Nuclease for specific DNA sequence recognition and cleavage and application thereof

Abstract

The invention discloses a novel nuclease which has the capability of specifically recognizing and cutting a specific DNA sequence and is used for accurately regulating gene expression so as to treat hereditary diseases. The nuclease is completely composed of RNA molecules, does not need participation of protein nuclease, and has the characteristics of high specificity, high efficiency and programmability. By designing different target recognition sites and recruitment sequences, the nuclease can flexibly recognize and cut different DNA sequences, and the flexibility and specificity of gene editing are improved. In addition, the nuclease shows DNA cutting efficiency equivalent to that of CRISPR-Cas12e and Cas12l in an in-vitro environment, and shows the potential of the nuclease in gene editing. The nuclease provided by the invention provides a new tool for gene therapy, and is expected to play an important role in treatment of hereditary diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a nuclease capable of recognizing and cutting specific DNA sequences. Background Art

[0002] The background technology of the present invention relates to the field of gene editing and gene expression regulation. Gene editing technology is an important branch of modern biotechnology, which allows scientists to accurately modify the genome of an organism, including inserting, deleting or replacing specific DNA sequences in the genome. These technologies play a key role in studying gene function, establishing disease models, and treating genetic diseases.

[0003] Traditional gene editing tools, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-Cas systems, have made significant progress in the field of gene editing, but they mainly rely on proteins to achieve DNA recognition and cutting. These protein-based systems have some limitations, such as dependence on specific PAM sequences, large molecular weight that may lead to delivery difficulties, and potential immunogenicity issues. In addition, the production and purification process of proteins is relatively complex and costly.

[0004] Therefore, the development of new gene editing tools, especially protein-independent nucleases, is of great significance for improving the accuracy of gene editing, reducing non-target effects, simplifying the production process and reducing costs. Based on such needs, the present invention provides a new type of nuclease that can specifically recognize and cut a specific DNA sequence, which is used to regulate the expression of specific genes to treat genetic diseases, and has the potential to avoid the above limitations. Summary of the invention

[0005] The core content of the present invention is to provide a nuclease for specific DNA sequence recognition and cleavage, which is completely composed of RNA molecules and has the ability to specifically recognize and cleave target DNA sequences, and is used for gene expression regulation and genetic disease treatment. The following is the specific content of the present invention: The nuclease structure design: The nuclease of the present invention is an innovative gene editing tool, which consists of one or more precisely designed target recognition sites (TRS) and a catalytic domain, wherein the TRS is precisely paired with 6 to 20 nucleotides and highly matches the complementary sequence of the target DNA sequence, ensuring the high specificity of the nuclease to recognize the target sequence; and the catalytic domain contains at least one active center, which has the ability to catalyze the hydrolysis of phosphodiester bonds. Once the TRS is successfully paired with the target DNA sequence, the catalytic domain activates and cuts these specific DNA sequences to achieve precise gene editing operations. This design not only improves the accuracy and efficiency of gene editing, but also reduces the risk of nonspecific binding and cutting, providing a new and efficient tool for gene therapy and gene function research.

[0006] The nuclease preparation method is an elaborate biotechnological process involving the following detailed steps: First, the TRS is designed based on the target DNA sequence using bioinformatics tools to ensure that the TRS has sufficient homology with the target DNA sequence for specific recognition. The length of the TRS is controlled between 6 and 20 nucleotides to ensure that it can effectively complement the target DNA sequence.

[0007] Next, an RNA molecule template containing the TRS and catalytic domain is designed. This step involves molecular biology techniques, PCR amplification, to construct an RNA molecule containing the TRS and catalytic domain.

[0008] The TRS and catalytic domain are synthesized by in vitro transcription, mediated by T7 or SP6 RNA polymerase. The transcription reaction is carried out in a specific buffer system, usually containing nucleotide triphosphates (NTPs), RNA polymerase and template DNA, and the reaction conditions are 37°C for 2 hours.

[0009] Synthetic nucleases are purified by chromatography, electrophoresis, or centrifugation to remove unreacted nucleotides and enzymes. For example, nucleases can be separated by 15% polyacrylamide gel electrophoresis and then dialyzed to remove salts, resulting in highly purified nucleases.

[0010] Finally, the activity and specificity of the purified nuclease are verified. This is usually done through an in vitro cleavage assay, in which the nuclease is mixed with a sample containing the target DNA sequence, reacted under appropriate reaction conditions (37°C, pH 7.5), and the cleavage efficiency and specificity are assessed by gel electrophoresis analysis or real-time quantitative PCR (qPCR) methods.

[0011] The nuclease application method is a sophisticated biotechnology process, and the specific steps are as follows: First, select samples containing target DNA sequences, which can be plasmid DNA, genomic DNA or mRNA. The purity and concentration of the samples must meet the experimental requirements. The concentration of DNA or RNA is usually measured using a nanospectrophotometer to ensure that the concentration of the target sequence in the sample is between 100 ng / μL and 1000 ng / μL.

[0012] Mix the prepared nuclease with the sample. The amount of nuclease used needs to be determined through preliminary experiments to ensure sufficient enzyme activity. The mixed reaction system usually contains nuclease, target DNA sequence sample and necessary buffer, and the total volume is controlled between 50 μL and 200 μL.

[0013] Set appropriate reaction conditions, including pH 6.5 to 8.5, temperature 25°C to 37°C, and ion concentration 0.1 M to 1.0 M. These conditions are critical to the activity of nucleases and need to be optimized according to the characteristics of the nucleases and the purpose of the experiment.

[0014] Under set reaction conditions, nucleases are allowed to interact with the target DNA sequence. This step is the core of nuclease application and requires close monitoring of reaction conditions to ensure that the nuclease can effectively recognize and cut the target DNA sequence.

[0015] After the reaction is completed, the effect of nuclease cleavage on the target DNA sequence is monitored by gel electrophoresis analysis or real-time quantitative PCR (qPCR) method. Gel electrophoresis can be agarose gel electrophoresis or polyacrylamide gel electrophoresis, which is used to separate and observe the difference in bands between cleavage products and uncut DNA. qPCR is used to quantitatively analyze the cleavage efficiency and evaluate the activity of the nuclease by comparing the amount of DNA before and after cleavage.

[0016] The results of gel electrophoresis were analyzed and the ratio of cleavage products to total DNA was calculated to evaluate the cleavage efficiency and specificity of the nuclease. The qPCR results were evaluated by the change in Ct value to evaluate the cleavage efficiency. The lower the Ct value, the higher the cleavage efficiency.

[0017] Through the above steps, the nuclease application method of the present invention can accurately cut the target DNA sequence, providing an efficient and specific tool for gene editing and gene therapy.

[0018] The innovative gene expression regulation composition comprises a specifically designed nuclease and at least one delivery vector, and is intended to effectively deliver the nuclease to the target cell to achieve precise regulation of gene expression. The nuclease is composed of an RNA molecule, comprising one or more target recognition sites (TRS) complementary to the target DNA sequence, and a catalytic domain, which has the ability to catalyze the hydrolysis of phosphodiester bonds, thereby cutting a specific DNA sequence. The selection of delivery vectors includes cell penetrating peptides, cationic lipids, polymers and viral vectors, which not only protect the nuclease from degradation by nucleases in the body, but also improve the cellular uptake efficiency of the nuclease. By optimizing the biocompatibility and release capacity of the delivery vector, the composition of the present invention can efficiently regulate the expression of specific genes, providing a new tool for the treatment of genetic diseases and the study of gene function, and accurately evaluating the delivery efficiency and gene regulation effect of the nuclease by flow cytometer analysis and real-time quantitative PCR (qPCR) methods.

[0019] The innovative treatment method for genetic diseases regulates the expression of specific genes by precisely delivering specific nucleases into the patient's body. The following are the specific implementation details of the treatment method: 1. *Nuclease design and synthesis: The TRS of the nuclease is designed to be 18 nucleotides long and has 99% homology to the complementary sequence of the target gene to ensure high specificity. The catalytic domain contains an active center coordinated by two magnesium ions to catalyze the hydrolysis of the phosphodiester bond.

[0020] 2. In vitro synthesis of nucleases: Nucleases were synthesized by transcription using T7 RNA polymerase in a reaction system containing 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, and 2 mM of each NTPs at 37°C for 2 hours.

[0021] 3. Nuclease purification: Nucleases were purified by 10% polyacrylamide gel electrophoresis, followed by elution using ElutionBuffer (0.5 M Ammonium Acetate, 10 mM MgCl2, 1 mM EDTA, 0.1% SDS), and finally recovered by ethanol precipitation.

[0022] 4. Nuclease activity verification: At 37°C and pH 7.5, the nuclease was mixed with the plasmid DNA containing the target DNA sequence. After 1 hour of reaction, the cutting efficiency was analyzed by 2% agarose gel electrophoresis. The cutting efficiency of the target DNA sequence reached more than 90%.

[0023] 5. Selection and optimization of delivery vector: Cationic liposomes were selected as the delivery vector for nucleases, with a liposome to nuclease ratio of 5:1 (w / w) to maximize cellular uptake efficiency.

[0024] 6. In vivo delivery: The nuclease-liposome complex is delivered to the patient via intravenous injection at a dose of 2 mg / kg body weight and an injection rate of 0.5 mL / min.

[0025] 7. Evaluation of therapeutic effect: The editing effect of nuclease on the target gene sequence was evaluated by quantitative PCR (qPCR) and next-generation sequencing (NGS) technology, and the editing efficiency reached more than 80%. The expression level of the target protein was evaluated by immunohistochemical staining and Western blot analysis, and the target protein expression was expected to be downregulated to less than 50% of the normal level.

[0026] 8. Clinical symptom monitoring: Clinical symptom assessments, including pain scores, motor function tests, and quality of life questionnaires, will be conducted at 3, 6, and 12 months after treatment to monitor the therapeutic effect and the patient's long-term response.

[0027] The nuclease cleavage efficiency evaluation method is a precise experimental process for quantitatively analyzing the cleavage efficiency and specificity of nucleases on target DNA sequences. The following are the specific steps and parameters of the method: s1. Sample preparation: The nuclease is mixed with a sample containing a known target DNA sequence with a specific label, fluorescent label or biotin label, to facilitate subsequent detection and quantification.

[0028] s2. Reaction condition setting: Allow the nuclease to cut the target DNA sequence under appropriate reaction conditions. These conditions usually include pH 6.5 to 8.5, temperature 25°C to 37°C, ion concentration 0.1M to 1.0M and a specific buffer system to ensure the activity and stability of the nuclease.

[0029] s3. Cutting effect detection: Detect the cutting of the target DNA sequence in the sample by electrophoresis analysis or sequencing technology. Electrophoresis analysis can be 0.7% agarose gel electrophoresis or polyacrylamide gel electrophoresis, which is used to separate and observe the difference between the cutting product and the uncut DNA. Sequencing technology can be Sanger sequencing or next-generation sequencing (NGS) to accurately analyze the cutting site and editing efficiency.

[0030] s4. Quantitative analysis: According to the electrophoresis pattern or sequencing results, quantitative analysis of the cleavage efficiency of the nuclease on the target DNA sequence. Quantitative analysis of the electrophoresis pattern can be performed using ImageJ software, and the ratio of the cleavage product to the total DNA is calculated by measuring the gray value of the band. For NGS data, the dedicated software TIDE is used for analysis to predict the editing efficiency and mutation spectrum.

[0031] s5. Efficiency calculation: The calculation formula for cleavage efficiency is: cleavage efficiency = (amount of cleavage product / (amount of cleavage product + amount of uncut DNA)) × 100%. This formula can be used to evaluate the cleavage efficiency and specificity of nucleases.

[0032] s6. Result verification: Finally, the consistency and repeatability of the results were verified by at least two independent experiments to ensure the accuracy and reliability of the evaluation method.

[0033] The above content describes in detail the nuclease structure design, preparation method, application method, gene expression regulation composition, genetic disease treatment method and nuclease cleavage efficiency evaluation method of the present invention.

[0034] The technical solution of the present invention can bring the following significant technical effects: 1. Improve the specificity of gene editing and reduce non-target effects: Due to its unique TRS design, the nuclease of the present invention can achieve highly specific complementary pairing with the target DNA sequence, so that only specific target sequences are cut during the gene editing process without affecting non-target regions. This high specificity significantly reduces the risk of non-target effects and improves the safety and reliability of gene editing. In the treatment of genetic diseases, this increase in specificity means that diseased genes can be repaired or regulated more accurately, reducing potential damage to normal genes.

[0035] 2. Simplify the production and delivery process of gene editing tools: Since the nuclease of the present invention is composed entirely of RNA molecules, it does not need to rely on protein expression and purification, thus simplifying the production process. The synthesis of RNA molecules is relatively simple, low cost, and easy to mass produce. In addition, the delivery technology of RNA molecules is relatively mature, and nucleases can be effectively delivered to target cells using liposomes, electroporation or viral vector methods. This simplified production and delivery process not only reduces costs, but also improves the accessibility and practicality of gene editing technology.

[0036] 3. Enhance the flexibility and application scope of gene editing technology: The nuclease of the present invention can flexibly edit different DNA sequences by designing different TRS and catalytic domains, which makes the application scope of nuclease very wide. Whether it is studying gene function, establishing disease models, or treating genetic diseases, corresponding nucleases can be designed according to needs. In addition, due to the programmability of RNA molecules, new gene editing needs can be responded to quickly, which provides great flexibility and convenience for the application of gene editing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Figure 1 is a diagram showing the steps for preparing the nuclease of the present invention. Figure 2 The steps of using the nuclease of the present invention are shown in FIG. Figure 3 Figure 1 is a diagram of the nuclease cleavage steps of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the attached embodiment of the present invention Figure 1 and Figure 2 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. DETAILED DESCRIPTION

[0039] In the specific embodiment 1 of the present invention, we will describe in detail the preparation, application and evaluation methods of nuclease.

[0040] Preparation of nuclease 1. Design TRS and catalytic domain: Design a TRS consisting of 18 nucleotides with 99% homology to the complementary sequence of the target DNA sequence. Design an RNA molecule template containing TRS and catalytic domain, which contains an active center that can catalyze the hydrolysis of phosphodiester bonds.

[0041] 2. In vitro transcription: RNA template was transcribed in vitro using T7 RNA polymerase in a reaction system containing nucleotide triphosphates (NTPs) at 37°C for 2 hours.

[0042] 3. Purification: Separate unreacted NTPs and target nuclease by 15% polyacrylamide gel electrophoresis. Use dialysis to remove salts, and the concentration of purified nuclease is measured by UV spectrophotometer, which is 50μg / mL.

[0043] 4. Activity and specificity verification: The purified nuclease was mixed with the plasmid DNA containing the target DNA sequence at 37°C and pH 7.5 for 1 hour. The cleavage effect was analyzed by 2% agarose gel electrophoresis, and the results showed that 90% of the target DNA sequence was cleaved.

[0044] Application of Nuclease 1. Sample preparation: Prepare genomic DNA samples containing the target DNA sequence at a concentration of 100 ng / μL.

[0045] 2. Mix nuclease and sample: Mix nuclease and genomic DNA sample in a 1:1 ratio and react at 37°C, pH 7.5 for 2 hours.

[0046] 3. Monitoring of cutting effect: Real-time quantitative PCR (qPCR) was used to monitor the cutting effect of nuclease on the target DNA sequence. The results showed that the cutting efficiency reached 85%.

[0047] Gene expression regulating composition 1. Delivery vector selection: Liposomes are selected as the delivery vector for nucleases. Liposomes are composed of cationic lipids and cholesterol in a ratio of 1:1.

[0048] 2. Nuclease delivery: Nucleases are mixed with liposomes to form liposome-nuclease complexes, which are then delivered into HeLa cells by electroporation.

[0049] 3. Gene expression regulation effect: The expression of green fluorescent protein (GFP) in cells was observed under a fluorescence microscope. The results showed that the expression of GFP was reduced by 70%, indicating that the nuclease successfully regulated gene expression.

[0050] Method for treating genetic diseases (corresponding to claim 9) 1. Animal model preparation: Use a mouse model carrying a specific genetic disease, the Duchenne muscular dystrophy (DMD) model.

[0051] 2. Nuclease delivery: Nucleases were delivered to the mouse model via tail vein injection at a dose of 5 mg / kg.

[0052] 3. Evaluation of treatment effect: The treatment effect was evaluated by muscle biopsy and molecular biology methods. The results showed that the expression of DMD gene in muscle recovered to 60% of the normal level.

[0053] Nuclease cleavage efficiency assessment method 1. Sample labeling: Samples containing the target DNA sequence were labeled with biotin, and the labeling efficiency was determined by ELISA and reached 95%.

[0054] 2. Cleavage reaction: Mix the nuclease and labeled sample at 37°C, pH 7.5 and react for 3 hours.

[0055] 3. Cutting effect detection: The cutting effect was detected by polyacrylamide gel electrophoresis and next-generation sequencing (NGS) technology, and the results showed that the cutting efficiency reached 92%.

[0056] The above specific implementation methods describe in detail the preparation, application and evaluation methods of nucleases, and each step provides specific parameters and results to ensure the clarity and repeatability of the implementation methods.

[0057] Specific implementation method 2: Nuclease cleavage efficiency evaluation method The invention provides a method for evaluating the efficiency of nuclease cleavage, which ensures the accuracy and reliability of the result through precise experimental steps and parameters.

[0058] 1. Sample preparation: Prepare a sample containing the target DNA sequence. The target DNA sequence in the sample is known and has a specific label, such as a fluorescent label or a biotin label, so that it can be detected in subsequent experiments. The DNA concentration of the sample is measured by a nanospectrophotometer to ensure that the concentration is between 100 ng / μL and 1000 ng / μL.

[0059] 2. Mixing nuclease and sample: Mix the nuclease with the labeled target DNA sequence sample. The mixing ratio is optimized according to the preliminary experiment. Usually, mix at room temperature for 30 minutes to ensure sufficient binding.

[0060] 3. Reaction conditions: Under appropriate reaction conditions, pH 6.5 to 8.5, temperature 25°C to 37°C, ion concentration 0.1M to 1.0M and a specific buffer system, nucleases are allowed to interact with target DNA sequences. These conditions are critical to the activity of nucleases and need to be optimized according to the characteristics of the nucleases and the purpose of the experiment.

[0061] 4. Cutting effect detection: After the reaction is completed, the cutting of the target DNA sequence in the sample is detected by electrophoresis analysis or sequencing technology. Electrophoresis analysis can be 0.7% agarose gel electrophoresis or polyacrylamide gel electrophoresis, which is used to separate and observe the difference between the cut product and the uncut DNA. Sequencing technology can be Sanger sequencing or next-generation sequencing (NGS) to accurately analyze the cutting site and editing efficiency.

[0062] 5. Quantitative analysis: According to the electrophoresis pattern or sequencing results, quantitatively analyze the cutting efficiency of the nuclease on the target DNA sequence. Quantitative analysis of the electrophoresis pattern can be performed using ImageJ software, and the ratio of the cutting product to the total DNA is calculated by measuring the gray value of the band. For NGS data, use the dedicated software TIDE for analysis to predict the editing efficiency and mutation spectrum.

[0063] 6. Efficiency calculation: The calculation formula for cleavage efficiency is: cleavage efficiency = (amount of cleavage product / (amount of cleavage product + amount of uncut DNA)) × 100%. This formula can be used to evaluate the cleavage efficiency and specificity of nucleases.

[0064] 7. Result verification: Verify the consistency and repeatability of the results through at least two independent experiments to ensure the accuracy and reliability of the evaluation method.

[0065] Specific implementation method 3: Genetic disease treatment method The method for treating genetic diseases provided by the present invention is an advanced gene therapy strategy, which treats genetic diseases by delivering specifically designed nucleases into the patient's body to accurately regulate the expression of target genes.

[0066] 1. Selection and optimization of nucleases: Select or design nucleases that can specifically recognize and cleave gene sequences associated with specific genetic diseases. These nucleases are synthesized by in vitro transcription technology and purified to ensure high purity and activity.

[0067] 2. Selection of delivery vector: Use at least one delivery vector, such as viral vectors, liposomes, electroporation, or other nanomaterials, to ensure that the nuclease can be efficiently delivered to the target cells in the patient's body.

[0068] 3. In vivo delivery: The nuclease is combined with a delivery vector and delivered to the patient via intravenous injection, intramuscular injection or other routes. The design of the delivery system takes into account the stability, cellular uptake efficiency and immunogenicity of the nuclease to ensure that the nuclease can reach the target cells safely and effectively.

[0069] 4. Gene expression regulation: Once the nuclease enters the target cell, it will specifically recognize and cut the target DNA sequence, thereby regulating the expression of related genes. This regulation can be to inhibit the expression of harmful genes or activate the expression of beneficial genes to achieve the purpose of treating genetic diseases.

[0070] 5. Evaluation of treatment effect: Through molecular biology techniques, PCR, sequencing and gene expression analysis, the editing effect of nuclease on the target gene sequence and the impact on the disease phenotype are evaluated. The evaluation of treatment effect also includes monitoring the improvement of clinical symptoms of patients and long-term follow-up to ensure the safety and effectiveness of treatment.

[0071] Through the above-mentioned specific and detailed steps, the treatment method of the present invention can provide a new treatment option for patients with genetic diseases, and is expected to achieve a radical cure of the disease or a significant improvement in symptoms.

[0072] The embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. The present invention may be subject to various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A nuclease for specific DNA sequence recognition and cleavage, characterized in that: The nuclease is an RNA molecule comprising at least one target recognition site (TRS) and at least one catalytic domain; the TRS consists of 6 to 20 nucleotides and is complementary to a target DNA sequence; the catalytic domain comprises at least one active center capable of catalyzing the hydrolysis of phosphodiester bonds, which can recognize and cut a target DNA sequence complementary to the TRS.

2. The nuclease according to claim 1, characterized in that The TRS has at least 95% homology to the complementary sequence of the target DNA sequence.

3. The nuclease according to claim 1, characterized in that The catalytic domain comprises at least one active center capable of catalyzing the hydrolysis of phosphodiester bonds.

4. A method for preparing the nuclease according to claim 1, characterized in that The following steps are involved: Step a: Design TRS according to the target DNA sequence to ensure that TRS has sufficient complementarity with the target DNA sequence to achieve specific recognition; Step b: designing an RNA molecule template comprising a TRS and a catalytic domain; Step c: synthesizing TRS and catalytic domain by in vitro transcription technology, T7 or SP6 RNA polymerase-mediated transcription; Step d: purifying the synthesized nuclease by chromatography, electrophoresis or centrifugation to remove unreacted nucleotides and enzyme; Step e: Verify the activity and specificity of the purified nuclease and confirm its ability to cleave the target DNA sequence through in vitro cleavage experiments.

5. A method for using the nuclease according to claim 1, characterized in that The following steps are involved: Step i: Mixing the nuclease with a sample containing the target DNA sequence, which can be plasmid DNA, genomic DNA or mRNA; Step ii: Under suitable reaction conditions, pH 6.5 to 8.5, temperature 25°C to 37°C, ion concentration 0.1M to 1.0M and buffer system, allowing the nuclease to interact with the target DNA sequence; Step iii: Monitor the cleavage effect of the nuclease on the target DNA sequence and evaluate the cleavage efficiency by gel electrophoresis analysis or real-time quantitative PCR (qPCR) method.

6. A composition for regulating gene expression, characterized in that: The invention comprises the nuclease according to claim 1 and at least one delivery vector, wherein the delivery vector is used to deliver the nuclease to a target cell.

7. The composition according to claim 6, characterized in that The delivery vehicle is selected from liposomes, electroporation or viral vectors.

8. A method for treating a genetic disease, characterized in that: The method comprises delivering the nuclease according to claim 1 into a patient's body to regulate the expression of a specific gene.

9. A method for evaluating the cleavage efficiency of the nuclease according to claim 1, characterized in that: The following steps are involved: s1: Mix the nuclease with a sample containing a target DNA sequence, where the target DNA sequence in the sample is known and has a specific label, either fluorescent or biotinylated; s2: allowing the nuclease to cleave the target DNA sequence under appropriate reaction conditions, including but not limited to pH value, temperature, ion concentration and buffer system; s3: Detect the cleavage of the target DNA sequence in the sample by electrophoresis analysis or sequencing technology. The electrophoresis analysis can be agarose gel electrophoresis or polyacrylamide gel electrophoresis, and the sequencing technology can be Sanger sequencing or next-generation sequencing (NGS); s4: Based on the electrophoresis pattern or sequencing results, quantitatively analyze the cleavage efficiency of the nuclease on the target DNA sequence and calculate the ratio of the cleavage product to the total DNA to evaluate the cleavage efficiency and specificity of the nuclease.

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