Application of beta-nicotinamide mononucleotide in regulation and control of gene editing efficiency

Abstract

The invention discloses application of beta-nicotinamide mononucleotide in regulation and control of gene editing efficiency, belongs to the field of gene editing treatment, and finds that the beta-nicotinamide mononucleotide (NMN) can efficiently inhibit the activity of CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13 systems in a broad-spectrum manner for the first time. The application comprises emergency blocking of off-target effect in gene editing clinical treatment, biological safety prevention and control of a virus vector gene editing system, and CRISPR activity regulation and control of in-vitro non-diagnostic purpose. Experiments show that NMN can inhibit CRISPR-mediated gene damage and cell death in a cell model, the inhibition efficiency in an in-vitro enzyme digestion system reaches 68.7%, and cell growth or transfection efficiency is not affected. The invention provides an innovative solution for safe application of CRISPR (clustered regularly interspaced short palindromic repeats) technology, and the NMN is approved to be taken orally as a health care product, so that the NMN has extremely strong clinical application potential. Compared with the existing CRISPR (clustered regularly interspaced short palindromic repeats)-resistant protein or synthetic small-molecule inhibitor, the NMN has the advantages of endogenous property, high biocompatibility, good oral safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of gene editing therapy, specifically relating to the application of a β-nicotinamide mononucleotide in regulating gene editing efficiency. Background Technology

[0002] The CRISPR-Cas system, with its precision and versatility, has revolutionized genome editing, enabling applications in fields such as genetic disease treatment, agricultural improvement, and synthetic biology. However, the clinical translation of this technology remains constrained by challenges such as off-target effects and uncontrollable nuclease activity. Current CRISPR inhibition strategies, including phage-derived anti-CRISPR proteins (Acr) and synthetic small molecule inhibitors, suffer from insufficient specificity and biological rejection. For example, AcrIIC1 and AcrIIA3, as two anti-Cas9 proteins, typically target only a single Cas protein through specific inhibitory mechanisms, and as exogenous biomolecules, they can trigger an immune response in vivo. Synthetic small molecule inhibitors often suffer from poor cell penetration, high cytotoxicity, long development cycles, and the potential for biological rejection, all of which severely hinder their clinical translation. These inhibitors cannot meet the expanding needs of the CRISPR toolkit—which now includes Cas12 for multiplex DNA editing and Cas13 for RNA knockdown.

[0003] Nicotinamide mononucleotide (NMN) is an endogenous metabolite of vitamin B3 and a key precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). NAD+ is an indispensable coenzyme for cellular energy metabolism and genome maintenance. NMN exhibits significant therapeutic potential due to its excellent pharmacokinetic properties. As an endogenous molecule, it has high bioavailability when administered orally, and clinical studies have confirmed its safety and tolerability in humans. Although it has been put into clinical use, the molecular mechanism by which NMN directly interacts with DNA remains unclear. Previous studies have focused on the role of NMN in promoting gene repair after conversion to the NAD+ precursor, but our understanding of whether it directly regulates DNA repair proteins or interacts with damaged DNA structures remains blank. This invention reveals for the first time that NMN can directly bind to nucleic acids and Cas proteins, thereby inhibiting various CRISPR-mediated gene editing processes. Through in-depth mechanistic studies, we discovered two pathways for inhibiting CRISPR. This means that we can utilize its endogenous properties to develop safer and more effective strategies to maintain genome integrity through gene therapy based on CRISPR gene editing technology. Summary of the Invention

[0004] Based on the technical problems existing in the prior art, the present invention provides an application of β-nicotinamide mononucleotide in regulating gene editing efficiency. It is a novel functional discovery and application of β-nicotinamide mononucleotide (NMN), which can interact with a variety of components efficiently and broadly, thereby inhibiting several common CRISPR systems.

[0005] According to a first aspect of the technical solution of the present invention, the present invention first provides the use of β-nicotinamide mononucleotide (NMN) in the preparation of inhibitors for inhibiting the activity of the CRISPR system.

[0006] Preferably, the CRISPR system is selected from at least one of CRISPR-Cas9, CRISPR-Cas12, or CRISPR-Cas13.

[0007] Preferably, the inhibition is concentration-dependent and / or incubation time-dependent.

[0008] According to a second aspect of the present invention, an application of β-nicotinamide mononucleotide (NMN) is provided, comprising one or more of the following: a) Activity regulation in CRISPR enzyme digestion experiments; b) Safety verification of gene editing tools; c) For emergency blocking of off-target effects during clinical gene editing treatment; d) Prevent biosafety issues arising from gene editing systems based on viral vectors.

[0009] Preferably, the application is for non-therapeutic or non-diagnostic purposes.

[0010] According to a third aspect of the technical solution of the present invention, the present invention provides a method for inhibiting the enzymatic activity of the CRISPR system in vitro, comprising co-incubating NMN with the substrate nucleic acid and / or Cas protein of the CRISPR system in vitro, wherein the incubation concentration is 1-100 mM and the incubation time is 10-60 minutes.

[0011] Preferably, the CRISPR system is CRISPR-Cas9.

[0012] Preferably, the method is for non-therapeutic purposes and is carried out in a buffer system with a pH of 7.0-7.5.

[0013] According to a fourth aspect of the present invention, the present invention also provides the use of β-nicotinamide mononucleotide (NMN) in the preparation of a drug for treating CRISPR-mediated gene damage, said use being achieved by inhibiting CRISPR activity.

[0014] Preferably, the drug is an oral formulation, and the purity of β-nicotinamide mononucleotide is ≥99.9%.

[0015] Compared with existing technologies, the application of β-nicotinamide mononucleotide in regulating gene editing efficiency of the present invention has the following beneficial effects: 1. This invention provides a novel functional discovery and application of β-nicotinamide mononucleotide, which interacts with multiple components in the CRISPR system to achieve efficient inhibition of three common CRISPR systems: CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13.

[0016] 2. Compared with existing CRISPR inhibition technologies based on phage proteins or synthetic compounds, this invention is the first endogenous small molecule compound CRISPR inhibitor, which has good adaptability to the body. NMN has high biocompatibility and has been approved for oral use, showing great potential for clinical application.

[0017] 3. The application of β-nicotinamide mononucleotide in regulating gene editing efficiency in this invention provides a new use for β-nicotinamide mononucleotide (NMN): gene editing blocking when off-target effects occur and cause serious consequences during gene editing clinical treatment; or for preventing biosafety issues caused by viral vector-based gene editing systems. Attached Figure Description

[0018] Figure 1 This is a schematic diagram based on the present invention; Figure 2A A schematic diagram illustrating cell death following CRISPR-Cas9 targeting Alu; Figure 2B Statistical data on cell confluence after CRISPR-Cas9 targeting Alu; Figure 2C Results of CRISPR-Cas9 system plasmid transfection and untransfected comet experiments targeting the Alu sequence; Figure 2D This refers to statistical data on the ratio of comet tail length to total comet length in comet experiments. Figure 2E Comparison of cell fusion rates after transfection with CRISPR-Cas9 system plasmids targeting the Alu sequence with and without NMN addition; Figure 2F Statistical data on cell fusion rates with and without NMN addition for CRISPR-Cas9 system plasmids targeting the Alu sequence; Figure 2G Comparison of the effects of adding NMN or not on cell growth 12 hours later; Figure 2H Statistical data on cell growth 12 hours after NMN was added; Figure 2I This demonstrates the effectiveness of normal transfection of plasmids containing the GFP protein sequence into cells. Figure 2J To assess the transfection effect of adding NMN to the culture medium when a plasmid containing the GFP protein sequence enters the cell; Figure 2K Add statistics on transfection efficiency before and after NMN; Figure 3A This indicates that the cleavage efficiency of the CRISPR-Cas9 in vitro enzyme digestion kit was inhibited after the addition of NMN to the system, while the enzyme digestion in the acidic control group was normal. Figure 3B This indicates that when NMN is pre-incubated with the corresponding substrate nucleic acid, the cleavage efficiency of the CRISPR-Cas9 in vitro enzyme digestion kit is inhibited, and different nucleic acid deposition patterns appear in the wells; Figure 3C This indicates that when NMN is pre-incubated with Cas9, the cleavage efficiency of the CRISPR-Cas9 in vitro enzyme digestion kit is inhibited, and different nucleic acid deposition patterns appear in the wells; Figure 3D This indicates that direct incubation of DNA with NMN does not cause abnormal electrophoretic migration; Figure 3E This indicates that when NMN is pre-incubated with different components of the corresponding substrate nucleic acid, the cleavage efficiency of the CRISPR-Cas9 in vitro enzyme digestion kit is inhibited, and different nucleic acid deposition patterns appear in the wells; Figure 3F This indicates that the cleavage efficiency of the CRISPR-Cas12 in vitro enzyme digestion kit was inhibited after NMN was pre-incubated with the corresponding substrate nucleic acid. Figure 3G This indicates that the cleavage efficiency of the CRISPR-Cas12 in vitro enzyme digestion kit was inhibited after NMN was pre-incubated with Cas12. Figure 3H This indicates that the cleavage efficiency of the CRISPR-Cas13 in vitro enzyme digestion kit was inhibited after NMN was pre-incubated with the corresponding substrate nucleic acid. Figure 3I This indicates that the cleavage efficiency of the CRISPR-Cas13 in vitro enzyme digestion kit was inhibited after NMN was pre-incubated with Cas13. Figure 4A The principle of qPCR detection of substrate residues; Figure 4BA statistical graph showing the Cq values ​​of residual substrates in qPCR detection results after 30 minutes of pre-incubation of different concentrations of NMN with corresponding substrates in the CRISPR-Cas9 system; Figure 4C The results of qPCR detection of residual substrates after pre-incubation of different concentrations of NMN with corresponding substrates in the CRISPR-Cas9 system for 30 minutes; Figure 4D A heatmap showing the specificity analysis results of the detection of wild-type LASV virus plasmid using a lateral flow chromatography test strip method; and the percentage of residual substrate after 30 minutes of pre-incubation of different concentrations of NMN with the corresponding substrates of the CRISPR-Cas9 system in qPCR detection results. Figure 4E A statistical graph showing the Cq values ​​of residual substrates in qPCR detection results of 40 mM NMN and corresponding substrates in the CRISPR-Cas9 system after different pre-incubation times; Figure 4F The results of qPCR detection of residual substrates after different pre-incubation times for 40 mM NMN and corresponding substrates in the CRISPR-Cas9 system; Figure 4G A heatmap showing the percentage of residual substrate in qPCR detection results for 40 mM NMN and the corresponding substrates in the CRISPR-Cas9 system after different pre-incubation times; Figure 4H A statistical graph showing the Cq values ​​of residual substrates detected by qPCR after pre-incubation of different concentrations of NMN with Cas9 for 30 minutes. Figure 4I The results of qPCR detection of residual substrates after pre-incubation of different concentrations of NMN with Cas9 for 30 minutes; Figure 4J A heatmap showing the percentage of residual substrate detected by qPCR after pre-incubating different concentrations of NMN with Cas9 for 30 minutes; Figure 4K A statistical graph showing the Cq values ​​of residual substrates in qPCR detection of 40 mM NMN and Cas9 after different pre-incubation times; Figure 4L The results of qPCR detection of residual substrates of 40 mM NMN and Cas9 after different pre-incubation times are presented.

[0019] Figure 4M A heatmap showing the percentage of residual substrate in qPCR detection of 40 mM NMN and Cas9 after different pre-incubation times; Figure 4NTo investigate the differences in qPCR detection efficiency after adding different concentrations of NMN to the same nucleic acid content of the test product system; Figure 4O The relationship between the in vitro enzyme cleavage inhibition efficiency of CRISPR-Cas9 and the concentration of NMN after pre-incubation of NMN with the corresponding substrate of the CRISPR-Cas9 system for 30 minutes; Figure 4P The relationship between the in vitro enzyme cleavage inhibition efficiency of 40 mM NMN and the corresponding substrates of the CRISPR-Cas9 system under different pre-incubation times was investigated. Detailed Implementation

[0020] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0021] The reagents used in the following examples and their sources are as follows: Biosafety cabinet (catalog number: NU-433-400S) was purchased from Nuaire, Inc., USA. Carbon dioxide incubator (catalog number: MCO-170AICDL-PC) was purchased from Pröhsch Ltd. Centrifuge (catalog number: 5702JL453003) was purchased from Eppendorf Ltd. Fluorescence inverted microscope (catalog number: CKX53) was purchased from Olympus Corporation. SCILANS adjustable mixer (catalog number: VB218XN0021453) was purchased from SCILOGEX, Inc., USA. MiniL-12G mini centrifuge (catalog number: MiNiL-12G-240514003) was purchased from Shanghai Maigao Scientific Instruments Co., Ltd. PHS-3E pH meter (catalog number: 600721N0022010214) was purchased from Shanghai Innasa Scientific Instruments Co., Ltd. The mini gel tray (catalog number: 1104241850) and PCR instrument application biosystems instrument (catalog number: A24812) were purchased from Thermo Fisher Scientific. The PowerPac Basic (catalog number: 041BR319570) was purchased from Bio-Rad Laboratories (Shanghai) Co., Ltd. The LightCycler 96 instrument (catalog number: 05815916001) was purchased from Roche Group, Germany. SpCas9 nuclease (Catalog No.: 32101), 10× HOLMESCas9 buffer (Catalog No.: 32041), TOLO Cas9 control target and sgRNA (Catalog No.: 32020), Cas9 control target double-stranded DNA (Catalog No.: 32020-TP1), Cas9 control sgRNA (Catalog No.: 32020-TP2), LbCas12a nuclease (Catalog No.: 32108-01), TOLO Cas12a control target and crRNA (Catalog No.: 32021-01), 10× HOLMES Cas12a buffer (Catalog No.: 32108-03), Cas12a control target DNA and crRNA (Catalog No.: 32021-01), LwaCas13a nuclease (Catalog No.: 32117-03), Cas13a control target RNA and crRNA (Catalog No.: 32024-01), TOLO Cas13a control target and crRNA (Catalog No.: 32024-01) were purchased from Tolo Biotechnology Co., Ltd. DMEM basal medium (1×) (Catalog No.: C11995500BT), TrypLE™ Expree (1×) (Catalog No.: 12605), LTX and Plus™ reagents (Catalog No.: 15338100), NativePAGE™ 20× running buffer (Catalog No.: BN2001), and NativePAGE™ 3-12% Bis-Tris gel (Catalog No.: BN1003BOX) were purchased from Thermo Fisher Scientific.PBS (1×) (Catalog No.: PB180327) was purchased from Wuhan Presby Biotechnology Co., Ltd. Nuclease-free water (Catalog No.: BL510B) was purchased from BlueJack Technology Co., Ltd. 50× TAE buffer (Catalog No.: B1110) was purchased from Beijing Anpu Biotechnology Co., Ltd. HiPure agarose (Catalog No.: MF-103-01) was purchased from Guangzhou Maigen Biotechnology Co., Ltd. M5 Gelred Plus nucleic acid dye (10000X) (Catalog No.: MF079-plus-01) was purchased from Meisi Biotechnology Co., Ltd. 2K Plus II DNA Marker (Catalog No.: BM121) was purchased from TransGen Biotech; 6× DNA loading buffer (blue / cyan) (Catalog No.: 1109566) was purchased from Tiangen Biotech (Beijing) Co., Ltd. 2× Super Pfx Master Mix (Catalog No.: CW2965M) was purchased from Jiangsu Kewen Biotechnology Co., Ltd.

[0022] The application of β-nicotinamide mononucleotide (NMN) in regulating gene editing efficiency in this invention is based on the rapid development of nucleic acid detection technology, especially next-generation molecular diagnostic technology based on clustered regularly interspaced short palindromic repeats (CRISPR). The inventors have developed a pathogen nucleic acid detection platform based on the CRISPR-Cas13a (CRISPR-associated protein 13) system, achieving a detection sensitivity reaching attomolar (aM). Combined with isothermal amplification techniques such as recombinase-aided amplification ((RT)RAA), it can detect single-copy pathogen nucleic acids. The CRISPR-Cas13a detection technology can be combined with lateral flow chromatography strips to achieve naked-eye visualization of the test results. This technology has advantages such as speed, simplicity, and the ability to perform on-site testing. Based on this nucleic acid detection technology, detection methods for various pathogens such as Ebola virus, Marburg virus, Zika virus, and COVID-19 infection can be developed.

[0023] According to a first aspect of the technical solution of the present invention, the present invention first provides the use of β-nicotinamide mononucleotide (NMN) in the preparation of inhibitors for inhibiting the activity of the CRISPR system.

[0024] In some embodiments, the CRISPR system is selected from at least one of CRISPR-Cas9, CRISPR-Cas12, or CRISPR-Cas13.

[0025] In some embodiments, the inhibition is a concentration-dependent and / or incubation time-dependent inhibition.

[0026] According to a second aspect of the present invention, applications of β-nicotinamide mononucleotide (NMN) are provided, wherein the applications include one or more of the following: a) Activity regulation in CRISPR enzyme digestion experiments; b) Safety verification of gene editing tools; c) For emergency blocking of off-target effects during clinical gene editing treatment; d) Prevent biosafety issues arising from gene editing systems based on viral vectors.

[0027] In some embodiments, the application is for non-therapeutic or non-diagnostic purposes.

[0028] According to a third aspect of the technical solution of the present invention, the present invention also provides a method for inhibiting the enzymatic activity of the CRISPR system in vitro, comprising co-incubating NMN with the substrate nucleic acid and / or Cas protein of the CRISPR system in vitro, wherein the incubation concentration is 1-100 mM and the incubation time is 10-60 minutes.

[0029] In some embodiments, the CRISPR system is CRISPR-Cas9.

[0030] In some embodiments, the method is for non-therapeutic purposes and is carried out in a buffer system with a pH of 7.0-7.5.

[0031] According to a fourth aspect of the present invention, the present invention also provides the use of β-nicotinamide mononucleotide (NMN) in the preparation of a drug for treating CRISPR-mediated gene damage, said use being achieved by inhibiting CRISPR activity.

[0032] In some embodiments, the drug is an oral formulation and the NMN purity is ≥99.9%.

[0033] The following specific embodiments further illustrate the technical solution for the application of β-nicotinamide mononucleotide (NMN) in regulating gene editing efficiency.

[0034] Example 1: Intracellular inhibition of the NMN-based CRISPR-Cas9 system.

[0035] Detection designs based on (RT)RAA / CRISPR, such as Figure 1 As shown, it includes the following steps: Step S1: Perform CRISPR-Cas9-based gene damage on the cells. HEK293 cells were cultured at 1 × 10⁻⁶ cells per well. 6Cells were seeded at a density of 1000 cells / well in 24-well plates. Cells were cultured in 1 mL of Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin-streptomycin. The culture plates were then incubated at 37°C for 24 hours under humidified conditions containing 5% CO2. Plasmids were transfected into the cells; the CRISPR-Cas9-Alu targeting plasmid was used under DNA damage conditions, while the px459 plasmid (empty vector control) was used as a control. Two transfection solutions were prepared: Solution A: 50 µL Opti-DMEM, 1 µg plasmid DNA, and 10 µL PLUS™ reagent, incubated at room temperature for 10 minutes. Solution B: 50 µL Opti-DMEM and 10 µL Lipofectamine™ LTX reagent, incubated at room temperature for 10 minutes. Solution A was then mixed with Solution B, gently stirred, and incubated at room temperature for another 10 minutes to form a complex. The complete transfection mixture (100 µL / well) was added to five different locations in each well containing cells and culture medium. The cells were then returned to an incubator at 37°C and 5% CO2. Forty-eight hours post-transfection, cell confluence was assessed and recorded using phase-contrast microscopy, and representative images were captured. Step S2: Comet assay to detect cellular gene damage. First, a basic agarose layer is prepared by melting 1% constant-melting-point agarose and pouring it into a molding tray. Glass slides are then immersed in the melt, followed by the removal of the agarose fragments on both sides (removing one-fifth of the outer width). The treated slides are air-dried overnight at room temperature for later use. For cell preparation, adherent cells are digested with trypsin, washed once with PBS, and centrifuged to precipitate the cells. The resulting precipitate is resuspended in PBS to a concentration of 1×10⁻⁶. 5Cells / mL. Separately, melt 0.75% low-melting-point agarose and keep it in a 37°C water bath to prevent solidification. Prepare a cell-agarose complex by gently mixing 30 μL of cell suspension with 50 μL of molten agarose. Add the mixture to the center of a pre-coated slide, cover with a coverslip to remove air bubbles, and then solidify in the dark at 4°C for 15 minutes. For lysis, remove the coverslip from the slide and immerse it in pre-chilled lysis buffer, incubating at 4°C in the dark for 2 hours. After lysis, wash the slide three times manually with gentle agitation in distilled water, 1 minute each time (do not mechanically shake). Place the washed slide parallel to the electric field in the electrophoresis tank, in pre-chilled alkaline electrophoresis buffer (pH>13), and incubate in the dark at 4°C for 20 minutes to develop DNA. Surround the electrophoresis tank with an ice bath. Electrophoresis is performed at 20 V (300 mA) in the dark for 25 minutes. The neutralization step involved three 1-minute washes with Tris-HCl buffer (0.4 M, pH 7.5), handled manually to prevent agarose detachment, followed by air drying at room temperature. For staining, 50 μL of ethidium bromide solution (500-fold dilution) was added to the gel, immediately covered with a coverslip to ensure even distribution, and incubated in the dark for 10 minutes. Immediately after staining, the gel was imaged using a fluorescence microscope at 200× magnification to capture the comet morphology.

[0036] Step S3, Cell Gene Damage Inhibition Assay. HEK293 cells were seeded in 24-well plates as described previously and cultured at 37°C in a humid environment with 5% CO2 for 24 hours. After removing the incubator, 500 µL of culture medium was aspirated from each well and discarded. Fresh culture medium was prepared as follows: DNA damage group: 500 µL of fresh culture medium (DMEM, supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin) was added to each well; NMN treatment group: 500 µL of fresh culture medium containing 20 mM NMN was added to each well, resulting in a final NMN concentration of 10 mM. Immediately after changing the culture medium, transfection was performed using the CRISPR-Cas9-Alu plasmid according to the transfection protocol described above. Cell confluence was assessed 48 hours after transfection, and representative phase-contrast microscopy images were taken.

[0037] Step S4: Exploring the effect of NMN on cell growth efficiency. HEK293 cells were seeded in 24-well plates as described previously and cultured at 37°C in a humidified environment with 5% CO2 for 24 hours. After removing the incubator, 500 µL of culture medium was aspirated from each well and discarded. Fresh culture medium was prepared as follows: Untreated control group: 500 µL of fresh culture medium (DMEM, supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin) was added to each well; NMN-treated group: 500 µL of fresh culture medium containing 20 mM NMN was added to each well, resulting in a final NMN concentration of 10 mM. Cell confluence was assessed and representative phase-contrast microscopy images were taken 12 hours after changing the culture medium.

[0038] Step S5: Exploring the effect of NMN on transfection efficiency. HEK293 cells were seeded in 24-well plates according to the previously described method and cultured at 37°C in a humid environment with 5% CO2 for 24 hours. After removing the incubator, 500 µL of culture medium was aspirated from each well and discarded. Fresh culture medium was prepared as follows: Untreated control group: 500 µL of fresh culture medium (DMEM, supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin) was added to each well; NMN-treated group: 500 µL of fresh culture medium containing 20 mM NMN was added to each well, resulting in a final NMN concentration of 10 mM. Immediately after changing the culture medium, transfection was performed using the PX458-GFP plasmid, following the transfection protocol described in the previous method / experimental steps. GFP fluorescence signal was evaluated 48 hours after transfection, and representative phase-contrast microscopy images were captured.

[0039] The results are as follows Figure 2A As shown, after CRISPR-Cas9 targets the Alu sequence, significant cell death occurs, resulting in a decrease in fusion rate. Figure 2B The cell confluence statistics show that the confluence of the target group decreased by about 20%, indicating that the CRISPR system induced cell death. Figure 2C and Figure 2D Results of the comet experiment: The ratio of comet tail length to total length increased by 12% in the transfected group, confirming DNA damage; the damage was reduced after the addition of NMN. Figure 2E and 2F The results showed that adding NMN increased cell fusion by 25%, and NMN did not affect cell growth. Figure 2G , Figure 2H ) or transfection efficiency ( Figures 2I-2K This demonstrates the specificity of the inhibition.

[0040] In summary, NMN inhibited CRISPR-Cas9-induced damage to cellular genes without affecting cell growth efficiency or transfection efficiency.

[0041] Example 2: Detection of the inhibition effect of enzyme digestion efficiency of various CRISPR systems, including the following: I. In vitro digestion using the CRISPR system. Assemble all in vitro lysis reaction mixtures into 50 μL PCR tubes kept on ice. For the standard cleavage reaction, the reaction mixture (5 μL final volume) contains: 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein, 0.25 μL positive control substrate (0.5 μL for Cas13), 4 μL nuclease-free water (3.75 μL for Cas13), and 0.5 μL HOMELESS reaction buffer; for the reaction co-incubated with NMN, the reaction mixture (5 μL final volume) contains: 0.25 μL Cas9 / Cas12 / Cas13 protein, 0.25 μL positive control substrate (0.5 μL for Cas13), 4 μL NMN (3.75 μL for Cas13) indicating concentration, and 0.5 μL HOMELESS reaction buffer; for the pre-incubation reaction with NMN-substrate, the reaction mixture (5 μL final volume) contains: 0.25 μL positive control substrate (0.5 μL for Cas13) and 4 μL HOMELESS reaction buffer. NMN premixed at an indicated concentration of 3.75 μL (Cas13: 3.75 μL) and incubated at room temperature for 30 min, followed by the addition of 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein and 0.5 μL HOMELESS reaction buffer. For the reaction premixed with NMN-protein, the reaction mixture (5 μL final volume) contained: 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein premixed with 4 μL NMN at an indicated concentration (Cas13: 3.75 μL) and incubated at room temperature for 30 min, followed by the addition of 0.25 μL positive control substrate (Cas13: 0.5 μL) and 0.5 μL HOMELESS reaction buffer. For the negative control (substrate control), the reaction mixture (5 μL final volume) contained: 0.25 μL positive control substrate (Cas13: 0.5 μL), 4.25 μL nuclease-free water (Cas13: 0.5 μL), and 4.25 μL nuclease-free water (Cas13: 0.5 μL). 4 μL of HOMELESS reaction buffer and 0.5 μL of HOMELESS reaction buffer. Note: This reaction is enzyme-deficient. After assembly, thoroughly mix all reaction mixtures by pipetting and incubate at 37°C for 30 minutes in a thermal cycler (PCR machine).

[0042] II. Agarose Gel Electrophoresis for DNA Visualization. Preparation of Agarose Gel: Accurately weigh a total of 1.5 g (1.5%) of agarose using an electronic balance and transfer it to an Erlenmeyer flask. Then, measure 100 mL of 1×TAE buffer solution using a graduated cylinder and add it to the flask. Thoroughly stir the mixture to ensure homogeneity, and then heat it in a microwave oven for 4 minutes. After heating, allow the solution to cool to a temperature range of 50–60 °C. At this point, incorporate 10 μL of 10000× nucleic acid dye into the solution. Then pour the mixture into a casting tray and insert a comb to form wells. The agarose gel solidifies at room temperature.

[0043] Sample loading: Before loading the sample into the gel, mix 1 μL of 6× DNA loading buffer with 5 μL of CRISPR enzyme digestion product. Adjust the voltage to 85 V and start electrophoresis for 40 min. Observation: After 40 min, remove the gel and observe the results in an AlphaImager HP gel imaging system.

[0044] III. RNA Visualization Detection via SDS-PAGE. Remove the SDS-PAGE gel (GeneScript) and place it in the SDS-PAGE electrophoresis tank. Before loading, mix 1.67 μL of 5× RNA loading buffer with 5 μL of CRISPR digestion product. Adjust the voltage to 85 V and electrophoresis for 60 minutes. Staining: After 60 minutes of electrophoresis, remove the gel and place it in a container. Add 100 mL of 1× nucleic acid dye and vortex for 120 minutes. Observation: Place the gel in an AlphaImager HP gel imaging system to observe the results.

[0045] The results are as follows Figure 3A As shown, the CRISPR-Cas9 cleavage efficiency was inhibited after the addition of NMN, while the acidic control group was normal, proving that the effect of NMN is not pH dependent. Figure 3B and 3C The results showed that NMN inhibited cleavage when pre-incubated with the substrate or Cas9, and abnormal nucleic acid deposition was observed in the wells, suggesting differences in binding mechanisms. Figure 3D It was confirmed that direct incubation of DNA with NMN did not cause abnormal migration, ruling out non-specific effects. Figures 3E-3I Extending to Cas12 and Cas13 systems: cleavage efficiency was suppressed after NMN pre-incubation, indicating broad-spectrum activity.

[0046] Conclusion: NMN has an inhibitory effect on multiple CRISPR systems in vitro, and the mechanism involves substrate or protein binding.

[0047] Example 3: Detection of the efficiency of NMN in inhibiting CRISPR-Cas9, which includes the following: I. In vitro digestion using the CRISPR system. Assemble all in vitro lysis reaction mixtures into 50 μL PCR tubes kept on ice. For the standard cleavage reaction, the reaction mixture (5 μL final volume) contains: 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein, 0.25 μL positive control substrate (0.5 μL for Cas13), 4 μL nuclease-free water (3.75 μL for Cas13), and 0.5 μL HOMELESS reaction buffer; for the reaction co-incubated with NMN, the reaction mixture (5 μL final volume) contains: 0.25 μL Cas9 / Cas12 / Cas13 protein, 0.25 μL positive control substrate (0.5 μL for Cas13), 4 μL NMN (3.75 μL for Cas13) indicating concentration, and 0.5 μL HOMELESS reaction buffer; for the pre-incubation reaction with NMN-substrate, the reaction mixture (5 μL final volume) contains: 0.25 μL positive control substrate (0.5 μL for Cas13) and 4 μL HOMELESS reaction buffer. NMN premixed at an indicated concentration of 3.75 μL (Cas13: 3.75 μL) and incubated at room temperature for 30 min, followed by the addition of 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein and 0.5 μL HOMELESS reaction buffer. For the reaction premixed with NMN-protein, the reaction mixture (5 μL final volume) contained: 0.25 μL Cas9 / Cas12 / Cas13 / dCas9 protein premixed with 4 μL NMN at an indicated concentration (Cas13: 3.75 μL) and incubated at room temperature for 30 min, followed by the addition of 0.25 μL positive control substrate (Cas13: 0.5 μL) and 0.5 μL HOMELESS reaction buffer. For the negative control (substrate control), the reaction mixture (5 μL final volume) contained: 0.25 μL positive control substrate (Cas13: 0.5 μL), 4.25 μL nuclease-free water (Cas13: 0.5 μL), and 4.25 μL nuclease-free water (Cas13: 0.5 μL). 4 μL of HOMELESS reaction buffer and 0.5 μL of HOMELESS reaction buffer. Note: This reaction is enzyme-deficient. After assembly, thoroughly mix all reaction mixtures by pipetting and incubate at 37°C for 30 minutes in a thermal cycler (PCR machine).

[0048] II. qPCR Detection of CRISPR Digestion Products. Prepare the reaction mixture according to the specified protocol as follows: 25 μL Taq Pro universal SYBR qPCR master mix, 2 μL per primer, 2 μL CRISPR digestion product, 19 μL deionized water, total volume 50 μL. The thermal cycling conditions are as follows: initial denaturation at 95℃ for 5 minutes; followed by 35 cycles, each cycle consisting of 95℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 45 seconds; and a final extension at 72℃ for 10 minutes. For each experimental group, add the reagents to the reaction tube, shake well to ensure thorough mixing, and then amplify using the ROUCH real-time PCR system. After the reaction is complete, evaluate the results based on the Cq values ​​and draw conclusions.

[0049] Figure 4A This is a schematic diagram illustrating the principle of qPCR. Figures 4B-4D The results showed that the inhibition rate was concentration-dependent when pre-incubated with NMN concentration gradients (20-80 mM) for 30 minutes, reaching 42.5% at 80 mM. Figures 4E-4G The results showed that the inhibition rate increased with time gradient of 40 mM NMN pre-incubation (10-35 minutes), reaching 40.0% at 35 minutes. Figures 4H-4M This indicates that NMN pre-incubation with Cas9 has a stronger inhibitory effect, with an average efficiency of 68.7%. Figures 4N-4P The relationship between inhibition efficiency and concentration and time was summarized, confirming the controllability.

[0050] Conclusion: The efficiency of NMN in inhibiting CRISPR-Cas9 depends on concentration and time, and the effect is optimal when it binds to the protein.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0052] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of β-nicotinamide mononucleotide in the preparation of an inhibitor for inhibiting the activity of a CRISPR system.

2. Use according to claim 1, wherein The CRISPR system is selected from at least one of CRISPR-Cas9, CRISPR-Cas12 or CRISPR-Cas13.

3. The use according to claim 1, wherein The inhibition of the activity of the CRISPR system is a concentration-dependent and / or incubation time-dependent inhibition.

4. Use of a β-nicotinamide mononucleotide, characterized in that, The use comprises one or more of the following: a) regulation of the activity of a CRISPR enzyme cleavage experiment; b) verification of the safety of a gene editing tool; c) emergency blocking of off-target effects in the process of clinical treatment of gene editing; d) prevention of biosafety problems caused by a gene editing system based on a viral vector.

5. The use of claim 4, wherein the CRISPR system is selected from the group consisting of Cas9, CaslO, Cpf1, and C2c3. 5 The use is for non-therapeutic or non-diagnostic purposes.

6. A method of inhibiting the activity of a CRISPR system enzyme in vitro, comprising contacting the enzyme with a compound of claim 1. The method comprises in vitro co-incubation of β-nicotinamide mononucleotide with a substrate nucleic acid and / or a Cas protein of a CRISPR system, at a concentration of 1-100 mM for 10-60 minutes.

7. The method of inhibiting CRISPR system enzymatic activity in vitro of claim 6, wherein, The CRISPR system is CRISPR-Cas9.

8. The method of inhibiting CRISPR system enzymatic activity in vitro of claim 6, wherein, The method is for non-therapeutic purposes, and the incubation is carried out in a buffer system at pH 7.0-7.

5.

9. Use of β-nicotinamide mononucleotide in the preparation of a drug for treating CRISPR-mediated gene damage, the use being achieved by inhibiting the activity of a CRISPR system.

10. Use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The drug is an oral preparation, and the purity of β-nicotinamide mononucleotide is ≥99.9%.

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