Application of pargyline hydrochloride, metadoxine and dimethyl fumarate compound in preparation of product for inhibiting combination or cutting of SpCas9 to substrate

Abstract

The invention belongs to the technical field of CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 gene editing, and particularly relates to application of a pargyline hydrochloride, metadoxine and dimethyl fumarate compound in preparation of a product for inhibiting combination or cutting of SpCas9 to a substrate. One or more of the pargyline hydrochloride, the metadoxine and the dimethyl fumarate are combined for use, and the effect of interfering the SpCas9 to cut the DNA is achieved by destroying the combination of the SpCas9 and the DNA.

Description

Technical Field

[0001] The present invention relates to the field of CRISPR Cas9 gene editing technology, and in particular to the use of pargyline hydrochloride, metadoxine and dimethyl fumarate compounds in the preparation of products that inhibit SpCas9 from binding to or cutting a substrate. Background Art

[0002] In 1987, Ishino et al. first discovered a large number of repetitive sequences containing regular intervals when studying the alkaline phosphatase gene of Escherichia coli in Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology 169, 5429-5433. However, due to the limitations of technology and knowledge at the time, no further research was conducted. Until the beginning of this century, after a large number of subsequent studies by researchers, it was found that this sequence was widely present in bacteria and archaea, and the tandem repeats arranged at intervals were named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

[0003] The CRISPR / Cas system is a complex phage defense mechanism composed of a CRISPR array and a series of related Cas proteins. The CRISPR array itself consists of three main parts: a leader sequence located at the front end of the array, a series of highly conserved repeat sequences, and multiple spacers. The leader sequence, as the promoter of the CRISPR array, regulates the expression of the CRISPR gene and is generally located at the start of the CRISPR array; the repeat sequence is 21 to 48 bp long, contains a palindromic sequence, and can form a hairpin structure (R-loop); the spacer sequence is integrated from exogenous DNA fragments, and they are the basis for the CRISPR system to recognize foreign genetic material. In order to better distinguish different CRISPR systems, researchers divided CRISPR / Cas into two categories and six types (Ⅰ-VI) according to the types and sequences of CRISPR associate (Cas) proteins and their roles in the bacterial immune defense process: Class 1 includes Type Ⅰ, Type Ⅲ and Type Ⅳ, which require multiple Cas effector proteins to form a complex to play the role of endonuclease; Class 2 includes Type Ⅱ, Type Ⅴ and Type Ⅵ, which only require a single Cas effector protein to perform functions. For example, the Type Ⅱ CRISPR / Cas system only requires one protein, Cas9, to effectively cut double-stranded DNA. It is this simple and efficient advantage that gives the Type Ⅱ CRISPR / Cas9 system a broad space for development, and its related research is also the most extensive and in-depth. According to the authoritative clinical trial website (ClinicalTrials), there are currently 70 clinical trials on CRISPR / Cas9 (as of May 2024), and the types of diseases treated cover single gene genetic diseases, ophthalmic diseases, AIDS and many other aspects. It is particularly worth mentioning that in December 2023, Casgevy (based on the CRISPR / Cas9 system), a gene editing therapy for the treatment of sickle cell disease jointly developed by the US biotechnology company Vertex Pharmaceuticals (VRTX.US) and the Swiss pharmaceutical company CRISPR Therapeutics (CRSPR.US), has been officially launched with the approval of the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA).

[0004] The CRISPR / Cas9-mediated genome editing process can be roughly divided into three key stages: first, the Cas9 protein binds to the single-stranded guide RNA (sgRNA) to form a complex; second, the complex recognizes and binds to the specific PAM (Protospacer Adjacent Motif) sequence on the target DNA under the guidance of the sgRNA; finally, the Cas9 protein cuts the target DNA to produce double-strand breaks (DSB). Different types of Cas proteins in the CRISPR system use their own specific PAM sequences to confirm the target site. Taking the CRISPR / Cas9 system as an example, its PAM sequence is usually NGG, where N can be any base. After Cas9 protein-mediated DNA cutting, cells will respond to DSBs through two main DNA repair mechanisms: one is the non-homologous end joining repair mechanism (NHEJ), which may cause small insertions or deletions (indels); the other is homology directed repair (HDR), which can accurately repair DSBs with the addition of exogenous repair templates.

[0005] Compared with previous gene editing technologies, the CRISPR / Cas9 system has several significant advantages: first, it can edit the genome accurately and efficiently; second, the CRISPR / Cas system is not restricted by species and is applicable to genome editing of a variety of organisms such as plants, bacteria, and animals; finally, the CRISPR / Cas system has the ability to edit multiple target sites on the genome at the same time. However, the CRISPR / Cas9 system also has many shortcomings. One of them is that DNA repair after DSB mainly depends on NHEJ. This repair mechanism may produce errors during the repair process, resulting in the loss of gene function or the acquisition of new unknown functions; another major problem is the off-target effect, that is, the gene editing tool may cut the wrong target DNA site, which is an unavoidable side effect of gene editing technology based on the principle of nucleic acid complementarity. With the application of the CRISPR / Cas system, the accumulation of off-target effects may lead to chromosome translocation and even induce genotoxic mutations.

[0006] In order to provide a good, effective, and safer medication window for clinical applications, and to provide an efficient CRISPR system switch for basic research, it is necessary to accurately control the CRISPR / Cas9 system, so the development of CRISPR / Cas9 system inhibitors becomes particularly important. Scientists have discovered a variety of protein molecules (Anti-CRISPR, ACR) that antagonize the CRISPR / Cas9 system, but these molecules have low affinity for the most commonly used Cas9 protein, SpCas9 (Cas9 from Streptococcus pyogenes). In addition, because ACR is essentially a protein molecule, it has strong immunogenicity and poor drugability, making it difficult to promote in clinical practice. Small molecule compounds can largely overcome these two deficiencies and are recognized as the most promising means to solve this problem. Amit Choudhary's research group at Harvard Medical School published two articles on small molecule inhibitors of CRISPR / Cas9 in Cell and Nature Cell Biology in 2019 and 2022, respectively: A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9. Cell. 2019 May. and A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Nat Cell Biol. 2022 Dec. They have successively developed two small molecule drugs, BRD0539 and BRD7586, but the in vivo safety of these two drugs has not been verified, and the synthesis process is complicated and the cost is high, which undoubtedly limits their further promotion. We hope to screen out small molecule inhibitors that can effectively control the CRISPR / (Sp)Cas9 system from the FDA-approved small molecule drug library, thereby avoiding the drawbacks of previous candidate drugs. There is no report on the binding or cleavage effect of pargyline hydrochloride, metadoxine and dimethyl fumarate on substrates, and there is no report on the binding or cleavage effect of pargyline hydrochloride, metadoxine and dimethyl fumarate on substrates when used in combination. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides the use of pargyline hydrochloride, metadoxine and dimethyl fumarate in the preparation of products that inhibit SpCas9 from binding or cutting substrates.

[0008] Use of pargyline hydrochloride, metadoxine and dimethyl fumarate in preparing products that inhibit SpCas9 from binding to or cleaving a substrate, wherein one or more of pargyline hydrochloride, metadoxine and dimethyl fumarate are used in combination;

[0009] The concentration of dimethyl fumarate was 1.56-500 μM;

[0010] The concentration of pargyline hydrochloride was 1.56-500 μM;

[0011] The concentration of metadoxine is 12.5-500 μM.

[0012] Preferably, the product comprises any of the compounds.

[0013] Preferably, the product also includes SpCas9 protein.

[0014] Preferably, the usage ratio of dimethyl fumarate to SpCas9 is 0.25-1mmol:5U.

[0015] Preferably, the dosage ratio of metadoxine to SpCas9 is 0.25-1mmol:5U.

[0016] Preferably, the usage ratio of dimethyl fumarate to SpCas9 is 0.25-1mmol:5U.

[0017] Preferably, pargyline hydrochloride, metadoxine and dimethyl fumarate are mixed in equal amounts.

[0018] Preferably, the product also includes SpCas9 protein.

[0019] Preferably, the total concentration of pargyline hydrochloride, metadoxine and dimethyl fumarate to the dosage ratio of SpCas9 protein is 0.25-1mmol:5U.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention provides the use of pargyline hydrochloride, metadoxine and dimethyl fumarate in inhibiting the binding or cutting of SpCas9 to a substrate, so that the "anti-CRISPR-Cas9" gene editing system can be more effectively controlled, and the binding of SpCas9 to DNA can be destroyed, thereby interfering with SpCas9 in cutting DNA.

[0022] 2. Pargyline hydrochloride has a significant antihypertensive effect and is a monoamine oxidase inhibitor. Its antihypertensive mechanism has not yet been fully elucidated. It may be due to the inhibition of monoamine oxidase, which changes the normal metabolism of tyramine in adrenergic nerve endings, producing 1-hydroxytyramine, which is a "pseudo-mediator" that can be stored, released and bound to receptors like norepinephrine, but because the reaction it causes is weak, it cannot play a role in the conduction of postganglionic sympathetic nerve impulses, resulting in vasodilation and lowering of blood pressure. Other monoamine oxidase inhibitors such as phenelzine also have antihypertensive effects, but as antihypertensive drugs, they have no practical value due to many side effects. It has not been reported that galactoside has an inhibitory effect on the binding or cutting of SpCas9 to the substrate.

[0023] Metadoxine is a water-soluble vitamin that can accelerate the excretion of ethanol and its metabolites acetaldehyde and ketone bodies through the kidneys. It is mainly used for acute alcohol poisoning. It is easily absorbed after oral administration, and its bioavailability is 60%-80%. The peak concentration appears in the blood about 1 hour after oral administration. It is widely distributed in the body, with the liver and kidney tissues being the highest. Pyridoxine and L-2-pyrrolidone-5-carboxylic acid in the blood and tissues still maintain an equal ratio. The half-life of blood concentration is 40-60 minutes. Metadoxine participates in the γ-glutamyl cycle and is metabolized, and the metabolites are finally excreted from urine (40%-45%) and feces (35%-50%).

[0024] Dimethyl fumarate (DMF) is an orally active Nrf2 activator that can penetrate the blood-brain barrier and induce upregulation of antioxidant gene expression. Dimethyl fumarate induces necrosis of colon cancer cells through the GSH depletion / ROS elevation / MAPKs activation pathway and induces cell autophagy. Dimethyl fumarate can be used in the study of multiple sclerosis.

[0025] Existing drugs have a low affinity for SpCas9, which means that the regulatory efficacy is not high and may not be able to achieve high-level regulation in vivo. However, the drugs provided in this application have been proven to be able to enter and regulate the human body under safe conditions and have a high affinity for SpCas9.

[0026] 3. In the previous literature review, it was found that the binding of pargyline hydrochloride, metadoxine and dimethyl fumarate to SpCas9 had no significant effect on its ability to bind or cut substrates. The application of pargyline hydrochloride, metadoxine and dimethyl fumarate and their combination in inhibiting SpCas9 binding or cutting substrates can extend the clinical application of SpCas9 and increase the clinical medication window, so as to improve doctors' application methods of SpCas9 drugs and continuously adjust clinical medication plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The electrophoresis diagrams of Pargyline Hydrochloride (3690), Metadoxine (5060) and Dimethyl Fumarate (2586) inhibiting CRISPR / Cas9 from cleaving target DNA, respectively, and BRD0539 is a positive control;

[0028] Figure 2 This is the electrophoresis diagram of the inhibition of CRISPR / Cas9 cleavage of target DNA by the concentration gradient of pargyline hydrochloride, and BRD0539 is the positive control;

[0029] Figure 3 This is a surface plasmon resonance (SPR) experimental result diagram of the binding of pargyline hydrochloride to the SpCas9 complex; the lines in the figure are cycle 13 (500 μM), cycle 12 (100 μM), cycle 11 (50 μM), cycle 10 (25 μM), cycle 9 (12.5 μM), cycle 8 (6.25 μM) from top to bottom, and cycles 8, 9, 10, 11, 12, and 13 overlap into one line;

[0030] Figure 4 This is the electrophoresis diagram of the dose-dependent inhibition of CRISPR / Cas9 cleavage of target DNA by metadoxine, and BRD0539 is the positive control;

[0031] Figure 5 This is a surface plasmon resonance (SPR) experimental result diagram of the binding of metadoxine to the SpCas9 complex; the lines in the figure are cycle 10 (25 μM), cycle 9 (12.5 μM), cycle 11 (50 μM), cycle 13 (500 μM), and cycle 12 (100 μM) from top to bottom.

[0032] Figure 6 This is the electrophoresis diagram of dimethyl fumarate dose-dependently inhibiting CRISPR / Cas9 from cleaving target DNA, and BRD0539 is the positive control;

[0033] Figure 7 The electrophoresis diagrams show that positive compounds BRD0539 and BRD7586 inhibit CRISPR / Cas9 from cutting target DNA.

[0034] Figure 8 This is the electrophoresis diagram of the positive compound BRD7586 dose-dependently inhibiting CRISPR / Cas9 from cutting the target DNA.

[0035] Fig. 9This is a surface plasmon resonance (SPR) experimental result diagram of the binding of the positive compound 1BRD0539 to the SpCas9 complex; the lines in the figure are cycle 13 (500μM), cycle 12 (100μM), cycle 11 (50μM), cycle 10 (25μM), cycle 9 (12.5μM), cycle 8 (6.25μM), cycle 7 (3.125μM) from top to bottom;

[0036] Fig.10 This is a surface plasmon resonance (SPR) experimental result diagram of the binding of the positive compound 2BRD7586 to the SpCas9 complex; the lines in the figure are cycle 9 (12.5μM), cycle 8 (6.25μM), cycle 7 (3.125μM), cycle 6 (1.56μM), cycle 5 (0.78μM), and cycle 4 (0.36μM) from top to bottom.

[0037] Fig.11 This is the electrophoresis diagram of the combined use of pargyline hydrochloride, metadoxine and dimethyl fumarate (ALL) to inhibit CRISPR / Cas9 from cutting target DNA.

[0038] Note: Figure 3 , Figure 5 , Fig. 9 and Fig.10 The digital symbols in the middle cycle indicate the detection of the coupling affinity of Cas9 protein and small molecule drugs under different drug concentration conditions. Graph 3690 is the drug Pargyline hydrochloride, 5060 is the drug Metadoxine, and 2586 is the drug Dimethyl Fumarate.

[0039] The DNA is the target DNA cut by CRISPR / Cas9 in the kit (Yinmao Shengye PC-1400). DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0041] Example 1

[0042] 1. Verification experiment of the inhibitory effect of compounds on CRISPR / Cas9 DNA cleavage

[0043] In order to verify the inhibitory effect of the drug on the DNA cutting of Cas9, we first carried out an enzyme cleavage reaction in a cell-free system, using the PC-1400 kit from Innovent Biologics and the method provided in the kit manual to perform an in vitro enzyme cleavage experiment. The enzyme cleavage efficiency was then verified by agarose gel electrophoresis, using the PCDNA and PC sgRNA in the kit.

[0044] First, use enzyme-free PCR tubes and enzyme-free pipette tips to conduct the experiment: add 5μL (1U / μL) of Cas9 protein, 5μL (1μg / μL) of PC sgRNA, 10μL (1μg / μL) of PC DNA, 1.2μL of small molecule compounds or solvents, and 2.4μL of 10×Cas9 Buffer to each sterile enzyme-free test tube, and then blow and mix. This process is carried out on ice. The specific groups are as follows:

[0045] A negative control group (solvent group) was set up: containing 1.2 μL of DMSO solvent, 5 μL of Cas9 protein (1 U / μL), 5 μL of PCsgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer. The mixture was then blown and mixed. The whole process was carried out on ice.

[0046] Positive control group 1 (BRD0539): contains 1.2 μL of 1 mM BRD0539, 5 μL of Cas9 protein (1 U / μL), 5 μL of PC sgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer. Then blow and mix well. The whole process was carried out on ice.

[0047] Positive control group 2 (BRD7586): contains 1.2 μL of 1 mM BRD7586, 5 μL (1 U / μL) of Cas9 protein, 5 μL (1 μg / μL) of PC sgRNA, 10 μL (1 μg / μL) of PC DNA, and 2.4 μL of 10×Cas9 Buffer. Then blow and mix well. The whole process is carried out on ice.

[0048] Target drug group: Add 1.2μL of the corresponding small molecule drug with a concentration of 1mM, 5μL of Cas9 protein (1U / μL), 5μL of PCsgRNA, 10μL of PC DNA, and 2.4μL of 10×Cas9 Buffer. Then blow and mix well. The whole process is carried out on ice. The small molecule drug is pargyline hydrochloride, metadoxine or dimethyl fumarate.

[0049] Combined drug group (ALL): Three small molecule drugs with a concentration of 1mM were added, 0.4μL of each, Cas9 protein 5μL (1U / μL), PC sgRNA 5μL, PC DNA 10μL, 10×Cas9 Buffer 2.4μL. Then blow and mix well, and the whole process was carried out on ice. The small molecule combined drug was a mixture of equal volumes of pargyline hydrochloride, metadoxine, and dimethyl fumarate.

[0050] Use a metal bath at 37°C for 30 minutes, then treat at 85°C for 10 minutes to complete the in vitro enzyme digestion reaction. After the enzyme digestion reaction, the product was detected and 5 μL of TIANGEN was added to each group. TM Protein K (20 mg / mL), this step was mixed on ice, and incubated in a metal bath at 56°C overnight. Prepare 2% agarose gel (add 4 μL YeaRed Nucleic Acid Gel Stain CAT: 10202ES76 (Yeasen Yisheng Biology) to every 40 mL of agarose gel solution).

[0051] Take 10 μL of the reaction product for electrophoresis detection, and take 3 μL of Takara DL2000 marker. When performing agarose gel electrophoresis, the condition is a constant voltage of 150V for 30 minutes. After the electrophoresis is completed, color development is performed, and the position of the gel block is adjusted by white light. Then, when the white light is turned off, ultraviolet color development is performed with an exposure time of 500ms, and photos are taken for analysis. Lane 1D software is used for gel analysis, the photos are inverted, and the width is adaptively adjusted according to the lane width, and the lanes of all experimental groups, solvent groups, and PC groups are selected.

[0052] After lane adjustment, the bands were selected, the location of the residual DNA bands was selected, the selection box was controlled to be of the same size, the grayscale of the bands was read and the results were output to an Excel table, the band results were statistically analyzed in the Excel table, and the remaining amount of uncut bands and the remaining ratio of uncut bands were calculated respectively.

[0053] Calculation of cleavage efficiency and inhibition efficiency

[0054] (1) Cutting efficiency calculation formula: E = A1 / B1 × 100%;

[0055] Among them, E represents the cutting efficiency; A1 represents the gray value of the cutting product; B1 represents the gray value of the original DNA cutting product.

[0056] (2) The relative ratio of inhibition efficiency was calculated as follows: C = (F1-D1) / (G1-D1) × 100%;

[0057] Wherein, C represents the inhibition efficiency, F1 represents the gray value of the cleavage product of the experimental group, D1 represents the gray value of the cleavage product of DMSO, and G1 represents the gray value of the cleavage product of the positive control BRD0539 (BRD0539).

[0058] The results are shown in Table 1.

[0059] Table 1 Comparison results of cutting efficiency and inhibition efficiency

[0060]

[0061] The cleavage inhibition effects of positive drug BRD0539 and positive drug BRD7586 are shown in Figure 7 After treatment with the positive compounds BRD0539 and BRD7586, the gray value of the uncut DNA band was larger than that of the solvent group, indicating that the two positive control compounds did have the effect of inhibiting Cas9 from binding to or cutting target DNA.

[0062] The inhibitory cleavage effect of the target drug group can be found in Figure 1 .right Figure 1 The grayscale of the uncut DNA bands in each group was analyzed. The results showed that compared with the positive control drug BRD0539, the grayscale of the uncut DNA was stronger after treatment with the candidate compounds pargyline hydrochloride (3690), metadoxine (5060) and dimethyl fumarate (2586), indicating that pargyline hydrochloride, metadoxine and dimethyl fumarate have stronger inhibitory effects on Cas9 binding or cutting target DNA than the positive compounds.

[0063] The effect of combined use of candidate compounds is as follows Fig.11 As shown, the ALL group displayed the electrophoresis results of uncut DNA after the combined use of pargyline hydrochloride (3690), metadoxine (5060) and dimethyl fumarate (2586). It can be seen that the combined use of the three candidate compounds has more uncut DNA residues than any positive control compound, indicating that the combined use of pargyline hydrochloride, metadoxine and dimethyl fumarate has a stronger inhibitory effect on Cas9 binding or cutting target DNA than the positive compounds.

[0064] Example 2

[0065] Drug concentration gradient experiment

[0066] In order to verify that the candidate compounds have a dose-dependent effect on the inhibition of SpCas9, we performed an enzymatic cleavage reaction after gradient drug treatment in a cell-free system, using the PC-1400 kit from Innovent Biologics and the method provided in the kit manual to perform in vitro enzymatic cleavage experiments. The enzymatic cleavage efficiency was then verified by agarose gel electrophoresis. PC DNA and PC sgRNA in the kit were used here.

[0067] First, use enzyme-free PCR tubes and enzyme-free pipette tips to conduct the experiment: add 5μL (1U / μL) of Cas9 protein, 5μL (1μg / μL) of PC sgRNA, 10μL (1μg / μL) of PC DNA, 1.2μL of small molecule compounds or solvents, and 2.4μL of 10×Cas9 Buffer to each sterile enzyme-free test tube, then blow and mix well. The sample addition process is carried out on ice. The specific groups are as follows:

[0068] A negative control group was set up: containing 1.2 μL of DMSO solvent, 5 μL of Cas9 protein (1 U / μL), 5 μL of PC sgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer. The mixture was then blown evenly, and the whole process was carried out on ice.

[0069] Positive control group 1 (BRD0539): contains 1.2 μL of 1 mM BRD0539, 5 μL of Cas9 protein (1 U / μL), 5 μL of PC sgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer. Then blow and mix well. The whole process is carried out on ice.

[0070] Positive control group 2 (BRD7586): contains 1.2 μL of 1 mM BRD7586, 5 μL of Cas9 protein (1 U / μL), 5 μL of PC sgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer. Then blow and mix well. The whole process is carried out on ice.

[0071] The target drug group was added with 1.2 μL of the corresponding small molecule drugs with concentrations of 1 mM, 750 μM, 500 μM, 250 μM, and 0 μM, 5 μL of Cas9 protein (1 U / μL), 5 μL of PC sgRNA (1 μg / μL), 10 μL of PC DNA (1 μg / μL), and 2.4 μL of 10×Cas9 Buffer into the reaction system. Then, the mixture was blown evenly, and the whole process was carried out on ice. Among them, the small molecule drugs were pargyline hydrochloride, metadoxine, and dimethyl fumarate; the corresponding concentrations of pargyline hydrochloride, metadoxine, and dimethyl fumarate in the reaction system were 50 μM, 37.5 μM, 25 μM, 12.5 μM, and 0 μM, respectively.

[0072] Use a metal bath at 37°C for 30 minutes, then treat at 85°C for 10 minutes to complete the in vitro enzyme digestion reaction. After the enzyme digestion reaction, the product was detected and 5 μL of TIANGEN was added to each group.TM Protein K (20 mg / mL), this step was mixed on ice, and incubated in a metal bath at 56°C overnight. Prepare 2% agarose gel (add 4 μL YeaRed Nucleic Acid Gel Stain CAT: 10202ES76 (Yeasen Yisheng Biology) to every 40 mL of agarose gel solution).

[0073] Take 10 μL of the reaction product for electrophoresis detection, and take 3 μL of Takara DL2000 marker. When performing agarose gel electrophoresis, the condition is a constant voltage of 150V for 30 minutes. After the electrophoresis is completed, color development is performed, and the position of the gel block is adjusted by white light. Then, when the white light is turned off, ultraviolet color development is performed with an exposure time of 500ms, and photos are taken for analysis. Use Lane 1D software for gel analysis, invert the photos, and then adjust the width of the lanes adaptively according to the lane width, and select the lanes of all experimental groups, solvent groups, and positive control groups.

[0074] After lane adjustment, the bands were selected, the location of the residual DNA bands was selected, the selection box was controlled to be of the same size, the grayscale of the bands was read and the results were output to an Excel table, the band results were statistically analyzed in the Excel table, and the remaining amount of uncut bands and the remaining ratio of uncut bands were calculated respectively.

[0075] The results of the concentration gradient experiment of the positive drug BRD7586 are as follows Figure 8 As shown in the figure, the drug concentration in BRD0539 and BRD7586 is 50 μM, and the concentration gradient at the back shows that the inhibitory efficiency continues to increase with the increase of BRD7586 concentration, that is, the inhibitory effect is positively correlated with the concentration of BRD7586, showing dose dependence.

[0076] The results of the target drug group concentration gradient experiment can be found in Figure 2 , Figure 4 and Figure 6 .

[0077] Specifically, Figure 2 It is shown that as the concentration of pargyline hydrochloride (3690) increases, the amount of uncut DNA shows an upward trend, suggesting that the inhibitory effect of pargyline hydrochloride on SpCas9 has a dose-dependent effect; Figure 4 It is shown that with the increase of the concentration of metadoxine (5060), the amount of uncut DNA shows an upward trend, indicating that metadoxine has a dose-dependent effect in inhibiting SpCas9; Figure 6 It is shown that with the increase of dimethyl fumarate (2586) concentration, the amount of uncut DNA showed an upward trend, indicating that dimethyl fumarate has a dose-dependent effect on the inhibition of SpCas9.

[0078] Example 3

[0079] Surface Plasmon Resonance (SPR) assay to detect affinity of Cas9 protein and candidate small molecule inhibitors

[0080] The instrument used for the SPR experiment was Biacore T200.

[0081] The binding test results of positive compound 1 (BRD0539) and positive compound 2 (BRD7586) with Cas9 protein are shown in Fig. 9 and Fig.10 It is not difficult to find that at most concentrations, the SPR results of BRD0539 and BRD7586 are a straight line close to the X-axis, indicating that their binding force with SpCas9 is very weak.

[0082] The test results of the binding affinity between the target drug group and the Cas9 protein are as follows Figure 3 , Figure 5 As shown in the figure, the gradient represents that as the concentration of small molecules increases, their inhibitory ability to the CRISPR / Cas9 system continues to increase. Figure 3 and Figure 5 The affinity of pargyline hydrochloride (3690) and metadoxine (5060) did not change with the drug concentration, and did not show an obvious binding gradient trend, and remained around 0. The affinity of positive control 1 BRD0539 and positive control 2 BRD7586 did not change with the drug concentration, showing a straight line and in the range of around 0; this shows that the affinity of pargyline hydrochloride and metadoxine to Cas9 is similar to that of positive compound 1 (BRD0539) and positive compound 2 (BRD7586) (compared to Fig. 9 and Fig.10 compared to).

[0083] Figure 3 , Figure 5 The results of SPR are the results of the drug binding to SpCas9 protein. From the figure, we can see the binding of drugs to SpCas9. The binding of drugs pargyline hydrochloride (3690) and metadoxine (5060) to Cas9 is similar to the previously reported Cas9 inhibitor BRD0539 ( Fig. 9 ) and BRD7586( Fig.10 ) have similar affinities, and their binding force curves are maintained around 0.

[0084] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0085] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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.

[0086] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of pargyline hydrochloride, metadoxine and dimethyl fumarate compounds in the preparation of products that inhibit SpCas9 from binding or cleaving substrates, characterized in that: One or more of the pargyline hydrochloride, metadoxine and dimethyl fumarate are used in combination; The concentration of dimethyl fumarate was 1.56-500 μM; The concentration of pargyline hydrochloride was 1.56-500 μM; The concentration of metadoxine was 1.56-500 μM.

2. The use according to claim 1, characterized in that: The product includes any one of pargyline hydrochloride, metadoxine and dimethyl fumarate.

3. The use according to claim 2, characterized in that: The product also includes SpCas9 protein.

4. The use according to claim 3, characterized in that: The dosage ratio of pargyline hydrochloride, metadoxine and dimethyl fumarate to SpCas9 is 0.25-1mmol:5U.

5. The use according to claim 1, characterized in that: The products include pargyline hydrochloride, metadoxine and dimethyl fumarate.

6. The use according to claim 5, characterized in that: The product also includes SpCas9 protein.

7. The use according to claim 6, characterized in that: The total dosage ratio of pargyline hydrochloride, metadoxine and dimethyl fumarate to SpCas9 is 0.25-1mmol:5U.

8. The use according to claim 7, characterized in that: When inhibiting binding or cleavage, use the PC-1400 kit from ImmunoSynthetics.

9. The use according to claim 1, characterized in that: The reaction system for inhibiting binding or cleavage is 5 μL of Cas9 protein, 5 μL of PC sgRNA, 10 μL of PC DNA, 1.2 μL of drug or solvent, and 2.4 μL of 10×Cas9 Buffer.

10. The use according to claim 1, characterized in that: When inhibiting binding or cleavage, incubate at 37°C for 30 minutes and at 85°C for 10 minutes for enzyme cleavage.

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