Method for constructing miR-181a-2 knockout mouse animal model based on CRISPR / Cas9 and application of miR-181a-2 knockout mouse animal model

Abstract

The invention discloses a method for constructing a miR-181a-2 knockout mouse animal model based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 and application of the method, and belongs to the technical field of transgenosis. According to the invention, two sgRNAs of a specific targeting non-coding gene miR-181a-2 are designed, the sgRNAs comprise an sgRNA 1 with a nucleotide sequence as shown in SEQ ID NO.2 and an sgRNA 4 with a nucleotide sequence as shown in SEQ ID NO.5, bidirectional knockout is carried out by combining Cas9 protein, and a miR-181a-2 knockout mouse strain capable of stably breeding is obtained. Experimental results show that the knockout method provided by the invention does not generate off-target in preparation of the mouse model, and has the advantages of accuracy, high efficiency, simplicity and convenience. An oral cancer model is constructed by using the knockout strain, so that the modeling time is shortened, the research cost is reduced, and the knockout strain has remarkable advantages in the aspects of scientific research efficiency and achievement transformation and provides great convenience for related treatment of oral cancer.

Description

Technical Field

[0001] The present invention relates to the field of transgenic technology, and in particular to a method for constructing a miR-181a-2 knockout mouse animal model based on CRISPR / Cas9 and an application thereof. Background Art

[0002] Gene editing technology is a technology that uses nucleases to modify genomic DNA in a targeted manner, which can achieve the deletion and replacement of specific DNA bases. At present, the three commonly used gene editing tools are: zinc finger nucleases (Zincfinger Nucleases, ZFNs), transcription activator-like effector nucleases (Transcription Activator-Like Effector Nucleases, TALENs) and CRISPR / Cas9 system. Among them, CRISPR / Cas9, as a new type of genome editing technology, has the advantages of simplicity, good specificity and high cutting efficiency. CRISPR / Cas9 technology has been applied in a variety of organisms, and a variety of genetically modified organisms have been successfully obtained. At present, CRISPR / Cas9 technology has formed an engineered system that only requires sgRNA and Cas9 protein to work together to achieve the editing of the target sequence. At present, CRISPR / Cas9 technology is widely used in the field of oncology research. For key genes in the development of tumors, gene-edited animal models are prepared by directly injecting Cas9 protein and sgRNA into fertilized eggs for the study of the pathogenesis of related diseases, providing more new model resources for the field of biomedical research. This technology has been used to construct a variety of mouse gene knockout models, providing an important tool for studying the functions of specific genes and disease mechanisms.

[0003] miRNA is a class of endogenous single-stranded small non-coding RNA with a length of 18-25 nucleotides. It is widely present in eukaryotic cells and has an important role in gene regulation. In recent years, the function of miRNA has become the focus of oncology research. It is differentially expressed in different types of tumors and directly participates in the regulation of key biological processes such as tumor cell cycle, differentiation, apoptosis, invasion and long-distance metastasis. Compared with protein expression, miRNA is more sensitive as a biomarker, which makes them have great application potential in the diagnosis and treatment of tumors. Studies have reported that the miR-181a family has been confirmed to play a key role in the process of malignant transformation. The members of this family mainly affect the expression of target genes through expression changes and act on related signaling pathways, ultimately affecting the biological behaviors of tumor cells such as growth, programmed cell death, differentiation, and metastasis. miR-181a-5p is a highly conserved member of the miR-181 family, located on the long arm of chromosome 9. miR-181a-5p is mainly transcribed from the precursor miR-181a-2, and is widely involved in regulating the proliferation and differentiation of many tissue cells in the body. The expression level of miR-181a-5p is inconsistent in different tumors and different tissue types of the same tumor. It has a dual regulatory role in the development of cancer and can act as a tumor suppressor or tumor promoter to affect the expression of target genes at the post-transcriptional level, which shows the complexity and diversity of the regulatory mechanism of miR-181a-5p. It has been reported that miR-181a-5p is upregulated in tumors such as breast cancer, thyroid cancer, multiple myeloma, and liver cancer, and it behaves as a cancer-promoting miRNA, promoting the occurrence, development, invasion and metastasis of tumors. At the same time, the expression of miR-181a-5p is reduced in malignant tumors such as non-small cell lung cancer, glioblastoma, oral cancer, and acute myeloid leukemia, and it behaves as a tumor suppressor miRNA, inhibiting the proliferation and invasion of tumor cells. In gastric cancer, miR-181a-5p can regulate cell autophagy and apoptosis. In hepatocellular carcinoma, miR-181a-5p can target Egr1 to inhibit the Egr1 / TGF-β / Smad signaling pathway and inhibit cell malignant proliferation. miR-181a-5p is also associated with PTEN / AKT, NF-κB, and Wnt signaling pathways, and is involved in the occurrence and development of tumors.

[0004] Given that miR-181a-5p plays a key role in the development of various tumors, further research on the function and role of miR-181a-5p is of great significance for clarifying the pathogenesis of tumors. How to quickly and accurately construct a miR-181a-5p knockout mouse model to promote the study of related diseases is an urgent problem that needs to be solved.

[0005] CRISPR / Cas9 technology has the following risks: ① off-target effect, that is, the Cas9 enzyme may cut non-target DNA sequences, leading to unexpected gene mutations; ② Protospacer Adjacent Motif (PAM) sequence restrictions; ③ mosaic phenomenon; ④ genomic instability; ⑤ homozygous lethality. In theory, about 1 / 3 of the genes will cause embryonic death or neonatal death in mice after homozygous knockout. The results of previous studies have shown that the survival rate of mice with systemic knockout of miR-181a is significantly reduced. This means that systemic knockout of miR-181a-1 / 2 may cause homozygous lethality. Therefore, it is urgent to consider this potential risk when preparing the miR-181a knockout mouse animal model and take appropriate measures to avoid or reduce the occurrence of this lethal phenomenon. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a miR-181a-2 knockout mouse animal model based on CRISPR / Cas9 and its application to solve the problems existing in the above-mentioned prior art. The present invention provides two sgRNAs that specifically target the non-coding gene miR-181a-2, and uses Cas9 protein to perform bidirectional knockout of miR-181a-2 to obtain a stably propagated miR-181a-2 knockout mouse animal model.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an sgRNA for knocking out mouse miR-181a-2, wherein the sgRNA consists of an sgRNA1 whose nucleotide sequence is shown in SEQ ID NO.2 and an sgRNA4 whose nucleotide sequence is shown in SEQ ID NO.5.

[0009] The present invention also provides an application of the sgRNA in preparing a product for knocking out mouse miR-181a-2.

[0010] Preferably, the product is a kit.

[0011] The present invention also provides a kit for knocking out mouse miR-181a-2, comprising the sgRNA.

[0012] The present invention also provides a method for constructing a miR-181a-2 knockout mouse animal model, comprising the step of performing gene knockout using the sgRNA or the kit.

[0013] Preferably, the method comprises the following steps:

[0014] The sgRNA and Cas9 mRNA are used to prepare a microinjection solution, and fertilized eggs are collected and microinjected to obtain surviving fertilized eggs after injection, and the surviving fertilized eggs after injection are transplanted into surrogate mother mice to obtain offspring mice, identify the genotype, and obtain F0 generation positive mice;

[0015] The F0 generation positive mice are mated with wild-type background mice to obtain F1 generation mice, and the genotypes are identified to obtain F1 generation heterozygous mice;

[0016] The male and female F1 heterozygous mice are mated to obtain F2 mice, and the genotypes are identified to obtain F2 homozygous mice, thereby obtaining the miR-181a-2 knockout mouse animal model.

[0017] Preferably, in the microinjection solution, the mass ratio of the sgRNA to the Cas9 mRNA is 1:2.

[0018] Preferably, the genotype identification comprises the steps of performing a PCR reaction using primers with nucleotide sequences as shown in SEQ ID NO. 11-12 and performing agarose gel electrophoresis on the products of the PCR reaction.

[0019] The present invention also provides an application of the miR-181a-2 knockout mouse animal model obtained according to the method in the development of anti-tumor drugs.

[0020] Preferably, the tumor is oral cancer.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses CRISPR / Cas9 technology to design two sgRNAs that specifically target the non-coding gene miR-181a-2, and combines Cas9 protein for bidirectional knockout, so as to obtain a stable breeding miR-181a-2 knockout mouse strain, providing an accurate, efficient and simple method, successfully avoiding the risk of homozygous lethality caused by simultaneous knockout of miR-181a-1 / 2, and facilitating researchers to quickly implement and repeat experiments. The present invention provides an important tool for exploring the function of miR-181a, helps promote the application of miR-181a in the study of related disease mechanisms and treatment strategies, while reducing the use of experimental animals, reducing research costs, and having significant advantages in scientific research efficiency and achievement transformation. At the same time, an oral cancer model was constructed using miR-181a-2 gene knockout mice, shortening the modeling time, and providing great convenience for oral cancer-related treatment research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Flow chart for the preparation of miR-181a-2 knockout mice;

[0025] Figure 2 Schematic diagram of bidirectional sgRNA knockout of miR-181a-2;

[0026] Figure 3 The sequence strategy diagram for knocking out miR-181a-2 by bidirectional sgRNA;

[0027] Figure 4 Agarose gel electrophoresis of F0 generation mice (No. 8852-8871);

[0028] Figure 5 This is the sequencing result of F0 generation positive mice (No. 8858 and 8865);

[0029] Figure 6 This is the agarose gel electrophoresis image of F1 generation mice;

[0030] Figure 7 This is a map of sgRNA off-target sites;

[0031] Figure 8 Agarose gel electrophoresis diagram for off-target identification of F1 generation miR-181a-2 knockout mice;

[0032] Fig. 9 This is the sequence map of the No. 1 off-target site of sgRNA1;

[0033] Fig.10 This is the sequence diagram of the No. 2 off-target site of sgRNA1;

[0034] Fig.11 This is the sequence diagram of the 3rd off-target site of sgRNA1;

[0035] Fig.12 This is the sequence diagram of the 4th off-target site of sgRNA1;

[0036] Fig.13 This is the sequence map of the No. 1 off-target site of sgRNA4;

[0037] Fig.14 This is the sequence map of the No. 2 off-target site of sgRNA4;

[0038] Fig.15 This is the sequence diagram of the No. 3 off-target site of sgRNA4;

[0039] Fig.16 This is the sequence diagram of the 4th off-target site of sgRNA4;

[0040] Fig.17 Schematic diagram of drug administration for the knockout mouse oral cancer model;

[0041] Fig.18 The figure shows the weight changes of wild-type mice and oral cancer model mice; **P<0.01;

[0042] Fig.19 The tongue appearance observation pictures of wild-type mice and miR-181a-2 knockout mice;

[0043] Fig. 20 Microscopic observation of pathological changes in tongue tissue of wild-type mice and miR-181a-2 knockout mice; the scale bar is 200 μm;

[0044] Fig.21 Statistical chart of the success rate of oral cancer modeling in wild-type mice and miR-181a-2 knockout mice. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] The experimental process of knockout mice of the present invention is as follows Figure 1 shown and summarized as follows:

[0051] 1. Target sequence confirmation and specific sgRNA design

[0052] Genbank was used to query the sequence of mouse miR-181a-2, and the online tool CRISPOR was used to analyze and design bidirectional sgRNA. Following the principles of sgRNA primer design, sgRNA sequences with specificity, high cutting efficiency and easy identification were screened.

[0053] 2. Microinjection Solution Preparation

[0054] Using NEB's HiScribe TM Transcribe in vitro using T7 Quick High Yield RNA Synthesis Kit. Prepare a microinjection mixture with dual sgRNA combination. Mix the injection solution according to Cas9:sgRNA (20ng / μL:10ng / μL), dispense into 5μL / tube, and store at -80℃. Thaw 1 tube for each microinjection. Avoid repeated freezing and thawing of RNP solution, which is easy to degrade. Operate on ice.

[0055] 3. Fertilized egg collection

[0056] The superovulated donor female mouse was placed in a cage with a normal male mouse. After mating, the fertilized eggs in the ampulla of the oviduct were taken out and cultured briefly in a 5% CO2 incubator before injection.

[0057] 4. In vitro Microinjection

[0058] Prepare the injection needle and the egg holding needle, load the Cas9 mRNA and sgRNA microinjection mixture into the injection needle, and then microinject it into the fertilized egg.

[0059] 5. Fertilized egg cell transplantation

[0060] The estrus female mice were caged with the ligated male mice and the surrogate female mice with positive vaginal plug were selected. The surrogate female mice were weighed and anesthetized, and placed under a stereomicroscope to expose the ovaries, fallopian tubes and uterus. The fimbria of the fallopian tube was clamped with micro forceps, and the transplant tube was inserted into the fallopian tube opening. The embryo was injected into the ampulla of the fallopian tube, the tissue was placed in the body cavity, and the wound was sutured.

[0061] 6. Genotyping of knockout mice

[0062] The fertilized eggs are transplanted into the surrogate mother mouse, and about 21 days later, the surrogate mouse will give birth to pups. The tails of the newborn mice born one week after birth are cut, and the digestion solution is added to lyse at 56℃ for 6-8h, and the genomic DNA of the newborn mice is extracted using a mouse tail DNA extraction kit. Using the mouse tail DNA as a template, appropriate primers are designed, and PCR reactions are performed to identify the genotypes of all the pups.

[0063] 7. Obtaining Homozygous Knockout Mice

[0064] The F0 generation positive mice grown to 8 weeks old were mated with wild-type background mice. After the F1 generation mice were born, the mouse genotypes were detected using primers to obtain F1 generation heterozygous mice. Then, the F1 generation positive female mice and male mice grown to 8 weeks old were mated together. After the F2 generation mice were born, the primers were used to detect them to obtain F2 generation homozygous mice.

[0065] 8. Detection of potential off-target sites

[0066] The CRISPOR website was used to predict the potential off-target sites of miR-181a-2-sgRNA, primers were designed for off-target sequences, and off-target identification was performed using agarose gel electrophoresis. The PCR amplification products were sent for sequencing, and the sequencing results were analyzed using SnapGene software.

[0067] Example 1 Construction of miR-181a-2 knockout mouse oral cancer susceptibility model

[0068] 1. Confirmation of the target sequence

[0069] Genebank was used to query the gene sequence of mouse miR-181a-2. The mouse miR-181a-2 gene is located on chromosome 2, the precursor RNA is 76 nt long, and the lengths of mmu-miR-181a-5p and mmu-miR-181a-2-3p are 23 nt and 22 nt respectively (NCBIACCESSION: NR_029568). sgRNA was designed for the precursor RNA of miR-181a-2 to achieve efficient knockout of the miR-181a-2 gene.

[0070] Among them, the precursor-RNA sequence of the miR-181a-2 gene is as follows:

[0071] 5'-CCATGGAACATTCAACGCTGTCGGTGAGTTTGGGATTCAAAAACAAAAAAACCACCGACCGTTGACTGTACCTTGG-3' (SEQ ID NO. 1).

[0072] 2. sgRNA Design

[0073] The mouse miR-181a-2 gene sequence was queried on the Genbank website, and the sequence to be knocked out was analyzed using the website https: / / zlab.bio / guide-design-resources. In accordance with the sgRNA primer design principles, sgRNAs were designed at the corresponding sites on both sides of the precursor-RNA of the miR-181a-2 gene. In order to ensure the cutting efficiency and facilitate the subsequent identification, the four sgRNAs with high scores on the screening website were selected as candidate sgRNAs (Table 1), and the designed sgRNA primer sequences were sent to Shanghai Bioengineering for synthesis. The schematic diagram of the bidirectional sgRNA knockout of the miR-181a-2 gene of the present invention is shown in Figure 2 The knockout sequence strategy is shown in Figure 3 (Take sgRNA1 and sgRNA4 as examples).

[0074] Table 1 sgRNA sequence information

[0075]

[0076] 3. Microinjection Solution Preparation

[0077] Using NEB's HiScribe TM T7 Quick High Yield RNA Synthesis Kit was used for in vitro transcription. Each sgRNA transcribed in vitro was purified and recovered, and a dual sgRNA microinjection mixture was prepared according to the concentration of Cas9 mRNA:sgRNA (20ng / μL:10ng / μL) (the selection of dual sgRNA includes: sgRNA1+sgRNA4, sgRNA2+sgRNA4 and sgRNA3+sgRNA4), and 5μL / tube was packaged and stored at -80℃.

[0078] Thaw one tube for each microinjection. Avoid repeated freezing and thawing as the RNP solution is easily degraded. Operate on ice. See Table 2 for the synthetic sequence information of sgRNA sequence.

[0079] Table 2 sgRNA primer information

[0080]

[0081]

[0082] 4. Preparation of ligated male mice

[0083] After weighing the C57BL / 6N male mice, 1.25% avertin anesthetic (injection dose is 0.2mL / 10g) was injected intraperitoneally, and the mice were placed supine on the operating table and disinfected with cotton balls soaked in 75% ethanol. A 1.5cm transverse incision was made at the vertical line of the upper end of the lower limbs, and an incision of the same size was made on the abdominal wall. The testicles on both sides can be accessed from this incision. Use blunt forceps to pull the testicular fat pad on one side out of the incision, and the testicle, epididymis and vas deferens will also be pulled out together. The vas deferens is located under the testicle, and there is a blood vessel next to it, which is easy to identify. Clamp the vas deferens with forceps and use another pair of red-hot forceps to burn it off about 1cm. Clamp the fat pad with small blunt forceps and carefully put the testicle back into the abdominal cavity. Repeat the above two steps to treat the testicle and vas deferens on the other side, suture the body cavity and body wall in turn, and place the mouse in a clean mouse cage after surgery. Select mature female mice over 8 weeks old to be caged together with the male mice, and the sterilized male mice that can see the female mice and are not pregnant are considered qualified.

[0084] 5. Preparation of Donor Mice

[0085] Select 6-week-old C57BL / 6N female mice, inject pregnant mare serum gonadotropin (5IU PMSG) intraperitoneally and put them back in the original mouse cage to promote superovulation of the donor female mice. After 48 hours, inject human chorionic gonadotropin (5IU HCG) intraperitoneally. Immediately after HCG injection, the female mice were caged with normal healthy male mice (cage ratio 1:1). After 20 hours of caged, the vaginal plugs of the female mice were checked and fertilized eggs were collected.

[0086] 6. Preparation of Surrogate Mothers

[0087] When the superovulated female mice were put into the cage, healthy C57BL / 6N female mice over 8 weeks old and in estrus were selected. If the female mice's vulva was moist and the vaginal opening was open, they were immediately put into the cage with the selected qualified sterilized male mice. The next day, the female mice with positive vaginal plug were selected for fertilized egg transplantation.

[0088] 7. Collection of fertilized eggs from donor mice

[0089] The donor female mice with positive vaginal plug were selected and killed by cervical dislocation. After spraying 75% alcohol on their backs, the abdominal cavity was opened, and the uterus-fallopian tube-ovary on both sides were removed. After rinsing with sterile PBS three times, the fallopian tubes were cut and placed in M2 culture medium. Subsequently, excess fat was removed under a stereomicroscope, transferred to M2 culture medium containing 0.1% hyaluronidase, and the enlarged ampulla of the fallopian tube was torn open with microtweezers to release the cumulus cell mass surrounding the fertilized egg. After digestion for 5 minutes, the fertilized egg was moved to M2 culture medium, washed 3 times, observed under an inverted microscope, and the number of fertilized egg cells was counted. Finally, it was moved to M16 culture medium and placed in a 37°C, 5% CO2 incubator for culture.

[0090] 8. In vitro Microinjection

[0091] Before injection, the injection needle and the egg-holding needle must be prepared. The injection needle is loaded with the Cas9 mRNA / sgRNA microinjection mixture. After checking whether there are bubbles in the oil circuit of the EppendorfTransfer ManNK2 micromanipulator, the egg-holding needle and the tail of the injection needle with injection solution are inserted into the needle holder filled with paraffin oil. Tighten the knob and install it on the micromanipulator, keeping the tip of the suction needle and the injection chamber parallel to the horizontal plane of the injection chamber under the 40x objective. After superovulation of C57BL / 6N female mice, fertilized eggs are collected and observed under an inverted microscope. Fertilized eggs with plump cells, clear zona pellucida, and clearly visible male pronuclei are selected for use. Subsequently, the injection needle fine-tuning knob is adjusted under the 40x objective to keep the liquid flowing out steadily. During the microinjection process, the flow rate is generally not adjusted unless the needle tip is blocked. Repeatedly and gently blow and suck the fertilized egg so that the focal plane of the male pronucleus is at the same level as the injection needle, which is the best position for injection. At this time, insert the injection needle into the male pronucleus of the fertilized egg until the male pronucleus slowly expands and exits the male pronucleus. The injection needle tip stays in the cytoplasm for a few seconds, and quickly exits when transparent vacuoles appear in the cytoplasm. Place the injected fertilized eggs and the uninjected fertilized eggs separately up and down to avoid confusion. After the injection, remove the failed or dead fertilized eggs (yellowing cells, ruptured cells or irregular shapes), and place the surviving fertilized eggs in a sterile incubator at 37°C for culture.

[0092] 9. Fertilized egg cell transplantation

[0093] After the injection, the fertilized oocytes were placed in a sterile incubator at 37°C for 2 hours, and then transferred into the transplant needle for use. After the surrogate mother was weighed and anesthetized, it was placed under a stereomicroscope together with the plastic culture dish. The fat pad was clamped with blunt forceps to pull out the attached left ovary, fallopian tube and uterus. The fat pad was clamped with a small spring clip and allowed to droop along the middle of the back, exposing the ovary and fallopian tube to the outside of the body cavity; the head was turned to the left, and the opening of the fallopian tube (fallopian tube umbrella) and the enlarged fallopian tube ampulla under the ovarian sac were found under the microscope. Carefully clamp the fallopian tube umbrella with blunt forceps and insert the transplant tube into the fallopian tube opening. Blow the embryo and air bubbles into the fallopian tube ampulla. If bubbles are seen in the fallopian tube, it means that the transplant has been successful; loosen the small spring clip, remove it from the microscope stage, clamp the fat pad with blunt forceps, and put the uterus, fallopian tube, ovary, etc. back into the body cavity. The body wall is sutured and the skin wound is sutured or clamped; the above three steps are repeated to transplant the embryo into the right fallopian tube; after the operation, the transplant recipient mouse is placed on a heating pad until it wakes up. Afterwards, each transplant recipient mouse is housed in a single cage.

[0094] 10. Genotyping of knockout mice

[0095] The fertilized egg is transplanted into the body of a surrogate mother mouse, and about 21 days later, the surrogate mouse will give birth to pups. Cut the tail of the newborn mouse one week old, add digestive fluid and lyse it at 56°C for 6-8 hours, and use a mouse tail DNA extraction kit to extract the genomic DNA of the newborn mouse. Using the mouse tail DNA as a template, design appropriate primers and send them to Shanghai Biotech to synthesize the primer sequence (primer sequence see Table 3). Take 100ng of genomic DNA for PCR reaction to identify the genotype of all pups, and then use 1.5% agarose gel electrophoresis to detect the PCR amplification product (genotype judgment criteria see Table 4). Cut the gel to recover and purify the PCR product, and then send it to Shanghai Biotech for sequencing to identify whether the sequence is missing (PCR reaction system and conditions see Table 5 and Table 6). The F0 generation mouse identified as positive in the above is the founder mouse, and the F0 generation mouse tail is identified. The PCR product gel results are shown in Figure 4 The sequencing results are shown in Figure 5 The information of F0 generation gene knockout mice is shown in Table 7.

[0096] Table 3 Primer sequence information

[0097]

[0098] Table 4 Genotype judgment criteria

[0099]

[0100] Table 5 PCR detection system

[0101]

[0102] Table 6 PCR reaction conditions

[0103]

[0104] Note: After microinjection, 20 F0 mice were produced. Agarose electrophoresis results initially showed that there were 11 positive founder mice, all of which were retained. Since the knockout sequence of each founder mouse was different, the 8854, 8858, 8865 and 8866 mice with a size of 431 bp in agarose identification results were sequenced. The sequencing results are as follows Figure 5 As shown, 8858 and 8865 positive mice were selected for co-housing.

[0105] Table 7 Information of F0 generation knockout mice

[0106]

[0107] 11. Obtaining Homozygous Knockout Mice

[0108] The F0 positive mice grown to 8 weeks old were backcrossed with wild-type mice (the information of the F0 knockout mice for mating is shown in Table 8). After the F1 mice were born, the genotype of the mice was detected using the primers in the previous step to obtain the F1 heterozygous mice with a single genotype (the results of the tail identification of the F1 mice are shown in Table 8). Figure 6 The information of F1 knockout mice is shown in Table 9). Then, the F1 heterozygous female mice and male mice grown to 8 weeks old were bred together, and after the F2 mice were born, the primers in the previous step were used for detection to obtain the F2 homozygous mice. The mice were the constructed miR-181a-2 knockout mouse model. The homozygous mice were mated and bred, and the experiment could be carried out after a certain number was obtained.

[0109] The miR-181a-2 knockout mouse model constructed in the present invention can be used to study the role and function of miR-181a-2 in the occurrence and development of various tumors.

[0110] Table 8 Information of F0 generation positive gene knockout mice

[0111]

[0112] Table 9 Information of F1 knockout mice

[0113]

[0114]

[0115] 12. Detection of potential off-target sites

[0116] In order to evaluate the potential off-target effects of miR-181a-2, a total of eight potential off-target sites of miR-181a-2-sgRNA1 / 4 were predicted using the CRISPOR (http: / / crispor.tefor.net / ) website, PCR primers were designed, and off-target identification was performed using agarose gel electrophoresis. The PCR amplification products were sent to Shanghai Bioengineering for sequencing, and the sequencing results were analyzed using SnapGene software. Figure 7 The primer sequences are shown in Table 10. Agarose gel electrophoresis and sequencing results showed that no off-target phenomenon occurred in the F1 generation miR-181a-2 knockout mice. Figures 8 to 16 .

[0117] Table 10 Off-target site primer sequences

[0118]

[0119] 13. miR-181a-2 knockout mice are susceptible to oral cancer

[0120] 8-10 week old C57BL / 6N mice were cultured with miR-181a-2 - / - Mice were randomly divided into four groups, namely, WT control group (n=6), WT 4NQO administration group (n=15), KO mouse control group (n=6), and KO mouse 4NQO administration group (n=15). DMSO solution was used as a solvent to dissolve 4NQO and prepare a 300μg / mL storage solution. When administering, it was diluted to a concentration of 50μg / mL with sterilized purified water. The modeling period was 24 weeks. After the mice in the administration group drank the 4NQO solution for 16 weeks, they were replaced with purified water for drinking until 24 weeks. The mice in the control group had a normal diet and drinking water. The administration process was as follows. Fig.17 .

[0121] 14. Evaluation of the oral cancer model in miR-181a-2 knockout mice

[0122] In the process of 4-NQO chemical induction modeling, miR-181a-2 - / - The body weight and water intake of the knockout mice and WT mice gradually decreased. At the eighth week, the knockout mice were the first to lose weight, which decreased by about 50% and then stabilized ( Fig.18 The tongue of normal mice was pink, smooth, soft and elastic. As the administration time increased, the knockout mice showed abnormal proliferation and white plaques earlier than the WT mice. At week 16, miR-181a-2 - / - Smaller growths appeared on the sides and middle third of the tongue of the mice. - / - Exogenous tumors were found in almost all tongue tissues of mice, while those in WT mice did not appear until the 24th week ( Fig.19 ).

[0123] As the drug exposure time increased, tongue tissues showed typical pathological changes, including simple epithelial hyperplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma. - / - Mice showed dysplasia at week 12, characterized by thickening of the stratum spinosum, increased basal cell layer, and loss of cell polarity. - / - All mice developed carcinoma in situ, while most WT mice did not develop lesions until week 24, and KO mice showed a faster rate of malignant transformation and early invasion tendency. This progression was marked by the disappearance of the basement membrane, the disorganization of epithelial cells, and the penetration of cancer cells into the submucosal layer, accompanied by severe cellular atypia and significant lymphocytic infiltration ( Fig. 20 ).

[0124] In summary, miR-181a-2 knockout mice showed higher susceptibility to oral cancer and higher success rate of oral cancer modeling ( Fig.21 ), indicating the key role of miR-181a-2 in the occurrence and development of oral cancer and the practical application of this gene knockout model.

[0125] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A sgRNA for knocking out mouse miR-181a-2, characterized in that: The sgRNA consists of sgRNA1 with a nucleotide sequence as shown in SEQ ID NO.2 and sgRNA4 with a nucleotide sequence as shown in SEQ ID NO.

5.

2. Use of the sgRNA as claimed in claim 1 in preparing a product for knocking out mouse miR-181a-2.

3. The use according to claim 2, characterized in that The product is a test kit.

4. A kit for knocking out mouse miR-181a-2, characterized in that: Comprising the sgRNA of claim 1.

5. A method for constructing a miR-181a-2 knockout mouse animal model, characterized in that: The method comprises the step of performing gene knockout using the sgRNA according to claim 1 or the kit according to claim 4.

6. The method according to claim 5, characterized in that The following steps are involved: The sgRNA and Cas9 mRNA described in claim 1 are prepared into a microinjection solution, and the fertilized eggs are collected and microinjected to obtain surviving fertilized eggs after injection, and the surviving fertilized eggs after injection are transplanted into surrogate mother mice to obtain offspring mice, identify the genotype, and obtain F0 generation positive mice; The F0 generation positive mice are mated with wild-type background mice to obtain F1 generation mice, and the genotypes are identified to obtain F1 generation heterozygous mice; The male and female F1 heterozygous mice are mated to obtain F2 mice, and the genotypes are identified to obtain F2 homozygous mice, thereby obtaining the miR-181a-2 knockout mouse animal model.

7. The method according to claim 6, characterized in that In the microinjection solution, the mass ratio of the sgRNA to the Cas9mRNA is 1:

2.

8. The method according to claim 6, characterized in that The genotype identification comprises the steps of performing a PCR reaction using primers with nucleotide sequences as shown in SEQ ID NO. 11-12 and performing agarose gel electrophoresis on the products of the PCR reaction.

9. Use of the miR-181a-2 knockout mouse animal model obtained according to the method according to any one of claims 5 to 8 in the development of anti-tumor drugs.

10. The use according to claim 9, characterized in that The tumor is oral cancer.

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