. The application of the detection system according to any one of claims 1-8 or the detection method of the detection system according to any one of claims 9-11 in the fields of microbial detection, viral variant detection, human functional SNP detection, crop germplasm detection and phenotypic prediction.

14. The application of the kit as described in claim 12 in the fields of microbial detection, viral variant detection, human functional SNP detection, crop germplasm detection, and phenotypic prediction.

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