Application of novel inhibitor to inhibition of Cas9 in-vivo and in-vitro activity and off-target efficiency thereof

Abstract

The invention discloses a small molecule inhibitor for antagonizing genome editing and application thereof, and relates to the technical field of biology, the small molecule inhibitor comprises a compound with the CAS number of 130-85-8, 563 90-09-1, 2227366-51-8, 742 1-40-1, 113 8549-36-6 or / and 925434-55-5; the small-molecule inhibitor is applied to inhibiting Cas9 enzyme digestion reaction in vitro, in bacteria and in cells; the small-molecule inhibitor is used as a drug primer and is applied to accurately controlling the in-vivo or in-vitro action time of Cas9. The small molecular compound screened by the invention is applied to gene editing systems such as Base Editing and Primer Editing as a small molecular probe, and can also be used as a drug primer for preventing and treating the problems of DNA damage, off-target and the like caused by Cas9 over-shearing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a small molecule inhibitor against CRISPR / Cas9 genome editing and its application. Background Art

[0002] Gene editing technology refers to the ability of humans to "edit" target genes, and genetic modifications such as knockout and insertion of DNA fragments can be achieved by knocking out or inserting specific DNA fragments at the genomic level. The ZFN gene editing technology first reported in 2002, the second-generation artificial nuclease TALENs technology, and the meganuclease after DNA binding specificity modification of naturally occurring homing endonucleases have been used for genome editing in human, animal, and plant cells respectively, but they still have many limitations. For example, off-target mutations are often introduced, the construction of editing vectors is very complex and laborious, and the modification is difficult. In recent years, the popular gene editing technology, the CRISPR / Cas system, is a revolutionary new gene editing technology, which has obvious advantages in speed, efficiency, and cost. Gene editing purposes targeting different DNAs can be achieved only by simply changing the short region of gRNA.

[0003] The CRISPR / Cas system is very diverse. According to the different effects of the Cas9 protein complex, it can be mainly divided into two different categories: the complex composed of multiple Cas protein subunits: Class 1, and the Cas protein with a relatively single effector: Class 2. Currently, among the members of these two known types of CRISPR / Cas systems, type II is the most common CRISPR / Cas system. Among them, the most widely and deeply studied gene editing tool, SpyCas9, belongs to the type II-A CRISPR / Cas system of Class 2. Only by designing an sgRNA matching the target gene can DNA cleavage be carried out at almost any DNA target site, and gene editing can be carried out through non-homologous end joining (NHEJ) or homology-directed repair (HDR). The amino acid sequence of SpyCas9 is shown in SEQ ID NO: 1. In recent years, the CRISPR / Cas9 system, including its derivative DNA editing systems such as Base Editing and Primer Editing, has been increasingly widely and deeply applied in gene modification and gene function research.

[0004] With the successful application of the CRISPR / Cas9 gene editing technology to the genomes of various organisms including humans, its drawbacks have gradually emerged, such as difficulties in packaging viral vectors, high complexity in delivery operations, obvious off-target effects, and low precision in gene editing, which are all safety issues. To precisely and effectively control the CRISPR / Cas9 system, naturally occurring anti-CRISPR proteins (Acr) have evolved in phages, which inhibit the activity of CRISPR by interfering with the loading of gRNA, competing with DNA binding, or inhibiting the cleavage activity of the endonuclease domain, thereby controlling side effects such as off-target effects, genotoxicity, and chromosomal translocations. However, there are problems such as large size, difficulty in entering cells, and easy hydrolysis in the body. Therefore, it is of great significance to screen for small molecule drugs that are stable, have a rapid onset of action, are easy to enter cells, are non-immunogenic, and can be synthesized on a large scale.

[0005] The known small molecule inhibitor of SpyCas9 is a synthetic small molecule compound (BRD0539) screened by Maji et al. in 2019 based on fluorescence polarization and high-throughput detection methods. It can inhibit the binding of SpyCas9 to substrate DNA and thus inhibit the cleavage of DNA by SpyCas9. However, small molecule inhibitors of SpyCas9 such as BRD0539, which are chemically synthesized lead compounds, cannot be directly used in humans. Their mechanism of action is unclear, and their pharmacological, pharmacokinetic, and other properties have not been verified. Therefore, there is a need to discover small molecule inhibitors with new structures and new mechanisms for the CRISPR / Cas9 gene editing system to act as probe tools to more precisely regulate the editing time and process of Cas9 at the cellular and animal levels.

[0006] Therefore, those skilled in the art are committed to developing an inhibitor of the CRISPR / Cas9 gene editing system and the application of this inhibitor as a small molecule probe in gene editing systems such as Base Editing and Primer Editing, and it can also be used as a drug lead to prevent and treat DNA damage, off-target effects, etc. caused by excessive cleavage of Cas9. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop an inhibitor of the CRISPR / Cas9 gene editing system and the application of this inhibitor as a small molecule probe in gene editing systems such as Base Editing and Primer Editing, and it can also be used as a drug lead to prevent and treat DNA damage, off-target effects, etc. caused by excessive cleavage of Cas9.

[0008] To achieve the above object, the present invention provides a small molecule inhibitor against CRISPR / Cas9 genome editing, including: pamolic acid, epirubicin, dalbavancin, sodium glycyrrhetate, sodium docusate, 2-(4-methyl-1H-1,4-diazepan-1-yl)-N-[(5-methyl-2-pyrazinyl)methyl]-5-oxo-5H-benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxamide or / and N-[2-[3-(1-piperazinylmethyl)imidazo[2,1-b]thiazol-6-yl]phenyl]-2-quinoxalinecarboxamide, and the corresponding CAS numbers are: 130-85-8, 56390-09-1, 2227366-51-8, 7421-40-1, 1138549-36-6 or / and 925434-55-5.

[0009] The present invention also provides an application of the small molecule inhibitor in inhibiting the Cas9 cleavage reaction in vitro.

[0010] Furthermore, the above application includes the following steps:

[0011] Step 1: Prepare an experimental buffer solution;

[0012] Step 2: Add the small molecule inhibitor into the experimental buffer solution obtained in Step 1 to obtain a compound inhibition system;

[0013] Step 3: Add an experimental substrate into the compound inhibition system in Step 2 to obtain an enzyme reaction mixture, and incubate it to obtain a reaction solution;

[0014] Step 4: Detect the application effect of the inhibitor on the reaction solution obtained in Step 3 by native polyacrylamide gel electrophoresis.

[0015] Furthermore, Step 1 further includes: configuring a DEPC-treated aqueous solution in an enzyme reaction container with a HEPES concentration of 20 mM, a KCl concentration of 150 mM, a MgCl2 concentration of 10 mM, a DTT concentration of 0.5 mM, an EDTA concentration of 0.1 mM, a pH of 7.5, adding a Tween-20 concentration of 0.01%, and an in vitro synthesized targeted sgRNA concentration of 400 nM.

[0016] Furthermore, Step 2 further includes preparing a compound inhibition system: preparing reaction mixtures with different concentrations of the small molecule compound and adding in vitro purified Cas9 protein into the experimental buffer solution prepared in Step 1 at a concentration of 375 nM to form an enzyme reaction system;

[0017] Furthermore, the incubation process in Step 3 is: closing the enzyme reaction container, incubating at 37 °C for 90 minutes, and then adding a DNA loading buffer containing SDS to terminate the reaction;

[0018] Further, in step 4, detection of the application effect of the small molecule compound as a Cas9 inhibitor:

[0019] Use 1% agarose gel and 20% non-denaturing polyacrylamide gel to detect the cleavage efficiency of Cas9 on DNA substrates and the inhibitory effect of the compound on Cas9.

[0020] Further, in the above method for inhibiting Cas9 cleavage reaction, the concentration of the compound in step 2 is 0 - 200 μM.

[0021] Further, in the above method for inhibiting Cas9 cleavage reaction, the compound and Cas9 protein are incubated for 15 min in step 2.

[0022] Further, in the above method for inhibiting Cas9 cleavage reaction, the concentration of the circular plasmid added in step 3 is 300 ng, the concentration of the linear substrate modified with the fluorophore FAM is 10 nM, and the reaction system is 20 μL.

[0023] Further, in the above method for inhibiting Cas9 cleavage reaction, in the DNA loading buffer termination solution (5×) added during the incubation in step 3, the concentration of EDTA is 250 mM, the concentration of SDS is 1.2%, and the volume ratio of glycerol is 30% (v / v).

[0024] Further, in the above method for inhibiting Cas9 cleavage reaction, the detection means for the inhibitory effect of the small molecule compound on Cas9 in step 4 is 1% agarose gel and 20% non-denaturing polyacrylamide gel. The 1% agarose gel contains ethidium bromide for staining the circular plasmid substrate for cleavage detection. The sample loading volume of the reaction solution is 15 μL. The electrophoresis buffer for the gel running condition is 0.5×TBE, the voltage is 120 V, and the time is 40 min. After electrophoresis, take out the agarose gel from the electrophoresis tank and observe the cleavage of the DNA band and the inhibitory effect of the compound under appropriate UV excitation light. The 20% non-denaturing polyacrylamide gel is suitable for the cleavage detection of small fragment linear DNA substrates. The sample loading volume of the reaction solution is 15 μL. The electrophoresis buffer for the gel running condition is 1×TBE. First, run at 150 V for 10 min to flatten the band, then run at 120 V for 1.5 h. After electrophoresis, take out the gel from the gel plate and observe the FAM-fluorescent DNA band under 488 nM excitation light.

[0025] Further, the in vitro activity detection of Cas9 and the detection of the inhibitory effect of the compound targeted by the present invention can also be applied to the in vitro cleavage reaction of SauCas9.

[0026] The present invention also provides an application of a small molecule inhibitor in inhibiting the Cas9 cleavage reaction in bacteria.

[0027] Further, the above application includes the following steps:

[0028] Step 1) Prepare pCas competent cells, electrotransform the sgRNA plasmid encoding the target SSEA gene into the bacterial competent cells, and at the same time add the small molecule inhibitor to the electrotransformed competent cells for co-incubation.

[0029] Step 2) After the co-incubated competent cells obtained in Step 1 are resuscitated at 32°C, perform plate coating or liquid culture to obtain a plate after coating culture or a bacterial liquid mixture after liquid culture.

[0030] Step 3) Statistically analyze the number of plate clones on the plate obtained in Step 2 through cloning technology software; or use the bacterial liquid mixture obtained in Step 2 as a PCR template for PCR program identification.

[0031] Further, for the plate coating experiment, the plate also contains the corresponding concentration of the compound, the plate is inverted and cultured overnight in a 32°C incubator, and the number of plate clones is statistically analyzed through cloning technology software the next day; for the liquid culture experiment, the liquid contains double the concentration of antibiotics and the corresponding concentration of the compound, and after shaking culture at 250 rpm at 32°C for 20 h, the bacterial liquid mixture is used as a PCR template for PCR program identification. After PCR is completed, 1% agarose gel electrophoresis is performed to determine the sample genotype.

[0032] Further, for the plate coating experiment, the small molecule compounds added in Step 1 have CAS numbers 130-85-8, 56390-09-1, 2227366-51-8, 7421-40-1, 1138549-36-6, 925434-55-5, and the final concentrations are 50-800 μM, 3.13-50 μM, 12.5-200 μM, 50-800 μM, 1-200 μM, 5-200 μM respectively; for the liquid culture experiment, the small molecule compounds added in Step 1 have CAS numbers 130-85-8, 56390-09-1, 2227366-51-8, 7421-40-1, 1138549-36-6, 925434-55-5, and the final concentrations are 12.5-800 μM, 1.6-100 μM, 3.13-200 μM, 3.13-200 μM, 3.13-200 μM, 3.13-200 μM respectively.

[0033] Further, the electroporation equipment used for the electroporation operation is Gene Pulser Xcell TMElectroporation system (Bio-Rad), and the experimental equipment is Gene Electroporation cuvette (Bio-Rad).

[0034] Furthermore, for the plate coating experiment, the Colony Counter software (Tanon) is used for plate counting.

[0035] Furthermore, for the liquid culture experiment, the PCR system is 20 μL. 1 μL of each primer (10 μM) designed for the upstream and downstream of the sgRNA of the target gene, 2 μL of the bacterial liquid obtained in Step 1, and after making up with deionized water, PCR reaction is carried out. The annealing temperature is 60 °C and the extension time is 1 min 30 s.

[0036] Furthermore, the detection method for the Cas9 gene editing activity inhibitor in bacteria of the present invention can also be applied to SauCas9.

[0037] The present invention also provides an application of a small molecule inhibitor in inhibiting the Cas9 cleavage reaction in cells.

[0038] Furthermore, the above application includes the following steps:

[0039] Step (1): Culture human HEK293FT cells in an amino acid nutrient solution. After incubating for one day, transfect the plasmid encoding Cas9 and the sgRNA targeting the target gene into the cells. At the same time, add the small molecule inhibitor to the amino acid nutrient solution for co-incubation; after one day, replace the amino acid nutrient solution with a medium containing 2.5 μg / mL puromycin antibiotic for screening culture; after 48 h, aspirate the upper layer of the medium, use trypsin to collect the cells, and after centrifuging the mixture on a centrifuge, collect the precipitated cells;

[0040] Step (2): Perform cell genome extraction operation on the precipitated cells collected in Step (1) to obtain the target gene of the cell genome;

[0041] Step (3): Use the target gene of the cell genome extracted in Step (2) as the PCR template for PCR program identification.

[0042] Furthermore, prepare a PCR system to amplify the target gene of the cell genome extracted in Step (2). After PCR is completed, perform 1% agarose gel electrophoresis to determine the purity and content of the target genome, and perform first-generation Sanger sequencing to analyze the editing efficiency of Cas9 and the inhibitory effect of the compound.

[0043] Further, when the compound is used as an intracellular Cas9 inhibitor, in step (1), the final concentrations of small molecule compounds 130-85-8, 2227366-51-8, 7421-40-1, and 1138549-36-6 are 20-200 μM, 5-50 μM, 10-100 μM, and 0.01-10 μM, respectively.

[0044] Further, the kit used for genomic extraction operation is Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (centrifugal column type).

[0045] Further, the PCR system is 20 μL. For each primer (10 μM) designed upstream and downstream of the target gene sgRNA, 1 μL is added, and 100 ng of the genomic template obtained by extraction in step (2) is used. After making up with deionized water, PCR reaction is carried out. The annealing temperature is 60 °C and the extension time is 40 s.

[0046] Further, after Sanger sequencing is performed after PCR, the Cas9 cleavage site downstream of sgRNA is analyzed, and the gene editing efficiency of Cas9 and the inhibitory effect of the compound can be calculated.

[0047] Further, the detection of Cas9 intracellular gene editing efficiency and the detection of compound inhibitory effect targeted by the present invention can also be applied to the intracellular experimental detection of SauCas9.

[0048] The present invention also provides a small molecule inhibitor as a drug lead, which is applied to precisely control the action time of Cas9 in vivo activity and off-target efficiency.

[0049] The present invention also provides a small molecule inhibitor as a drug lead, which is applied to precisely control the action time of Cas9 in vitro activity and off-target efficiency.

[0050] In the preferred embodiment 1 of the present invention, the process of in vitro inhibition of Cas9 cleavage reaction by small molecule compounds is described in detail;

[0051] In another preferred embodiment 2 of the present invention, the application of small molecule compounds as inhibitors of Cas9 gene editing activity in bacteria is described in detail;

[0052] In another preferred embodiment 3 of the present invention, the application of small molecule compounds as inhibitors of Cas9 gene editing activity in cells is described in detail;

[0053] In another preferred embodiment 4 of the present invention, the application of small molecule compounds as inhibitors of Cas9 gene editing activity in a mouse model is described in detail.

[0054] The beneficial technical effects of the present invention are as follows:

[0055] Based on the in vitro Cas9 activity detection method, effective inhibitors were screened, which can inhibit Cas9 editing activity at the in vitro, bacterial, cellular, and animal levels, and can be used as drug leads to precisely control the action time of Cas9 in vivo and in vitro.

[0056] By expanding the effective inhibitor test method of SpyCas9 to homologous SauCas9, it was found that this small molecule inhibitor can inhibit SauCas9 editing activity at the in vitro, bacterial, and cellular levels.

[0057] The editing activity of mice injected with Cas9 via the tail vein under high pressure was inhibited by intraperitoneal injection of a small molecule compound. This effective small molecule compound can inhibit the in vivo editing activity of SpyCas9 in a dose-dependent manner in mice.

[0058] Based on the method for detecting the enzymatic activity of in vitro purified Cas9, the drugs approved by the FDA were screened to obtain effective inhibitors, the conditions for the in vitro enzymatic activity detection experiment of Cas9 were optimized, and a method for screening Cas9 inhibitors was established.

[0059] According to the effective inhibitor screening means for Cas9 provided by the present invention, other compounds or other drugs approved by the FDA can be screened for activity to obtain more potential effective inhibitors. At the same time, it can also provide methods and new ideas for the development of activity inhibitors for other types of target proteins.

[0060] The small molecule compounds provided by the present invention have a certain inhibitory effect on Cas9 in vitro, in bacteria, in cells, and in mice. Especially in animals, they can effectively inhibit the editing efficiency of Cas9 on endogenous sites. Subsequently, group modification or appropriate drug modification can be carried out on the small molecule compounds, and the inhibitory effect of the drugs on gene therapy patients can be evaluated, which has an inhibitory and therapeutic effect on in vivo gene editing off-target and other side effects.

[0061] The concept, specific structure, and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features, and effects of the present invention. Description of the Drawings

[0062] Figure 1 It is the diagram of the activity determination method based on pure enzyme and the inhibitory effect of small molecule compounds on in vitro Cas9 in a preferred Embodiment 1 of the present invention;

[0063] Figure 2 It is the diagram of the inhibitory effect of small molecule inhibitors on Cas9-mediated SSEA gene editing in bacteria in a preferred Embodiment 2 of the present invention;

[0064] Figure 3The inhibitory effect of a small molecule inhibitor of a preferred embodiment 3 of the present invention on the gene editing of COSMC and EMX1 by Cas9 or BE4 in cells;

[0065] Figure 4 The inhibitory effect of a small molecule inhibitor of a preferred embodiment 4 of the present invention on the gene editing of PCSK9 by Cas9 in mice. Detailed implementation manners

[0066] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0067] Example 1 In vitro inhibition of Cas9 cleavage reaction by small molecule compounds

[0068] The experiment on the effect of small molecule compounds on the activities of SpyCas9 or SauCas9 in cleaving circular plasmids and linear substrates is as follows: Incubate a specified concentration of pamolic acid, epirubicin, daptomycin, carbenoxolone, 2-(4-methyl-1H-1,4-diazepan-1-yl)-N-[(5-methyl-2-pyrazinyl)methyl]-5-oxo-5H-benzo[ d]thiazolo[3,2-a][1,8]naphthyridine-6-carboxamide or N-[2-[3-(1-piperazinylmethyl)imidazo[2,1-b]thiazol-6-yl]phenyl]-2-quinoxalinecarboxamide with the complex of SpyCas9 (375 nM) and sgRNA (400 nM), and incubate at 37 °C for 90 min to test the in vitro cleavage activity of SpyCas9. The test conditions for the inhibitory effect of AcrIIA4 on SpyCas9 are 22 °C. In the in vitro DNA cleavage assays of SpyCas9 or SauCas9, epirubicin and daptomycin showed better inhibitory effects than the previously discovered BRD series compounds. The IC50 values of all inhibitors on the linear substrate ranged from 10 to 50 μM, showing certain advantages. Based on the pure enzyme activity assay method and the inhibitory effect of the compound on in vitro Cas9 as Figure 1As shown, part A shows the effects of the compound and AcrIIA4 on the cleavage activity of SpyCas9 on linear oligonucleotide substrates; part B shows the effects of the compound and AcrIIA4 on the cleavage activity of SauCas9 on linear oligonucleotide substrates; part C shows the effects of 2-(4-methyl-1H-1,4-diazepan-1-yl)-N-[(5-methyl-2-pyrazinyl)methyl]-5-oxo-5H-benzo[d]thiazolo[3,2-a][1,8]naphthyridine-6-carboxamide on the cleavage activity of SpyCas9 on linear oligonucleotide substrates; part D shows the effects of N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide on the cleavage activity of SpyCas9 on linear oligonucleotide substrates.

[0069] Application of small molecule compounds as inhibitors of Cas9 gene editing activity in bacteria

[0070] The experiment on the inhibitory effect of the inhibitor on the genome editing of the SSEA locus by SpyCas9 or SauCas9 in bacteria is as follows: In the dual-plasmid-based bacterial survival assay, the Cas9 inhibitor has an inhibitory effect on SpyCas9 activity. After electroporation of the compound, plasmid and competent cells, they were incubated at 32 °C for 1.5 hours, and then spread on an LB plate with kanamycin and spectinomycin antibiotics. After incubation overnight, the clones on the culture plate were photographed and the number of colonies was quantified using colony counter software. Compared with the control group, the number of colonies on the plate in the compound-treated group was significantly restored. In the presence of a homologous recombination repair template, the Cas9 inhibitor has an inhibitory effect on the genome editing activity of SpyCas9 or SauCas9. The compound, plasmid and competent cells were electroporated and incubated at 32 °C for 20 hours. The SSEA in bacteria was amplified by PCR and the genomic types were analyzed by 1% agarose gel electrophoresis. Compared with the existing BRD small molecule compounds, our inhibitor shows a more significant inhibitory effect on Cas9 at the bacterial level. The inhibitory effect of the inhibitor on Cas9-mediated SSEA gene editing in bacteria is as Figure 2 shown, where part A shows the inhibitory effect of the compound on the activity of SpyCas9 in the dual-plasmid bacterial survival experiment; part B shows the inhibitory effect of the compound on the genome editing activities of SpyCas9 and SauCas9 in the presence of a homologous recombination repair template.

[0071] Application of small molecule compounds as inhibitors of Cas9 gene editing activity in cells

[0072] The experiments on the inhibition of SpyCas9, SauCas9 or BE4 from editing endogenous COSMC or EMX1 loci in HEK293FT cells by inhibitors were as follows: Cells were transfected with the PX459-SpyCas9 plasmid encoding sgRNAs against COSMC and EMX1, treated with the specified compounds for 24 hours, and the target DNA was amplified by PCR for Sanger sequencing. The gene editing efficiency of Cas9 was analyzed using the TIDE or EditR method. Compared with existing compounds, our inhibitor was the first compound to study the inhibitory effect of SauCas9 at the cellular level and showed a better inhibitory effect than existing inhibitors in cells. The inhibitory effects of the inhibitor on Cas9 or BE4-mediated gene editing of COSMC and EMX1 genes in cells were as Figure 3 shown. Part A shows the inhibitory effect of the compound on SpyCas9 editing endogenous COSMC or EMX1 in HEK293FT cells; Part B shows the inhibitory effect of the compound on the BE4 base editor for base editing of C5 and C6 of EMX1 in HEK293FT cells; Part C shows the effect of the compound on SauCas9 editing COSMC in HEK293FT cells.

[0073] Example 4 Application of small molecule compounds as inhibitors of Cas9 gene editing activity in a mouse model

[0074] The experimental protocol for intraperitoneal injection of the compound and hydrodynamic tail vein injection of the SpyCas9-mediated anti-PCSK9 mouse model was as Figure 4 shown in Part A of

[0075] Step 1: On the first day, different doses of the compound were intraperitoneally injected. On the second day, the plasmid encoding Cas9 and sgRNA targeting the target gene was hydrodynamically injected into the tail vein. On the third day, blood was collected from the orbital cavity as the first-round sample. This was repeated three times. After the last blood collection from the orbital cavity, the mice were decapitated and the livers were taken and stored at -80 °C for later use.

[0076] Step 2: An ELISA kit was used to measure the content of PCSK9 in the plasma of the blood samples collected from the orbital cavity; a total cholesterol assay kit was used to measure the total cholesterol content in the plasma of the blood samples collected from the orbital cavity; T7E1 was used to measure the mismatch frequency generated by random repair after gene editing on the mouse liver genome; next-generation sequencing was used to measure the base deletions, insertions, etc. in the mouse liver.

[0077] When the compound was used as a Cas9 inhibitor in mice, the final concentrations of the small molecule compounds pamolic acid, daptomycin, and carbenoxolone injected in Step 1 were all 10 mg / kg, 20 mg / kg, 40 mg / kg, 80 mg / kg, and 160 mg / kg.

[0078] Among them, the ELISA kit used to measure the PCSK9 content in plasma of orbital blood samples is a mouse PCSK9 assay kit (R&D). The restoration effect of the compound on the Cas9 at the PCSK9 locus in mice is as Figure 4 shown in part B of

[0079] In the anti-PCSK9 mouse model based on high-pressure tail vein injection, the effect of the inhibitor on antagonizing Cas9-mediated genome editing. A SpyCas9-mediated anti-PCSK9 mouse model was constructed by high-pressure tail vein injection. On days 0, 3, and 6, the compound was intraperitoneally injected into BALB / c mice at a dose of 10, 20, 40, 80, or 160 mg / kg, and then the PX459-sgPCSK9 plasmid encoding SpyCas9 was administered. Blood was collected by orbital blood sampling on days 2, 5, and 8, and the amount of plasma PCSK9 was measured using a mouse PCSK9 ELISA kit. Our compound is the first inhibitor targeting the SpyCas9 protein in mice, and it has innovation and advantages compared with existing studies.

[0080] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning, or limited experiments should be within the protection scope determined by the claims.

Claims

1. A small molecule inhibitor that antagonizes CRISPR / Cas9 genome editing, characterized in that: Including pamoic acid, epirubicin, dalbavancin, carbenoxolone sodium, docusate sodium, 2-(4-methyl-1H-1,4-diazepan-1-yl)-N-[(5-methyl-2-pyrazinyl)methyl]-5-oxo-5H-benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxamide or / and N-[2-[3-(1-piperazinemethyl)imidazo[2,1-b]thiazol-6-yl]phenyl]-2-quinoxalinecarboxamide.

2. Use of the small molecule inhibitor as claimed in claim 1 to inhibit Cas9 enzyme cleavage reaction in vitro.

3. The use according to claim 1, characterized in that The application comprises the following steps: Step 1, preparing an experimental buffer solution; Step 2, adding the small molecule inhibitor into the experimental buffer obtained in step 1 to obtain a compound inhibition system; Step 3, adding the experimental substrate to the compound inhibition system in step 2 to obtain an enzymatic reaction mixture, and incubating to obtain a reaction solution; Step 4: The reaction solution obtained in step 3 is subjected to electrophoresis to detect the effect of the inhibitor application.

4. The method according to claim 3, characterized in that The step 1 also includes: preparing a DEPC-treated aqueous solution with a HEPES concentration of 20 mM, a KCl concentration of 150 mM, a MgCl2 concentration of 10 mM, a DTT concentration of 0.5 mM, and an EDTA concentration of 0.1 mM in an enzymatic reaction container, the pH value is 7.5, Tween-20 is added at a concentration of 0.01%, and the concentration of the in vitro synthesized targeting sgRNA is 400 nM.

5. Use of the small molecule inhibitor as claimed in claim 1 to inhibit Cas9 enzymatic cleavage reaction in bacteria.

6. The use according to claim 5, characterized in that The application comprises the following steps: Step 1) preparing pCas competent cells: electroporating the sgRNA plasmid encoding the target SSEA gene into the bacterial competent cells, and adding the small molecule inhibitor to the electroporated competent cells for co-incubation; Step 2) After the competent cells obtained in step 1) are recovered at 32° C., they are plate-coated or liquid-cultured to obtain a plate-coated or liquid-cultured bacterial liquid mixture; Step 3) counting the number of clones on the plate obtained in step 2) after the coating culture by using cloning technology software; or using the liquid culture mixture obtained in step 2) as a PCR template for PCR program identification.

7. Use of the small molecule inhibitor as claimed in claim 1 to inhibit Cas9 enzyme cleavage reaction in cells.

8. The use according to claim 7, characterized in that The application comprises the following steps: Step (1) Human HEK293FT cells are cultured in an amino acid nutrient solution, and after one day of incubation, Cas9 encoding and sgRNA targeting the target gene are transfected into the cells, and the small molecule inhibitor is added to the amino acid nutrient solution for co-incubation; one day later, the amino acid nutrient solution is replaced with a purine antibiotic medium containing 2.5 μg / mL for screening and culture; after 48 hours, the upper culture medium is aspirated, the cells are collected using trypsin, the mixed solution is centrifuged in a centrifuge, and the precipitated cells are collected; Step (2) performing a cell genome extraction operation on the precipitated cells collected in step (1) to obtain a target gene of the cell genome; In step (3), the target gene of the cell genome extracted in step (2) is used as a PCR template for PCR identification.

9. The small molecule inhibitor as claimed in claim 1 is used as a drug lead to accurately control the activity and off-target efficiency of Cas9 in vivo.

10. The small molecule inhibitor as claimed in claim 1 is used as a drug lead to precisely control the activity and off-target efficiency of Cas9 in vitro.

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