Method for modifying and regulating gene editing based on sgRNA

Abstract

The invention discloses a sgRNA-based method for modifying and regulating gene editing, which comprises the following steps: carrying out POC acylation modification on a 2 '-OH site of sgRNA to introduce an alkynyl marker to obtain sgRNA with alkynyl; the sgRNA with alkynyl and azide are subjected to copper-catalyzed azide-alkynyl cycloaddition reaction under the action of a copper catalytic reagent to form a stable chemical cross-linked structure, so that the activity of the sgRNA is quickly closed and adjusted. According to the method, alkynyl is introduced to sgRNA and is subjected to a CuAAC reaction with multi-azide to form stable crosslinking, so that the activity of the CRISPR-Cas9 system is accurately controlled. The innovative method not only can effectively control the gene editing activity on the basis of not destroying the original structure and function of RNA, but also can significantly improve the target specificity of the CRISPR system and reduce the risk of genome damage caused by non-specific cleavage.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for modifying and regulating gene editing based on sgRNA. Background Art

[0002] The CRISPR-Cas9 system, a revolutionary gene-editing tool, is widely used in biomedical research and disease treatment due to its efficient and precise gene manipulation capabilities. However, the system's application has certain limitations, particularly its potential for nonspecific targeting (i.e., "off-target effects"), which can lead to unintended gene modifications and increase the risks of gene therapy. To address this issue, both academia and industry are seeking methods to more precisely control the activity of the CRISPR-Cas9 system.

[0003] At present, the control of the CRISPR system mainly focuses on inhibiting the activity of the Cas9 protein, covering a variety of strategies, including the use of antigen-induced Cas9 protein degradation systems, light-sensitive regulatory elements, and chemically induced dCas9 activity regulation systems. For example, Chinese patent document No. CN117487831A discloses a method for controlling gene editing using a new CRISPR-Cas9 inhibitory protein AcrIIA31; another patent (CN116102508A) describes a small molecule inhibitor of the Cas protein. Although these methods can achieve conditional activation and shutdown of the CRISPR system, they generally have challenges such as complex operation, long reaction time, and difficulty in achieving precise control. In particular, these technologies often involve the use of the same regulatory strategy for all target genes, and the operation steps are complicated and cumbersome.

[0004] Another strategy, less widely explored, is to use small chemical molecules to directly regulate the activity of sgRNAs. This approach offers a significant advantage by avoiding complex protein engineering steps, thereby streamlining the entire gene editing process. By precisely controlling the activity of sgRNAs, this approach not only increases operational flexibility but also facilitates the targeted shutoff of specific gene expression, greatly enhancing the precision and efficiency of gene editing.

[0005] Therefore, it is necessary to develop a method to regulate sgRNA activity with small chemical molecules and apply it to precise and controllable gene editing, which is of great significance to promoting the innovation and application of gene editing technology. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for modifying and regulating gene editing based on sgRNA, which not only enhances the application flexibility of CRISPR technology, but also provides a safer method for high-risk gene operations.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a method for modifying and regulating gene editing based on sgRNA is provided, the method comprising:

[0009] Step S1, performing POC acylation modification at the 2'-OH site of the sgRNA to introduce an alkyne tag to obtain an sgRNA with an alkyne group;

[0010] Step S2: The sgRNA with an alkyne group undergoes a copper-catalyzed azide-alkyne cycloaddition reaction with an azide in the presence of a copper catalytic reagent to form a stable chemical cross-linked structure, thereby achieving rapid shut-down regulation of the sgRNA activity.

[0011] In the above technical solution, when targeting a specific target, step S1 is performed before the cutting experiment, and step S2 is performed before the cutting experiment.

[0012] Furthermore, the POC acylation modification is performed at the 2'-OH site of the sgRNA to introduce an alkyne tag to obtain an sgRNA with an alkyne group, specifically comprising:

[0013] The sgRNA sample was incubated in a solution containing POC (full name: 2-propyn-1-yl 1H-imidazole-1-carboxylate), and then a stop solution was added to terminate the reaction to obtain an sgRNA with an alkyne group.

[0014] Furthermore, the solution containing POC includes 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and 180-220 mM DMAP aqueous solution, wherein the volume ratio of DMAP to water in the DMAP aqueous solution is 1:9-11.

[0015] The structural formula of the POC is:

[0016]

[0017] The preparation method of the POC can be performed by adding N,N'-carbonyldiimidazole (1.94g, 12mM) to a solution of prop-2-yn-1-ol (560mg, 10mM) in anhydrous dichloromethane (50ml) at 0°C. The reaction solution was stirred at room temperature for 12 hours. After the reaction was completed, the reaction mixture was diluted in dichloromethane (100ml) and washed with brine (3×150ml), dried over sodium sulfate, filtered, and concentrated under vacuum to obtain a pale white crystalline solid product ROC (1.35g, 90% yield).

[0018] Furthermore, the incubation conditions include 37°C, 200 rpm

[0019] Furthermore, the mass volume ratio of the sgRNA sample to the solution containing POC is 1-3 μM / μL.

[0020] Furthermore, the azide is q-N3 (tetrakis(azidomethyl)methane).

[0021] The structural formula of q-N3 is

[0022] The preparation method of q-N3 can be carried out by dissolving 1,3-dibromo-2,2-bis(bromomethyl)propane (383 mg, 1.0 mM) in 20 ml of N,N-dimethylformamide (DMF). Sodium azide (325 mg, 5.0 mM) is added to the solution. The reaction mixture is stirred at 80°C for 12 hours. The reaction mixture is diluted with 200 ml of water and extracted three times with 150 ml of diethyl ether. The organic layers are combined, dried over sodium sulfate, and the solvent is removed under reduced pressure. The crude product is purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain q-N3 (188 mg, 80% yield) as a white solid.

[0023] Furthermore, the copper catalytic reagent is CuSO4 / BTTAA / Sodium Ascorbate, the molar ratio of CuSO4, BTTAA and Sodium Ascorbate is 1:2:10, and the dosage range of the copper catalytic reagent is 8-32 μM.

[0024] Furthermore, the mass volume ratio of the sgRNA with an alkyne group to the azide is in the range of 5-10 μM / μL.

[0025] In a second aspect of the present invention, a modified sgRNA having a chemically cross-linked structure obtained by the method is provided.

[0026] In a third aspect of the present invention, provided is the use of the modified sgRNA having a chemically cross-linked structure in the preparation of products for regulating gene editing.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] The present invention provides a method for modifying and regulating gene editing based on sgRNA. Through precise chemical modification, it not only enhances the functional control ability of the CRISPR system, but also significantly improves the safety and accuracy of gene editing operations. Through a series of in vitro experiments and intracellular application verification, the present invention has been demonstrated to provide significant advantages in controlling the activity of the CRISPR-Cas9 system and reducing off-target effects, demonstrating its potential for broad application in gene therapy, disease model construction, and gene function research. The development of this new technology is expected to promote the development of precision medicine and biotechnology, and provide a more reliable and efficient technical platform for the practical application of gene editing technology in the future.

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] (1) Precise control: Through chemical cross-linking, the activity of sgRNA can be quickly shut down and the operational accuracy of the CRISPR-Cas9 system can be improved.

[0031] (2) Reduce non-target effects: By conditionally controlling the activity of the CRISPR system, non-target gene editing events can be significantly reduced, thereby improving the safety of gene editing.

[0032] (3) Simplified operation: Compared with the CRISPR system control method based on protein engineering, the method of using small molecules to modify sgRNA is simple to operate, easy to implement, and suitable for a variety of biological and medical research.

[0033] Broad application prospects: This strategy is not only applicable to the CRISPR-Cas9 system, but can also be extended to other types of CRISPR systems, such as Cas12a or Cas13, and has broad application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 These are the structures of two small chemical molecules used to regulate gene editing.

[0036] Figure 2In vitro DNA cleavage assay targeting the SLX4IP gene. (A) Assessment of Cas9 tolerance to POC-modified sg-SLX4IP. (B) CRISPR-Cas9 inhibition depends on POC modification of sg-SLX4IP. (C) Dose-dependent effect of q-N3 on CRISPR / Cas9 inhibition of POC-modified sg-SLX4IP. Statistical analysis was performed using an unpaired Student's t-test. Error bars represent biological replicates ± SD (n = 3, ns indicates not significant, **p < 0.01 and ****p < 0.0001).

[0037] Figure 3 In vitro DNA cleavage assay targeting the GAPDH gene. (A) Assessment of Cas9 tolerance to POC-modified sg-GAPDH. (B) CRISPR-Cas9 inhibition is dependent on POC modification of sg-GAPDH. (C) Dose-dependent effect of q-N3 on CRISPR / Cas9 inhibition of POC-modified sg-GAPDH. Statistical analysis was performed using an unpaired Student's t-test. Error bars represent ±SD of biological replicates (n = 3, ns indicates not significant, **p < 0.01 and ****p < 0.0001).

[0038] Figure 4 In vitro DNA cleavage assay targeting the HBEGF gene. (A) Assessment of Cas9 tolerance to POC-modified sg-HBEGF. (B) CRISPR-Cas9 inhibition depends on POC modification of sg-HBEGF. (C) Dose-dependent effect of q-N3 on CRISPR / Cas9 inhibition of POC-modified sg-HBEGF. Statistical analysis was performed using an unpaired Student's t-test. Error bars represent ±SD of biological replicates (n = 3, ns indicates not significant, **p < 0.01 and ****p < 0.0001).

[0039] Figure 5 Denaturing PAGE characterization of POC-modified sgRNAs (sg-SLX4IP). (A) POC-modified sg-SLX4IP. (B) POC-modified sg-SLX4IP treated with q-N3.

[0040] Figure 6 Denaturing PAGE characterization of the reaction between q-N3 and POC-modified sgRNAs (sg-SLX4IP).

[0041] Figure 7Denaturing PAGE characterization of POC-modified sgRNAs (sg-GAPDH). (A) POC modification of sg-GAPDH. (B) POC-modified sg-GAPDH treated with q-N3.

[0042] Figure 8 Denaturing PAGE characterization of the reaction between q-N3 and POC-modified sgRNAs (sg-GAPDH).

[0043] Figure 9 Denaturing PAGE characterization of POC-modified sgRNAs (sg-HBEGF). (A) POC-modified sg-HBEGF. (B) POC-modified sg-HBEGF treated with q-N3.

[0044] Figure 10 Denaturing PAGE characterization of the reaction between q-N3 and POC-modified sgRNAs (sg-HBEGF).

[0045] Figure 11 This CuAAC-based sgRNA chemical crosslinking method is used to control gene editing in HeLa-OC cells stably expressing the Cas9 enzyme. (A) Design of an intracellular Cas9 cleavage experiment. sgRNAs were pre-coated prior to delivery to HeLa-OC cells. Four hours after transfection, cells were treated with q-N3 and a catalytic reagent. 28 hours later, INDEL rates were analyzed using T7E1 technology to quantify gene editing efficiency. (B) Intracellular CRISPR-Cas9 functional regulation depends on the extent of POC modification of sg-SLX4IP. (C) Intracellular CRISPR-Cas9 functional regulation depends on q-N3 concentration.

[0046] Figure 12 Schematic diagram of the present invention, (A) is a reaction schematic diagram (B) is a reaction diagram. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0048] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0050] In order to solve the above technical problems, the overall idea of ​​the present invention is as follows:

[0051] First, a specific site (2'-OH) of the sgRNA was subjected to POC acylation modification to introduce an alkyne group. This modification is intended to ensure that the structural integrity and function of the sgRNA are not affected.

[0052] Subsequently, a small molecule q-N3 carrying four azide groups was used to undergo an efficient CuAAC reaction with alkyne groups under the catalysis of Cu(I), forming a stable chemical cross-link, thereby achieving precise "off" control of the CRISPR-Cas9 system, effectively reducing non-target editing events, and significantly improving the accuracy and safety of gene editing.

[0053] As a specific implementation method,

[0054] This technical solution not only enhances the application flexibility of CRISPR technology, but also provides a safer method for high-risk genetic operations.

[0055] The sequences of all oligonucleotides used in the examples of the present invention are shown in Table 1.

[0056] Table 1. All oligonucleotide sequences used in the examples of the present invention

[0057]

[0058]

[0059] The present application will be described in detail below with reference to embodiments, comparative examples and experimental data.

[0060] Example 1: Modification of sgRNA

[0061] 1. POC-modified sg-SLX4IP

[0062] (1) Synthesize and obtain sgRNA targeting the SLX4IP gene: the sequence of sg-SLX4IP is shown in SEQ ID NO.1.

[0063] (2) The sgRNA sample (20 μM) was incubated in 10 μL of a solution containing 100 mM HEPES (pH adjusted to 7.5 at 25°C), 6 mM MgCl2, 100 mM NaCl, 60% DMSO, 1 M POC, and a 1:10 dilution of a freshly prepared 200 mM DMAP (4-dimethylaminopyridine) aqueous solution. After incubation at 37°C with shaking at 200 rpm for the specified time, 2.0 μL of 3 M NaOAc, 75 μL of ethanol, and 1.0 μL of glycogen (5 g / L) were added to the mixture to terminate the reaction. Subsequently, the sample was stored at -80°C for 3 hours and then centrifuged at 12,000 g for 2 hours. The product was purified by ethanol precipitation. Finally, the POC-modified RNA was collected and resuspended in nuclease-free water to a concentration of approximately 10 μM.

[0064] 2. CuAAC-based sgRNA chemical crosslinking

[0065] CuAAC reactions were performed in an aqueous solution (10 μL) containing POC-modified sgRNA (50 ng) and the indicated concentrations of q-N3 and catalytic reagents, and incubated at 37 °C for 30 min.

[0066] 3. Characterization

[0067] 1. Denaturing PAGE characterization results of POC modification of sg-SLX4IP are as follows Figure 5 As shown in A, it shows that under the catalysis of DMAP, the POC reagent can efficiently react with sgRNA and successfully introduce the alkyne group into the sgRNA molecule.

[0068] 2. The denaturing PAGE characterization results of POC modified sg-SLX4IP treated with q-N3 are as follows Figure 5 As shown in B, the POC-modified sgRNA undergoes a click reaction with q-N3 in the presence of a catalyst to form a stable chemical cross-linking structure.

[0069] 3. Denaturing PAGE characterization of the reaction between q-N3 and POC-modified sgRNAs (sg-SLX4IP) Figure 6 As shown, the click reaction has a clear q-N3 concentration dependence.

[0070] Example 2: Method for Modifying and Regulating Gene Editing Targeting the SLX4IP Gene

[0071] 1. POC modification

[0072] sg-SX4IP (20 μM) was added to a solution containing POC and incubated at 37°C with shaking at 200 rpm for the specified time. The POC solution consisted of 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and a 180-220 mM DMAP aqueous solution, where the volume ratio of DMAP to water was 1:9-11. The reaction mixture was terminated by adding 2.0 μL of 3 M NaOAc (sodium acetate), 75 μL of L ethanol, and 1.0 μL of glycogen (5 g / L). The sample was then stored at -80°C for 3 hours and then centrifuged at 12,000 g for 2 hours. The product was purified by ethanol precipitation. Finally, the POC-modified sg-SX4IP was collected and resuspended in nuclease-free water to a concentration of approximately 10 μM.

[0073] 2. In vitro Cas9 cleavage assay

[0074] The same volume of POC modified sg-SX4IP and Cas9 complex was mixed with 1×NEBuffer containing target DNA fragment (sequences are shown in the table below) (50ng). TM 3.1 The buffer solution was quickly mixed to initiate the Cas9-mediated DNA cleavage reaction and incubated at 37°C for 0.5 hours. TM 3.1 Buffer solution contains 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 and 100 μg / ml BSA (pH 7.9).

[0075] Table 2

[0076]

[0077]

[0078] 3. Evaluation of Cas9 tolerance to POC-modified sg-SLX4IP

[0079] The digestion system was denatured at 75°C for 3 minutes to terminate the reaction. SDS containing loading dye was added, and the samples were loaded on a 1.5% agarose gel containing 1.5× Super Gel Red for visualization (100V, 1.5 hours). In these experiments, GeneRuler 100-bp DNA Ladder was used as a DNA molecular weight marker. Bands were quantified using Image J software.

[0080] The results are as follows Figure 2As shown in Figure A, it can be seen that Cas9 can normally cleave POC-modified sg-SLX4IP within 60 minutes, indicating that sgRNA exhibits good tolerance to POC modifications within a moderate range. POC acylation modification is performed at a specific site (2'-OH) in the sgRNA to introduce an alkyne group. This modification is intended to ensure the structural integrity and function of the sgRNA.

[0081] The results are as follows Figure 2 As shown in Figure B, the inhibition of CRISPR-Cas9 depends on the modification time of sg-SLX4IP by POC. The longer the modification time, the more obvious the inhibitory effect.

[0082] 4. CuAAC-based sgRNA chemical cross-linking to regulate gene editing,

[0083] q-N3 and catalytic reagent sodium ascorbate were added to the cleavage system in step 3 and incubated at 37° C. for 30 min.

[0084] The results of the dose-dependent effect of q-N3 on the inhibition of CRISPR / Cas9 by POC-modified sg-SLX4IP are shown in Figure 2. Figure 2 As shown in Figure C, q-N3 inhibited the activity of sg-SLX4IP in a concentration-dependent manner, and the inhibitory effect gradually increased with increasing q-N3 concentration. The corresponding EC50 value was calculated to be 1.026 μM.

[0085] Example 3: Method for Modifying and Regulating Gene Editing Targeting the GAPDH Gene

[0086] 1. POC modification

[0087] sg-GAPDH (sequence shown in SEQ ID NO. 2) (20 μM) was added to a solution containing POC and incubated at 37°C with shaking at 200 rpm for a specified time. The POC solution included 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and a 180-220 mM DMAP aqueous solution, wherein the volume ratio of DMAP to water in the DMAP aqueous solution was 1:9-11. The reaction was terminated by adding 2.0 μL of 3 M NaOAc (sodium acetate), 75 μL of L ethanol, and 1.0 μL of glycogen (5 g / L). The sample was then stored at -80°C for 3 hours and then centrifuged at 12,000 g for 2 hours. The product was purified by ethanol precipitation. Finally, the POC-modified sg-GAPDH was collected and resuspended in nuclease-free water to a concentration of approximately 10 μM.

[0088] 2. In vitro Cas9 cleavage assay

[0089] The same volume of POC modified sg-GAPDH and Cas9 complex was mixed with 1×NEBuffer containing target DNA fragment (sequences are shown in the bold and underlined sequence in the table below) (50ng). TM 3.1 The buffer solution was quickly mixed to initiate the Cas9-mediated DNA cleavage reaction and incubated at 37°C for 0.5 hours. TM 3.1 Buffer solution contains 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 and 100 μg / ml BSA (pH 7.9).

[0090] Table 3

[0091]

[0092] 3. Evaluate Cas9 tolerance to POC-modified sg-GAPDH

[0093] The digestion system was denatured at 75°C for 3 minutes to terminate the reaction. SDS containing loading dye was added, and the samples were loaded on a 1.5% agarose gel containing 1.5× Super Gel Red for visualization (100V, 1.5 hours). In these experiments, GeneRuler 100-bp DNA Ladder was used as a DNA molecular weight marker. Bands were quantified using Image J software.

[0094] The results are as follows Figure 3 As shown in Figure A, Cas9 can normally cleave POC-modified sg-GAPDH within 40 minutes, indicating that sgRNA exhibits good tolerance to POC modifications within a moderate range. POC acylation is performed at a specific site (2'-OH) in the sgRNA to introduce an alkyne group. This modification is intended to ensure the structural integrity and function of the sgRNA.

[0095] The results are as follows Figure 3 As shown in Figure B, the inhibition of CRISPR-Cas9 depends on the modification time of sg-GAPDH by POC. The longer the modification time, the more obvious the inhibitory effect.

[0096] 4. CuAAC-based sgRNA chemical cross-linking to regulate gene editing,

[0097] q-N3 and catalytic reagent sodium ascorbate were added to the cleavage system in step 3 and incubated at 37° C. for 30 min.

[0098] The results of the dose-dependent effect of q-N3 on the inhibition of CRISPR / Cas9 by POC-modified sg-GAPDH are shown in Figure 2. Figure 3 As shown in Figure C, q-N3 inhibited the activity of sg-GAPDH in a concentration-dependent manner, and the inhibitory effect gradually increased with increasing q-N3 concentration. The corresponding EC50 value was calculated to be 5.10 μM.

[0099] Example 4: Method for Modifying and Regulating Gene Editing Targeting the HBEGF Gene

[0100] 1. POC modification

[0101] sg-HBEGF (sequence shown in SEQ ID NO. 3) (20 μM) was added to a solution containing POC and incubated at 37°C with shaking at 200 rpm for a specified time. The POC solution consisted of 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and a 180-220 mM DMAP aqueous solution, wherein the volume ratio of DMAP to water was 1:9-11. The reaction mixture was terminated by adding 2.0 μL of 3 M NaOAc (sodium acetate), 75 μL of L ethanol, and 1.0 μL of glycogen (5 g / L). The sample was then stored at -80°C for 3 hours and then centrifuged at 12,000 g for 2 hours. The product was purified by ethanol precipitation. Finally, the POC-modified sg-HBEGF was collected and resuspended in nuclease-free water to a concentration of approximately 10 μM.

[0102] 2. In vitro Cas9 cleavage assay

[0103] The same volume of POC modified sg-HBEGF and Cas9 complex was mixed with 1×NEBuffer containing target DNA fragment (sequences are shown in the table below) (50ng). TM 3.1 The buffer solution was quickly mixed to initiate the Cas9-mediated DNA cleavage reaction and incubated at 37°C for 0.5 hours. TM 3.1 Buffer solution contains 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 and 100 μg / ml BSA (pH 7.9).

[0104] Table 4

[0105]

[0106] 3. Evaluate Cas9 tolerance to POC-modified sg-HBEGF

[0107] The digestion system was denatured at 75°C for 3 minutes to terminate the reaction. SDS containing loading dye was added, and the samples were loaded on a 1.5% agarose gel containing 1.5× Super Gel Red for visualization (100V, 1.5 hours). In these experiments, GeneRuler 100-bp DNA Ladder was used as a DNA molecular weight marker. Bands were quantified using Image J software.

[0108] The results are as follows Figure 4 As shown in Figure A, Cas9 can normally cleave POC-modified sg-HBEGF within 40 minutes, indicating that sgRNA exhibits good tolerance to POC modifications within a moderate range. POC acylation modification is performed at a specific site (2'-OH) in the sgRNA to introduce an alkyne group. This modification is intended to ensure the structural integrity and function of the sgRNA.

[0109] The results are as follows Figure 4 As shown in Figure B, the inhibition of CRISPR-Cas9 depends on the modification time of sg-HBEGF by POC. The longer the modification time, the more obvious the inhibitory effect.

[0110] 4. CuAAC-based sgRNA chemical cross-linking to regulate gene editing

[0111] q-N3 and catalytic agent sodium ascorbate were added to the cleavage system in step 3 and incubated at 37°C for 30 minutes. The results of the dose-dependent effect of q-N3 on the inhibition of CRISPR / Cas9 by POC-modified sg-HBEGF are shown in Figure 2. Figure 4 As shown in Figure C, q-N3 inhibited the activity of sg-HBEGF in a concentration-dependent manner, and the inhibitory effect gradually increased with increasing q-N3 concentration. The corresponding EC50 value was calculated to be 3.09 μM.

[0112] Example 5: DNA cleavage experiment in HeLa-OC cells

[0113] 1. POC modification of sgRNA before delivery to HeLa-OC cells

[0114] The specific method is shown in Example 1 to obtain POC-modified sg-SLX4IP.

[0115] 2. Transfection

[0116] Human HeLa-OC cells were purchased from Gibco TMThe cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose medium (ThermoFisher Scientific) containing 10% (v / v) Gibco TM Fetal bovine serum (FBS, ThermoFisher Scientific) and 1% penicillin / streptomycin (Invitrogen) were placed in a humidified atmosphere at 37°C and 5% CO2.

[0117] Cells (2×10 per well) 5 Cells were seeded into 12-well plates and incubated overnight before transfection. Before the experiment, cells were washed twice with DPBS (Dulbecco's PBS) and 300 μL of prewarmed DMEM was added to each well. sg-SLX4IP (1.25 μg, at a concentration of 0.5 μg / μL) was mixed in 50 μL of DMEM. Lipofectamine 3000 transfection reagent (3.3 μL, Thermo Fisher Scientific) was added to 50 μL of DMEM per well, which was then added to the diluted sg-SLX4IP. The mixture was incubated at room temperature for 15 minutes to form the sg-SLX4IP:Lipofectamine complex and then transferred to the wells containing cells and growth medium.

[0118] 3. To determine the effect of this method on transfected sg-SLX4IP, two sets of experiments were performed.

[0119] First, sg-SLX4IP was transfected without q-N3. After 4 hours of incubation, the transfection medium was replaced with fresh complete medium.

[0120] Secondly, sg-SLX4IP was transfected without q-N3. After 4 h of incubation, the medium was replaced with fresh complete medium containing different concentrations of q-N3 (8, 16, and 32 μM) and catalytic reagent (CuSO4 / BTTAA / sodium ascorbate, 32 / 64 / 320 μM).

[0121] After 28 hours, the INDEL rate was analyzed using T7E1 technology to quantify the gene editing efficiency. Figure 11As shown, although the gene-editing activity of the modified sg-SLX4IP was reduced, it remained above 74% of the activity of the unmodified sg-SLX4IP, demonstrating that the POC-modified sgRNA can effectively induce targeted indel generation in HeLa-OC cells. Treatment with q-N3 and a catalyst significantly reduced the editing efficiency of sg-SLX4IP at various modification times. In particular, the editing efficiency of sg-SLX4IP modified for 30 minutes decreased from 49.7% to 5.4%, indicating that chemical cross-linking almost completely inhibited the activity of sg-SLX4IP in living cells. Meanwhile, q-N3 treatment had no significant effect on the gene-editing activity of the unmodified sgRNA. When q-N3 was added at varying concentrations 4 hours after transfection, the DNA cleavage efficiency of the CRISPR-Cas9 system decreased with increasing q-N3 concentration, indicating that q-N3 inhibits the function of the cellular CRISPR-Cas9 system in a concentration-dependent manner.

[0122] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0123] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0124] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for modifying and regulating gene editing based on sgRNA, characterized in that: The method comprises: Perform POC acylation modification at the 2'-OH site of sgRNA to introduce an alkyne tag to obtain an sgRNA with an alkyne group; The sgRNA with an alkyne group undergoes a copper-catalyzed azide-alkyne cycloaddition reaction with an azide in the presence of a copper-catalyzed reagent to form a stable chemical cross-linking structure, thereby achieving rapid shut-down regulation of the sgRNA activity.

2. The method according to claim 1, characterized in that The method of performing POC acylation modification at the 2'-OH site of the sgRNA to introduce an alkynyl label to obtain an sgRNA with an alkynyl group specifically includes: The sgRNA sample was incubated in a solution containing POC (2-propyn-1-yl 1H-imidazole-1-carboxylate), and then a stop solution was added to terminate the reaction to obtain an sgRNA with an alkyne group.

3. The method according to claim 2, characterized in that The POC-containing solution includes 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and 180-220 mM DMAP aqueous solution, wherein the volume ratio of DMAP to water in the DMAP aqueous solution is 1:9-11.

4. The method according to claim 2, characterized in that: The mass volume ratio of the sgRNA sample to the solution containing POC is 1-3 μM / μL.

5. The method according to claim 1, characterized in that The azide is tetrakis(azidomethyl)methane.

6. The method according to claim 1, characterized in that The copper catalytic agent is CuSO4 / BTTAA / Sodium Ascorbate, and the molar ratio of CuSO4, BTTAA and Sodium Ascorbate is 1:2:(9-11).

7. The method according to claim 1, characterized in that The mass volume ratio of the sgRNA with an alkyne group to the azide is in the range of 5-10 μM / μL.

8. A modified sgRNA having a chemical cross-linked structure obtained by the method according to any one of claims 1 to 7.

9. Use of the modified sgRNA having a chemical cross-linking structure according to claim 8 in the preparation of products for regulating gene editing.

10. A complete set of additive products for regulatory gene editing based on sgRNA modification, characterized in that: The product comprises: a solution containing POC, tetrakis(azidomethyl)methane and a copper catalytic agent, wherein the solution containing POC comprises 90-110 mM HEPES, 5-7 mM MgCl2, 90-110 mM NaCl, 50%-70% (v / v) DMSO, 1 M POC, and a 180-220 mM DMAP aqueous solution, wherein the volume ratio of DMAP to water in the DMAP aqueous solution is 1:9-11.

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