Preparation method and application of non-human mammal model of hyperuricemia

Abstract

The invention provides a preparation method and application of a non-human mammal model of hyperuricemia. Specifically, the invention provides application of the gene editing reagent and preparation of the kit for constructing the non-human mammal model of hyperuricemia, the gene editing reagent comprises a reagent for introducing an additional second initiation codon ATG at the upstream of an initiation codon ATG of a UOX gene, and when the second initiation codon ATG is introduced, the wild type mRNA transcript level and / or the wild type UOX protein expression level of the UOX gene are / is reduced. The animal model provided by the invention is an effective animal model for simulating human hyperuricemia, and has the advantages of no renal toxicity and longer survival time. The method can be used for long-term stable research of related diseases such as hyperuricemia and can be used for screening and testing of specific drugs.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and biomedical technology, and in particular to a method for preparing a non-human mammal model of hyperuricemia and its application. Background Art

[0002] Hyperuricemia (HUA) is a chronic metabolic disease caused by purine metabolism disorder. In recent years, with the improvement of living standards, the prevalence of HUA has been rising year by year. It has become the "fourth most common disease" after hypertension, hyperlipidemia, and hyperglycemia, and the second largest metabolic disease after diabetes, which seriously threatens human health. Hyperuricemia is not only the main cause of gout, but also closely related to a variety of metabolic diseases such as obesity, hypertension, diabetes, and cardiovascular disease.

[0003] Existing hyperuricemia animal models are mainly constructed by increasing the source of uric acid in the body, reducing the excretion of uric acid by the kidney, inhibiting the activity of urate oxidase, etc., through drug modeling or gene knockout. Traditional drug inducer modeling can increase the serum uric acid level of animal models in a short period of time, but it is difficult to maintain a long-term stable high uric acid level, and some drugs may cause more serious liver or kidney damage to the animal model during modeling. It cannot simulate the long-term stable pathological conditions of hyperuricemia patients, and there are large differences between individuals, and subjective interference is strong.

[0004] Although the gene knockout model can provide a long-term stable high serum uric acid environment, the knockout of some uric acid metabolism-related genes may lead to a higher adult mortality rate or embryonic lethality. The organ-specific knockout animal model cannot pass the trait of high serum uric acid to offspring, and the single preparation process is complex and costly.

[0005] Currently, the animal models used for hyperuricemia research are poultry and rodents. The purine metabolic pathway of poultry is similar to that of humans. It is only necessary to add uric acid precursors such as hypoxanthine and yeast or adenine to the basic feed to affect uric acid excretion to induce a stable hyperuricemia model. However, due to the large differences in anatomical structure and genetic information between poultry and humans, rodents are often selected as animal models in model construction. However, the urate oxidase gene of humans has been silenced during evolution, and the urate oxidase of rodents is still highly active and can decompose uric acid into allantoin and excrete it from the body. Therefore, it is difficult to ensure long-term and continuous high-concentration uric acid exposure of tissues and organs using drug inducers to model rodents.

[0006] Therefore, there is an urgent need in this field to develop a heritable non-human mammalian model that can better simulate the pathological characteristics of human hyperuricemia and maintain a long-term stable hyperuricemia state, which is of great significance for the research and treatment of hyperuricemia. Summary of the invention

[0007] The purpose of the present invention is to provide a non-human mammal model of hyperuricemia that can maintain long-term stability and effectiveness.

[0008] In a first aspect of the present invention, there is provided a use of a gene editing reagent for preparing a kit for constructing a non-human mammal model of hyperuricemia.

[0009] Wherein, the gene editing reagent includes a reagent for introducing an additional second start codon ATG (also called a competitive start codon) upstream of the start codon ATG (also called a major codon or a first start codon) of the UOX gene, and when the second start codon ATG is introduced, the wild-type mRNA transcript level and / or the wild-type UOX protein expression level of the UOX gene is downregulated.

[0010] In another preferred embodiment, the second start codon and the main effect codon are in different reading frames (off-frame).

[0011] In another preferred embodiment, the second initiation codon is not in-frame with the main codon.

[0012] In another preferred embodiment, the length L of the spacer sequence between the second start codon and the main effect codon is 1-100 nt, and the remainder of L / 3 is not 0 (ie, 1 or 2).

[0013] In another preferred embodiment, the length L is 10-20 nt, and the remainder of L / 3 is not 0 (ie, 1 or 2).

[0014] In another preferred embodiment, the remainder of L / 3 is 2.

[0015] In another preferred embodiment, under the reading frame of the second start codon, the spacer sequence between the second start codon and the main effect codon does not contain a stop codon.

[0016] In another preferred embodiment, the sequence for introducing the additional second start codon ATG is a donor fragment sequence as shown in SEQ ID NO:3.

[0017] In another preferred example, the length L of the spacer sequence between the second start codon and the main effect codon is within the reading frame of the second start codon and does not contain a stop codon.

[0018] In another preferred embodiment, the gene editing reagent is selected from:

[0019] (i) sgRNA targeting UOX gene;

[0020] (ii) a vector carrying the sgRNA coding sequence;

[0021] In another preferred embodiment, the vector is a viral vector.

[0022] In another preferred embodiment, the sgRNA sequence is selected from the following group:

[0023] (i) the sequence shown in SEQ ID NO.2;

[0024] (ii) a polynucleotide complementary to the sequence defined in (i);

[0025] In another preferred example, the gene editing reagent also includes a donor fragment, and the sequence of the donor fragment is shown in SEQ ID NO: 3.

[0026] In a second aspect of the present invention, a method for preparing a non-human mammal model of hyperuricemia is provided, comprising the following steps:

[0027] (a) providing non-human mammalian cells, and down-regulating the mRNA transcription level or protein expression of a uric acid metabolism-related gene UOX gene in the cells to obtain non-human mammalian cells in which the mRNA transcription level or protein expression of the UOX gene is down-regulated; and

[0028] (b) using the non-human mammalian cells in which the wild-type mRNA transcript level of the UOX gene and / or the wild-type UOX protein expression level obtained in step (a) is downregulated, to prepare a non-human mammalian model of hyperuricemia in which the mRNA transcription level of the UOX gene or its protein expression is downregulated.

[0029] In another preferred embodiment, the down-regulation of the mRNA transcription level of the UOX gene or its protein expression means that the coding sequence and transcription level of the UOX gene are not affected, while the expression level of the UOX protein is reduced.

[0030] In another preferred embodiment, the mRNA transcription level of the UOX gene refers to reducing the transcription level or translation level of the target gene UOX through a gene editor, thereby reducing the expression level of the protein encoded by the gene in the cell.

[0031] In another preferred embodiment, the down-regulation of the mRNA transcription level of the UOX gene or its protein expression includes a decrease in the expression of the UOX gene or its protein by 30%, preferably 50%, more preferably 70%, more preferably 90%, more preferably 100%.

[0032] In another preferred embodiment, the non-human mammal model of hyperuricemia is an animal model of non-induced spontaneous hyperuricemia or a disease related thereto.

[0033] In another preferred embodiment, the non-human mammal model of hyperuricemia exhibits pathological symptoms associated with hyperuricemia, including but not limited to elevated serum uric acid levels and mild chronic inflammation.

[0034] In another preferred embodiment, the non-human mammal model of hyperuricemia has a high survival rate and can survive for at least 65 weeks, preferably at least 90 weeks, and more preferably at least 124 weeks.

[0035] In another preferred embodiment, the non-human mammal model of hyperuricemia has mild renal impairment or no renal toxicity.

[0036] In another preferred embodiment, the uric acid metabolism-related genes also include the glucose transporter gene GLUT9.

[0037] In another preferred embodiment, the genome sequence of the hyperuricemia mouse in which the mRNA transcription level or protein expression of the UOX gene is downregulated is as shown in SEQ ID NO:9.

[0038] In another preferred embodiment, in step (a), the following steps are included:

[0039] (a1) Using gene editing technology, the non-coding region of the UOX gene is knocked into a donor fragment (uATG sequence) to obtain non-human mammalian cells in which the mRNA transcription level or protein expression of the UOX gene is downregulated.

[0040] In another preferred embodiment, the non-coding region includes but is not limited to: 5'UTR, 3'UTR and intron region, preferably 5'UTR.

[0041] In another preferred embodiment, the insertion position of the donor fragment (uATG sequence) is upstream of the start codon ATG of the UOX gene, and is located in a different reading frame from the start codon ATG.

[0042] In another preferred example, the sequence of the donor fragment (uATG sequence) is shown in SEQ ID NO:3.

[0043] In another preferred embodiment, the gene editing technology includes but is not limited to: zinc finger nuclease gene editing technology, transcription activator-like effector nuclease gene editing technology and CRISPR gene editing technology.

[0044] In another preferred embodiment, the gene editing technology is CRISPR-Cas9 gene editing technology.

[0045] In another preferred embodiment, the step (a1) comprises injecting the mixture of sgRNA and cas9 mRNA into the nucleus of non-human mammalian cells.

[0046] In another preferred embodiment, the step (a1) includes transfecting a vector carrying sgRNA and cas9 mRNA coding sequences into the nucleus of a non-human mammalian cell.

[0047] In another preferred embodiment, the vector carrying the sgRNA and cas9 mRNA coding sequences is a viral vector.

[0048] In another preferred embodiment, after the sgRNA enters the nucleus of a non-human mammalian cell, it can target the UOX gene, and under the action of cas9, the UOX gene is edited, resulting in a specific decrease in the expression of the UOX gene.

[0049] In another preferred embodiment, the target site of the sgRNA is upstream of the start codon ATG of the uric acid metabolism-related gene UOX.

[0050] In another preferred embodiment, the sgRNA sequence is selected from the following group:

[0051] (i) the sequence shown in SEQ ID NO.2;

[0052] (ii) a polynucleotide complementary to the sequence defined in (i).

[0053] In another preferred embodiment, the non-human mammalian cell is a fertilized egg cell.

[0054] In another preferred embodiment, the fertilized egg cell is in a single cell stage.

[0055] In another preferred embodiment, the non-human mammal is a rodent or a primate, preferably including a mouse, a rat, a rabbit and / or a monkey.

[0056] In another preferred embodiment, in step (b), the following steps are also included:

[0057] (b1) preparing a chimeric non-human mammal using the non-human mammal cells in which the mRNA transcription level of the UOX gene or the protein expression thereof obtained in step (a) is downregulated;

[0058] (b2) mating and breeding the chimeric non-human mammal obtained in step (b1) with a normal wild-type non-human mammal, and screening the offspring to obtain heterozygous non-human mammals in which the mRNA transcription level of the UOX gene or the protein expression thereof is downregulated; and

[0059] (b3) obtaining a non-human mammal model of hyperuricemia in which the mRNA transcription level of the UOX gene or its protein expression is downregulated by mating the heterozygous non-human mammals obtained in step (b2) with each other to obtain homozygous non-human mammals in which the mRNA transcription level of the UOX gene or its protein expression is downregulated.

[0060] In another preferred embodiment, the non-human mammal model of hyperuricemia in which the mRNA transcription level of the UOX gene or its protein expression is downregulated obtained in step (b) has one or more characteristics selected from the following group compared with the wild-type control animal:

[0061] (t1) UOX gene expression decreased;

[0062] (t2) UOX protein expression decreased;

[0063] (t3) Increased serum uric acid levels;

[0064] (t4) The number of infiltrating lymphocytes increases;

[0065] (t5) the number of infiltrating macrophages increased;

[0066] (t6) serum creatinine and blood urea nitrogen levels increased slightly;

[0067] (t6) increased mRNA levels of cytokine F4 / 80 in the kidney;

[0068] (t7) The mRNA level of interleukin-1β increased.

[0069] In another preferred embodiment, the reduction refers to an expression level that is significantly lower than a baseline value.

[0070] In another preferred embodiment, the increase refers to that the content, infiltration quantity or mRNA level is significantly higher than the baseline value.

[0071] In another preferred embodiment, the slightly increased content refers to a content higher than the reference value, but without significance.

[0072] In another preferred embodiment, the significantly lower than refers to an expression level ≤ 2 / 3 of the benchmark value, preferably ≤ 1 / 2 of the benchmark value, and more preferably ≤ 1 / 3 of the benchmark value.

[0073] In another preferred embodiment, the significantly higher refers to an expression level ≥ 4 / 3 of the benchmark value, preferably ≥ 3 / 2 of the benchmark value, and more preferably ≥ 2 times of the benchmark value.

[0074] In the third aspect of the present invention, there is provided a use of a non-human mammal model prepared by the method described in the second aspect of the present invention, wherein the model is used as an animal model for studying hyperuricemia or related diseases.

[0075] In another preferred embodiment, the non-human mammal model of hyperuricemia is an animal model of non-induced spontaneous hyperuricemia or a disease related thereto.

[0076] In another preferred embodiment, the non-human mammal model of hyperuricemia exhibits pathological symptoms associated with hyperuricemia, including but not limited to elevated serum uric acid levels and mild chronic inflammation.

[0077] In another preferred embodiment, the non-human mammal model of hyperuricemia has a high survival rate and is non-nephrotoxic.

[0078] In the fourth aspect of the present invention, there is provided a use of a non-human mammal model prepared by the method described in the second aspect of the present invention for screening or identifying substances (therapeutic agents) that can alleviate or treat hyperuricemia or its related diseases.

[0079] In a fifth aspect of the present invention, a method for screening or determining a potential therapeutic agent for preventing and / or treating hyperuricemia or a disease related thereto is provided, comprising the steps of:

[0080] (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing UOX for a period of time T1, and detecting the expression level E1 of the UOX gene or its protein in the culture system of the test group; and / or the activity level V1 of the UOX protein;

[0081] And in a control group without the test compound and with the same other conditions, detecting the expression level E2 of the UOX gene or its protein in the culture system of the control group; and / or the activity level V2 of the UOX protein; and

[0082] (b) comparing E1, E2, and / or V1, V2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for preventing and / or treating hyperuricemia or its related diseases;

[0083] Wherein, if E1 is significantly higher than E2; and / or V1 is significantly higher than V2, it indicates that the test compound is a potential therapeutic agent for preventing and / or treating hyperuricemia or its related diseases.

[0084] In another preferred embodiment, the “significantly higher than” refers to E1 / E2 ≥ 2, preferably ≥ 3, and more preferably ≥ 4.

[0085] In another preferred embodiment, the “significantly higher than” refers to V1 / V2 ≥ 2, preferably ≥ 3, and more preferably ≥ 4.

[0086] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0087] In another preferred embodiment, the method includes step (c), applying the potential therapeutic agent determined in step (b) to the non-human mammal model prepared by the method described in the first aspect of the present invention, so as to determine its effect on hyperuricemia or related diseases in the animal model.

[0088] In a sixth aspect of the present invention, a non-human mammal model is provided, wherein the model is prepared by the method described in the first aspect of the present invention.

[0089] In another preferred embodiment, the non-human mammal model is heterozygous or homozygous for down-regulation of the mRNA transcription level or protein expression of the UOX gene.

[0090] In another preferred embodiment, the down-regulation of the mRNA transcription level of the UOX gene or its protein expression means that the coding sequence and transcription level of the UOX gene are not affected, while the expression level of the UOX protein is reduced.

[0091] In a seventh aspect of the present invention, a use of a cell is provided, wherein the UOX gene or its protein is downregulated, for preparing a biological agent for constructing an animal model of hyperuricemia or related diseases in non-human mammals.

[0092] In another preferred embodiment, the biological agent is a liquid preparation.

[0093] In an eighth aspect of the present invention, there is provided a use of a down-regulator of the wild-type mRNA transcript level of a UOX gene and / or the wild-type UOX protein expression level for preparing a preparation for constructing an animal model of hyperuricemia or related diseases in non-human mammals.

[0094] In another preferred embodiment, the wild-type mRNA transcript level of the UOX gene and / or the wild-type UOX protein expression level down-regulator includes a member selected from:

[0095] (i) sgRNA targeting UOX gene;

[0096] (ii) a vector carrying the sgRNA coding sequence;

[0097] In another preferred embodiment, the vector is a viral vector.

[0098] In another preferred embodiment, the down-regulator includes an inhibitor.

[0099] In another preferred embodiment, the down-regulator refers to reducing the expression of the UOX gene or its protein by 30%, preferably 50%, more preferably 70%, more preferably 90%, more preferably 100%.

[0100] In another preferred embodiment, the down-regulator is selected from the group consisting of antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0101] In the ninth aspect of the present invention, a down-regulator for the wild-type mRNA transcript level and / or the wild-type UOX protein expression level of a UOX gene is provided, wherein the down-regulator comprises an sgRNA and a donor fragment, and the sgRNA sequence is selected from the following group:

[0102] (i) the sequence shown in SEQ ID NO.2;

[0103] (ii) a polynucleotide complementary to the sequence defined in (i);

[0104] The sequence of the donor fragment is shown in SEQ ID NO:3.

[0105] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The figure is a schematic diagram of inhibiting the expression of uric acid metabolism-related proteins by knocking in uATG in the present invention; wherein Promoter refers to promoter, Target gene refers to target gene, ATG refers to the start codon of target gene, uATG refers to the knocked-in upstream ATG, 5'UTR is the region where uATG is knocked in by gene editing, and the knocked-in uATG will generate an upstream uORF before the main effect ORF of the target gene, affecting the translation of the main effect ORF into protein.

[0107] Figure 2 The present invention is a schematic diagram of the process of constructing a mouse with knockdown of uric acid metabolism-related genes; female mice are injected with HCG to stimulate ovulation and mated with male mice, and cumulus cell complexes are collected from the oviducts of mated female mice. Cas9 mRNA, sgRNA and donor fragments are injected into the cumulus cell complex. Embryos are cultured to the two-cell stage under appropriate conditions, pseudo-pregnant female mice are selected as surrogate mice, and the developed embryos are transplanted into the uterus of surrogate mice. The mouse genotype is detected 3 weeks after birth.

[0108] Figure 3Figure 1 is a schematic diagram of the knockdown of uATG in the urate oxidase gene UOX and the results of the UOX gene expression in the mutant liver; Figure A shows the schematic diagram of the introduction of uATG in the 5'UTR of the UOX gene. The white box represents the 5'UTR, the green square and green font represent the major ORF of the UOX gene, the red font in the sequence represents the PAM sequence of Cas9, the green highlight represents the start codon ATG of the UOX gene, and the red highlight represents the knocked-in uATG. The Sanger sequencing peak diagram shows that the UOX-KD mouse is a homozygous knock-in mutant, the underlined part represents the position of the introduced uATG, and the red box represents the major ATG position of the UOX gene. Figure B shows the results of the liver expression of the UOX gene determined by quantitative real-time PCR in wild-type (WT) and homozygous UOX knockdown (KD) mice; Figure C shows the liver expression of the UOX gene determined by Western Blot analysis in wild-type (WT) and homozygous UOX knockdown (KD) mice. The two-sided Student t test was used for statistical analysis: ns indicates not significant; Figure D shows that after the uATG1 sequence was inserted into the original start codon of UOX (at the horizontal line), the transcript (mRNA) transcribed from uATG1 resulted in the production of a 55aa mutant protein.

[0109] Figure 4 Figure 2 is the pathological test result of urate oxidase gene UOX knockdown mice; Figure A shows the serum uric acid level between wild-type (WT) and homozygous UOX knockdown (KD) mice. Figure B shows the blood urea nitrogen and serum creatinine levels of wild-type (WT) and homozygous UOX knockdown (KD) mice. Figure C shows the infiltration of lymphocytes and macrophages in the kidney determined by immunostaining with anti-CD3 and anti-CD68 antibodies, respectively. Figure D shows the renal expression of F4 / 80 and interleukin-1β determined by quantitative real-time PCR. F4 / 80 is a special glycoprotein expressed on the surface of macrophages; data are mean ± SD; statistical analysis was performed using a two-sided Student t test: *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0110] After extensive and in-depth research, the inventors unexpectedly developed a non-human mammal model with elevated blood uric acid levels and long survival time (referred to as "the non-human mammal model of the present invention" for the first time). Specifically, the inventors unexpectedly discovered that by introducing an interfering or competitive second start codon ATG upstream of the original start codon ATG (also referred to as the "first start codon") of the UOX gene, not only can the level of the wild-type protein of the UOX gene be significantly reduced efficiently, but also without causing toxic and side effects such as nephrotoxicity, and without causing the death of non-human model animals. The present invention was completed on this basis.

[0111] Terminology

[0112] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0113] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0114] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0115] As used herein, the term "downregulation of the mRNA transcription level of a gene or its protein expression" refers to reducing the transcription level or translation level of a target gene through a gene editor, thereby reducing the expression level of the protein encoded by the gene in the cell.

[0116] UOX gene and protein

[0117] As described herein, "UOX gene" and "gene related to uric acid metabolism" refer to the urate oxidase gene, which encodes a 303-amino acid urate oxidase; it is mainly expressed in kidney and liver cells, and is also expressed in other organs of the urinary system and digestive system. The ORF sequence of the mouse UOX gene is shown in SEQ ID NO: 1:

[0118] ATGGCCCATTACCATGACAACTACGGAAAGAATGATGAAGTGGAATTTGTCCGAACTGGCTATGGGAAAGACATGGTCAAAGTTCTCCATATTCAGAGAGATGGAAAATACCACAGCATCAAAGAGGTGGCAACTTCGGTCCAGTTGACTCTGAGGTCCAAGAAGGATTACCTACACGGTGATAATTCCGACATCATCCCTACAGACACCATCAAGAACACTGTGCACGTCCTGGCGAAGCTCAGAGGGATCAGAAACATCGAGACCTTTGCAATGAACATCTGTGAGCACTTCCTCTCTTCTTTTAACCATGTCACTCGAGCCCACGTCTACGTGGAAGAGGTCCCCTGGAAACGATTTGAAAAGAATGGGATCAAACACGTCCATGCATTCATCCACACCCCGACTGGAACGCACTTCTGTGAGGTGGAGCAGATGAGAAACGGACCTCCCGTCATTCACTCTGGAATCAAAGACCTCAAGGTCTTGAAAACAACCCAGTCTGGGTTTGAAGGGTTCCTCAAGGACCAGTTCACTACCCTCCCTGAGGTGAAGGACCGATGCTTTGCCACTCAAGTGTACTGCAAGTGGCGCTACCAGAGACGGGACGTGGACTTCGAGGCTATCTGGGGCGCTGTCCGGGACATTGTCCTGCAGAAATTTGCTGGGCCCTATGACAAAGGTGAATACTCACCTTCCGTGCAGAAGACCCTCTATGATATACAAGTGCTGTCCCTGAGCCAGCTTCCTGAGATAGAAGACATGGAAATCAGCCTTCCGAACATTCACTACTTTAACATCGACATGTCCAAAATGGGACTGATCAACAAGGAAGAGGTTTTGCTGCCTCTCGACAATCCCTACGGCAAAATAACAGGGACAGTCAAGAGGAAGCTGCCTTCCAGGCTGTGA(SEQ ID NO:1)

[0119] Positions 1 to 3 of SEQ ID NO: 1 are the original start codon ATG of the wild-type UOX transcript or wild-type UOX protein, also referred to as the "dominant codon" or the "first codon".

[0120] In the present invention, the terms "UOX knockdown mouse animal model", "non-human mammal model of hyperuricemia with downregulated UOX gene expression", "non-human mammal model of hyperuricemia with downregulated mRNA transcription level of UOX gene or its protein expression" and "UOX-KD mouse" can be used interchangeably, all referring to the non-human mammal model of hyperuricemia with downregulated mRNA transcription level of UOX gene or its protein expression described in the first aspect of the present invention.

[0121] Second initiation codon (uATG) and major effect codon

[0122] As used herein, the terms "uATG", "second start codon", "competitive upstream start codon" and the like are used interchangeably to indicate an exogenous start codon introduced upstream of the major codon of the UOX gene by genetic engineering.

[0123] See also Figure 1 ,The inventors constructed a non-human mammalian model of hyperuricemia in which the mRNA transcription level or protein expression of the UOX gene was downregulated based on CRISPR / Cas9 gene editing technology, and knocked in uATG in the non-coding region of the UOX gene.

[0124] like Figure 1 As shown, insertion of uATG upstream of the major start codon (and off-frame from the major start codon ATG) can result in reduced expression levels of the desired UOX protein, rather than no expression of the UOX protein at all.

[0125] The animal model constructed by the present invention can better simulate the human hyperuricemia through a normal diet, and can reduce the serious lethality and nephrotoxicity and other side effects caused by UOX gene knockout mice (i.e., mice that do not express UOX protein); the model animal provided by the present invention only induces an increase in the blood uric acid level in the body without nephrotoxicity, and has a long survival time, and can better simulate the human hyperuricemia in the aging state, providing more accurate and rich experimental animal subjects for the study of hyperuricemia.

[0126] Gene Editor

[0127] In the present invention, the gene editor includes a DNA gene editor and an RNA gene editor. In a preferred embodiment, the gene editor of the present invention includes a gene editing protein and an optional sgRNA.

[0128] Gene editing proteins

[0129] In the present invention, the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence. In a preferred embodiment of the present invention, the gene editing protein includes, but is not limited to, Cas13 (such as CasRx), Cpf1, SaCas9, Cas13a, Cas13b, and Cas13c.

[0130] CRISPR / Cas system

[0131] The CRISPR / Cas system (Clustered regularly interspaced short palindromicrepeats / CRISPR-associated protein) is an acquired immune defense mechanism in prokaryotes that resists the invasion of foreign genes. It has evolved from bacteria and archaea in the process of resisting the invasion of foreign viruses and bacteriophages. The system can integrate DNA fragments from foreign invaders into the CRISPR site, and then guide the Cas endonuclease to cut the foreign DNA sequence through the corresponding CRISPR RNAs (crRNAs), thereby resisting the invasion of viruses or bacteriophages. The CRISPR / Cas gene cluster consists of a series of genes encoding Cas proteins (Cas1, Cas2, Cas4 and effector proteins such as Cas9, Cpf1, etc.) and a CRISPR sequence.

[0132] The CRISPR sequence consists of a leader, many short and conservative repeat regions, and a spacer. The repeat region contains a palindromic sequence that can form a hairpin structure. The spacer is the exogenous DNA sequence captured by the host. These captured exogenous DNA sequences are equivalent to the "blacklist" of the immune system. When these exogenous genetic materials invade the host again, the bacteria begin to transcribe CRISPR to form a primary transcription product, pre-crRNA, which is then cut by ribonucleases or Cas proteins within the repeat sequence site to form mature crRNA, which forms a ribonucleoprotein complex with specific CRISPR effector proteins to recognize and cut exogenous DNA that can complementarily pair with crRNA, causing double-strand breaks and triggering self-repair of host cells.

[0133] According to the composition of the Cas gene and the number of effector proteins, CRISPR is divided into 2 categories and 5 types, with a total of 16 subtypes. Category 1 is a CRISPR / Cas system that uses multiple effector protein complexes to interfere with target genes, including types I, III and IV; Category 2 is a CRISPR / Cas system that uses a single effector protein to interfere with target genes, including types II and V. Currently, the most widely studied and used is type II, namely the CRISPR / Cas9 system. This system successfully achieved gene editing in mammalian cells in 2013. The type II system can use a single Cas9 nuclease to accurately and fully cut the DNA target site through the guidance of crRNA. The system is simple to operate, has a short experimental cycle, high efficiency, and is widely applicable to multiple species. The system requires the design of a special guide RNA, namely sgRNA (single guide RNA). The sequence design of sgRNA is a 20nt nucleotide sequence in the PAM (NGG) region of the genome sequence. Under the guidance of sgRNA, Cas9 protein can achieve site-specific cutting of the genome, causing double-strand breaks in DNA and activating the cell's non-homologous end joining (NHEJ) or homologous recombination (HR) repair mechanisms, thereby achieving gene knockout, random fragment deletion or insertion, or using specific template repair to achieve permanent modification of the genome.

[0134] hyperuricemia

[0135] Hyperuricemia (HUA) is a chronic metabolic disease caused by purine metabolism disorder. In recent years, with the improvement of living standards, the prevalence of HUA has been rising year by year. It has become the "fourth most common disease" after hypertension, hyperlipidemia, and hyperglycemia, and the second largest metabolic disease after diabetes, which seriously threatens human health. Hyperuricemia is not only the main cause of gout, but also closely related to a variety of metabolic diseases such as obesity, hypertension, diabetes, and cardiovascular disease.

[0136] Animal Model of Hyperuricemia

[0137] In the present invention, a non-human mammal model of hyperuricemia with downregulated UOX gene expression is constructed based on CRISPR / Cas9 gene editing technology. The technical solution adopted by the present invention is:

[0138] 1. Determine the specific target site sgRNA of the UOX gene to be knocked in and transcribe it into mRNA in vitro with Cas9 nuclease;

[0139] 2. Microinject active sgRNA and Cas9 mRNA into mouse fertilized eggs to obtain UOX gene knockout mice; the sgRNA is shown in SEQ ID NO: 2.

[0140] In one embodiment of the present invention, the method further comprises the following steps:

[0141] (1) Inducing ovulation and in vitro fertilization of mice to cultivate fertilized eggs;

[0142] (2) Microinjection of active sgRNA and Cas9 mRNA into mouse fertilized eggs;

[0143] (3) In vitro culture of fertilized eggs, implantation of receptors, and cultivation of targeted gene-modified animals.

[0144] In one embodiment of the present invention, the method comprises the following steps:

[0145] (1) Determine the target site of the UOX gene to be knocked in, and transcribe the sgRNA and Cas9 nuclease mRNA in vitro;

[0146] (2) Ovulation induction, in vitro fertilization, and fertilized egg microinjection in mice;

[0147] (3) The fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice to produce mice, which are the F0 generation mice;

[0148] (4) Extract the tail DNA of F0 mice, amplify by PCR and send the product for sequencing;

[0149] (5) mating the positive mice with wild-type mice of the opposite sex to obtain F1 heterozygous mice;

[0150] (6) F1 heterozygous mice are hybridized to obtain F2 homozygous mice, which are the mouse animal model.

[0151] In one embodiment of the present invention, the method further comprises the step of identifying a mouse animal model in which the mRNA transcription level of the UOX gene or its protein expression is downregulated.

[0152] In one embodiment of the present invention, the mouse animal model for identifying the down-regulation of the mRNA transcription level or protein expression of the UOX gene is specifically:

[0153] (1) The fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice to produce mice, which are the F0 generation mice;

[0154] (2) Extract the tail DNA of F0 mice, amplify by PCR and send the product for sequencing;

[0155] (3) The positive mice were mated with wild-type mice of the opposite sex to obtain F1 heterozygous mice;

[0156] (4) F1 heterozygous mice are hybridized to obtain F2 homozygous mice, which are the mouse animal model.

[0157] The technical solution of the present invention has the following beneficial effects:

[0158] 1. The present invention provides a non-human mammal model of hyperuricemia, which has the same pathological manifestations as human hyperuricemia and high serum uric acid levels, and is an ideal model for studying hyperuricemia diseases.

[0159] 2. The difference between the present invention and the existing models is that the present model optimizes the modeling scheme, which has no nephrotoxicity on the one hand and prolongs the survival period of the model animals on the other hand.

[0160] 3. The present invention provides an optimized modeling scheme. On the one hand, the model yield is high, the overall embryo quality is good, and it has the advantage of a high knockout rate. On the other hand, the mouse model is fed with a normal diet and is more similar to the human hyperuricemia phenotype.

[0161] 4. The UOX-KD transgenic modeling scheme provided by the present invention affects the translation level of the mouse uric acid metabolism-related protein UOX without changing the transcription level of the UOX gene.

[0162] 5. The present invention provides a convenient, reliable and economical animal model for studying the relationship between UOX and hyperuricemia, and provides a reliable theoretical basis for its research. The detection of UOX protein expression shows that after the UOX gene is knocked into uATG, it neither fully expresses the UOX protein nor does not express the UOX protein, which can better simulate the human hyperuricemia; this model can better reduce the side effects of UOX gene knockout mice (i.e. mice that do not express UOX protein). For example, the model mice have no nephrotoxicity and survive longer, which better simulates the human hyperuricemia in the aging state, and provides more accurate and rich experimental animal subjects for the study of hyperuricemia.

[0163] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0164] Example 1 Selection and feeding of mice

[0165] The experimental animals were wild-type C57BL / 6 female mice (SPF grade, 3-4 weeks old) and wild-type male C57BL / 6 mice (SPF grade, 6-8 weeks old), purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The above mice were in good health and mental condition, without trauma or disability. The breeding environment was SPF grade environment, 12h alternating day and night, temperature (22±4)℃, humidity (55±10)%. The adaptation period was 1 week, during which the mice were given standard feed and clean drinking water, and the drinking water, bedding and feed were changed every day.

[0166] Example 2 Preparation of Cas9 mRNA, sgRNA and donor fragments

[0167] 2.1sgRNA design

[0168] The sgRNA target site was designed in the 5'UTR of the UOX gene. The designed sgRNA target site sequence was: 5'-TTCCCTCAGCCTTTAACTTC AGG -3' (SEQ ID NO: 2), where the underline is the PAM sequence of Cas9. sgRNA was synthesized by Integrated DNA Technologies (IDT).

[0169] 2.2 Donor fragment design

[0170] Figure 3 A is the molecular experimental design process for the construction of UOX-KD mice. The donor fragment was designed based on the UOX gene of wild-type mice. The donor fragment introduced an additional uATG upstream of the major ATG (referring to the start codon of the UOX gene itself) of the UOX gene. The donor fragment was knocked into the genome by CRISPR-Cas9 to achieve the introduction of uATG and complete the construction of UOX-KD mice. The donor fragment was synthesized in GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.).

[0171] like Figure 3 As shown in A, the donor fragment was designed according to the 5'UTR sequence of the wild-type mouse UOX gene and the upstream and downstream sequences of the designed sgRNA target site. The length of the 5' homology arm is 45 bp, and the length of the 3' homology arm is 42 bp. The specific donor fragment sequence designed is as follows:

[0172] (1) uATG-1 sequence:

[0173] 5'-CCACTCTTCGCTTCACACCATTCCGTCTCATTCTTGAAATCGAAC TGAAAAGGCTGAGGGAAAA GCCCATTACCATGACAACTACGGAAAG GTGA-3' (SEQ ID NO: 3, where the underlined part is uATG and the bold font is the major effect ATG)

[0174] like Figure 3 D, wherein the horizontal line is the position of the original start codon ATG, and the transcript (mRNA) transcribed from uATG1 leads to the production of a 55aa mutant protein, the sequence of which is shown in SEQ ID NO: 10: MKRLREKWPITMTTTERMMKWNLSELAMGKTWSKFSIFRE MENTTASKRWQLRSS (SEQ ID NO: 10)

[0175] (2) uATG-2 sequence:

[0176] 5'-CCACTCTTCGCTTCACACCATTCCGTCTCATTCTTGAAATCGAAC A TG AACGTTAAAGGCTGAGGGAAAA GCCCATTACCATGACAACTACG GAAAGGTGA-3' (SEQ ID NO: 4, where the underlined part is uATG and the bold font is the main effect ATG)

[0177] The transcript (mRNA) formed by transcription of uATG-2 results in the production of a short peptide of 5 aa (sequence not shown).

[0178] Through screening, this example found that the UOX protein expression level of UOX knockdown mice after insertion of the donor sequence uATG-1 was significantly lower than that of UOX knockdown mice after insertion of the donor sequence uATG-2, so the uATG-1 sequence was selected as the donor sequence.

[0179] 2.3 Preparation of Cas9 mRNA

[0180] The forward and reverse primers below were used to add amplification reagents and Cas9 IVT vectors to amplify Cas9 DNA by PCR. The reaction conditions were 98°C pre-denaturation for 5 min; 35 cycles according to the cycle program (98°C 30s, 60°C 30s, 72°C 30s); 72°C extension for 5 min. The reaction products were subjected to 2% agarose gel electrophoresis. The in-gel Cas9 PCR product was purified using FastPure Gel DNA Extraction MiniKit (purchased from Vazyme) and T7 High Yield RNASynthesis Kit (purchased from NEB) was used to transcribe Cas9 mRNA in vitro. The primer sequences are as follows:

[0181] Cas9-ivt-F: 5'-GTAAAACGACGGCCAGTGAATTCG-3' (SEQ ID NO: 5)

[0182] Cas9-ivt-R: 5'-CAGGAAACAGCTATGACCATGA-3' (SEQ ID NO: 6)

[0183] 2.4 Obtaining and transplanting UOX knockdown mouse embryos

[0184] like Figure 2 As shown, several female C57BL / 6 mice were injected with 10IU PMSG, and the next day they were injected with 10IU HCG, and then cohabited with male C57BL / 6 mice overnight. The female mice with thrombosis were euthanized, and the cumulus cell complexes were collected from the oviduct. The cumulus cell complexes were washed several times in M2+ hyaluronidase medium, placed in KSOM medium at 35°C to 38°C, and cultured in a 5% CO2 incubator. The microinjection needles were pulled, bent, and fixed using a needle puller and a needle breaker.

[0185] Under a microscope, Cas9 mRNA, sgRNA and donor fragments were injected into pronuclear embryos with a microinjection needle, including sgRNA 10ng / μL, Cas9 mRNA 20ng / μL, donor fragment 10ng / μL, and injection volume 5-8pL; embryos were cultured in KSOM medium for 24 hours until they became two-cell embryos, and the cleavage and blastocyst development of the embryos were observed. Embryos microinjected with Cas9 protein and UOX-sgRNA were transplanted into two-cell embryos.

[0186] The ligated male mice were placed in a cage with the estrus C57BL / 6 female mice. The next day, the female mice with 0.5 days of thrombosis were selected as surrogate mothers. Under a stereo microscope, the fallopian tube was found from the back of the surrogate mother, and the embryo was transferred from the fallopian tube into the uterus. The surrogate mother mice were sutured surgically and placed in the culture room for observation. The F0 generation mice were heterozygous mice with normal appearance and good health, and no trauma or infection occurred.

[0187] Identification of 2.5UOX knockdown mice

[0188] The surrogate mother mice gave birth to F0 mice 3 weeks later. Male and female F0 mice were raised separately 3 weeks after birth. The F0 mice 3 weeks after birth were taken, and the tail tip of about 7 mm was cut off. Genomic DNA was extracted using TIANamp Genomic DNA Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the gene knockdown was verified by PCR and Sanger sequencing. The primer sequences are as follows:

[0189] UOX-F: 5'-GTGAGCACTTCCTTCTCTTCTTT-3' (SEQ ID NO: 7)

[0190] UOX-R: 5'-GGACGTGTTTGATCCCATTCT-3' (SEQ ID NO: 8)

[0191] The genomic sequence of the UOX knockdown mouse is shown in SEQ ID NO: 9 (wherein the underlined portion is uATG and the bold font is the major effect ATG).

[0192] AAAGGAATTCCTGACAAACAGATGTTGAGTAAGGTGTGTTCCGTGTTTCTTTTCTCTTCTAGTTGACTATCCGTACGTGTAAATGCATGTAATTCATACATTAGCTAGAAACAACAAAGCCTTTGCCTTCTTAATAATTAGGATCTGTCCTATAGTAGCCTATGTAATAGGTTAATAACCTATAATAACCCCTGTCTTTAAGGGAATAACTTCTAACCACTATTCTGAAGGT TGCTTCAAACTAAGAGCAAATTCAATTGAGTTTCAGCAAAAATGTTGACATTATCTTCCACACACCCTGGTCTGACCACTCAGCGAGGCATTTGGCTTTGAAAGGCCTTTGTGTCTCACTGTCCACTATGCAATCAGGGCAGTATTGATCGCCTACTCTTCGGGGTTAAATAAAACCCTTTACCAACCACTCTTCGCTTCACACCATTCCGTCTCATTCTTGAAATCGAAC ATG AAAAGGCTGAGGGAAAA GCCCATTACCATGACAACTACGGAAAGGTGAGAGGAGGGAAAGGTAGATAGATACACATACATCTGCAAGTATTTTTTTCACTTTTCTCGAATCTACACTCAGCTGTCATGCTTAGTCACTTCATGCCTGGGAATCACGGTGGCTCTGCTCTGTCTCTCCATG ATTATAGACTCAGATTGGAGTCATCTTGGAGACCCAGACCAAACCAGAGCCCTGAAGATCAGGTCACTTCTATGGACTAGCCTTGCTGGTATTCTTCAGGACTCTCATTTGTAGGAAAGGAAGGGCTTGTGGCTCTGTTTCTCCTAAGACAGAGGACCAA(SEQ ID NO:9)

[0193] 2.6 Construction of homozygous UOX knockdown mice and survival test

[0194] The knockdown mice of the F0 generation were mated with wild-type mice to obtain the offspring F1, and mutation analysis was performed on the F1 generation mice. The mutant F1 generation heterozygous female mice were selected to mate with male mice to obtain the offspring F2, and mutation analysis was performed on the F2 generation mice. The homozygous mice in the F2 generation were mated to construct the UOX gene knockdown homozygous mouse strain (abbreviated as UOX-KD mice).

[0195] After further mating, the present invention finally detected the survival of UOX-KD mice of the F6 generation as shown in Table 1 below:

[0196] Table 1

[0197]

[0198]

[0199] It has been reported in the prior art that 65% of UOX homozygous knockout mice die at around 4 weeks of age. Only 40% of UOX homozygous knockout mice can survive to 64 weeks. The results in Table 1 above show that the average survival days of the UOX-KD mice prepared by the present invention can reach 687 days (about 98 weeks), among which the higher can reach 868 days, about 124 weeks; far higher than the survival lifespan of UOX homozygous knockout mice in the prior art, indicating that the UOX knockdown mice prepared by the present invention can significantly prolong the lifespan of hyperuricemia animal model mice. It is more conducive to the long-term and stable study of hyperuricemia.

[0200] 2.7 Pathological detection of homozygous UOX knockdown mice

[0201] In order to observe the modeling effect of hyperuricemia mice, the present invention conducted a series of pathological tests on UOX knockdown homozygous mice.

[0202] The results of qPCR showed that there was no significant change in the UOX mRNA level in UOX-KD mice compared with wild-type mice ( Figure 3 B) while the expression level of UOX protein was significantly decreased ( Figure 3 C);

[0203] Serum biochemical analysis showed that the serum uric acid (SUA) level of UOX-KD mice increased sharply from 50 μmol / L to over 300 μmol / L ( Figure 4 A), while serum creatinine and blood urea nitrogen only changed slightly, indicating that renal function was less impaired ( Figure 4 B).

[0204] Hyperuricemia is often associated with severe chronic inflammation, which directly contributes to the high mortality rate in mouse models of the disease. + ) and macrophages (CD68 + )'s infiltration condition ( Figure 4 C) and detection of mRNA levels of cytokines F4 / 80 and interleukin-1β (IL-1β) in kidneys ( Figure 4 D) only showed mild cortical medullary inflammation, which may contribute to the extended lifespan of UOX-KD mice and the increased SUA levels. These results indicate that the present invention has successfully created a better hyperuricemia mouse model, which will be helpful for further research related to hyperuricemia.

[0205] discuss

[0206] Upstream ORF is an important post-transcriptional regulatory element for protein translation. About half of human and mouse transcripts contain one or more uATG-initiated upstream ORFs, which have been reported to cause extensive gene expression inhibition, highlighting the importance of their research. In the present invention, the inventors verified the inhibitory effect of uATGs on downstream mORFs protein expression. Then different forms of uATG were introduced into the 5'UTR region of the CCL19 and IL12A genes, and protein and mRNA levels were detected in the dual luciferase system and K562 cell line. Although the transcription levels of different mutants did not change, the protein levels decreased to varying degrees. This result is reasonable because the regulation of uATG or uORF is driven by multiple factors, such as the distance between uATG and mATG, the context of uATG, and even the number of uATGs.

[0207] In addition, the inventors also used the CRISPR / Cas9 system to genetically engineer the 5'UTR of the UOX gene and successfully obtained a hyperuricemia mouse model, providing a new method for functional studies of essential genes and animal model construction. Previous studies have shown that uORFs are enriched in oncogenes and that loss-of-function mutations in uORFs are closely related to human malignant tumors. Therefore, the strategy developed in this study also provides new ideas for tumor treatment.

[0208] RNAi and CRISPRi are two widely used gene knockout methods, but are not suitable for producing long-term gene silencing effects in vivo. Although shRNA-mediated RNAi can be used to construct stable knockout cell lines and mice, potential toxicity and off-target effects limit its application. In the present invention, the CRISPR / Cas9 system was used to create uATG upstream of the mORF, and stable cell lines and mice with reduced target gene expression were successfully generated. Nowadays, another knock-in tool called prime editing has been developed, which has higher efficiency and precision and has also been used to introduce uORFs in rice and adjust plant phenotypes. Therefore, it will also be interesting to use this tool to upgrade the strategy of the present invention in mammalian cells and animal models, which will definitely improve the efficiency of knock-in and broaden the scope of application of the method of the present invention.

[0209] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a gene editing reagent, characterized in that: A kit for preparing a non-human mammal model for constructing hyperuricemia, Wherein, the gene editing reagent includes a reagent for introducing an additional second start codon ATG (also called a competitive start codon) upstream of the start codon ATG (also called a major codon or a first start codon) of the UOX gene, and when the second start codon ATG is introduced, the wild-type mRNA transcript level and / or the wild-type UOX protein expression level of the UOX gene is downregulated.

2. The use according to claim 1, characterized in that The length L of the spacer sequence between the second start codon and the main effect codon is 1-100 nt, and the remainder of L / 3 is not 0 (ie, 1 or 2).

3. The use according to claim 1, characterized in that In the reading frame of the second start codon, there is no stop codon in the spacer sequence between the second start codon and the main effect codon.

4. The use according to claim 1, characterized in that The sequence for introducing the additional second start codon ATG is a donor fragment sequence as shown in SEQ ID NO:

3.

5. A method for preparing a non-human mammal model of hyperuricemia, characterized in that: The following steps are involved: (a) providing non-human mammalian cells, and down-regulating the mRNA transcription level or protein expression of a uric acid metabolism-related gene UOX gene in the cells to obtain non-human mammalian cells in which the mRNA transcription level or protein expression of the UOX gene is down-regulated; and (b) using the non-human mammalian cells in which the wild-type mRNA transcript level of the UOX gene and / or the wild-type UOX protein expression level obtained in step (a) is downregulated, to prepare a non-human mammalian model of hyperuricemia in which the mRNA transcription level of the UOX gene or its protein expression is downregulated.

6. The preparation method according to claim 5, characterized in that: The down-regulation of the mRNA transcription level of the UOX gene or its protein expression means that the coding sequence and transcription level of the UOX gene are not affected, while the expression level of the UOX protein is reduced.

7. The preparation method according to claim 5, characterized in that: The non-human mammal model of hyperuricemia can survive for at least 65 weeks, preferably at least 90 weeks, and more preferably at least 124 weeks.

8. Use of the non-human mammal model prepared by the method according to claim 5 for screening or identifying substances (therapeutic agents) that can alleviate or treat hyperuricemia or its related diseases.

9. A method for screening or determining a potential therapeutic agent for preventing and / or treating hyperuricemia or a disease related thereto, characterized in that: Includes steps: (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing UOX for a period of time T1, and detecting the expression level E1 of the UOX gene or its protein in the culture system of the test group; and / or the activity level V1 of the UOX protein; And in a control group without the test compound and with the same other conditions, detecting the expression level E2 of the UOX gene or its protein in the culture system of the control group; and / or the activity level V2 of the UOX protein; and (b) comparing E1, E2, and / or V1, V2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for preventing and / or treating hyperuricemia or its related diseases; Wherein, if E1 is significantly higher than E2; and / or V1 is significantly higher than V2, it indicates that the test compound is a potential therapeutic agent for preventing and / or treating hyperuricemia or its related diseases.

10. A down-regulator of the wild-type mRNA transcript level of a UOX gene and / or the wild-type UOX protein expression level, characterized in that: The down-regulator comprises sgRNA and a donor fragment, and the sgRNA sequence is selected from the following group: (i) the sequence shown in SEQ ID NO.2; (ii) a polynucleotide complementary to the sequence defined in (i); The sequence of the donor fragment is shown in SEQ ID NO:3.

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