Mouse model capable of specifically degrading p300 protein and construction method thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a mouse model capable of specifically degrading p300 protein and a construction method thereof, and the construction method comprises the following steps: sequentially arranging a 5'homologous arm, 3 * GGGGS, an FKBP1A gene synonymous mutation sequence, P2A, a green fluorescent protein EGFP sequence and a 3 'homologous arm on a Donor vector skeleton to obtain a Donor recombinant vector; designing gRNA (guide Ribonucleic Acid) on the basis of the 31st exon of the Ep300 gene; mixing the gRNA, the Cas9 protein and the Donor recombinant vector, and co-injecting the mixture into a mouse fertilized egg so as to knock the synonymous mutation sequence of the FKBP1A gene and a green fluorescent protein (EGFP) sequence into a mouse genome; carrying out in-vitro culture to obtain a transplantable embryo, and transplanting the transplantable embryo into the uterus of the pregnant mouse to continuously develop until a young mouse is born; and hybridizing the F0 generation mouse with a wild type mouse to obtain a mouse for expressing the p300-FKPB-P2A-EGFP fusion protein, namely obtaining the mouse model for specifically degrading the p300 protein. According to the invention, dTAG administration is carried out on the obtained knocked-in mouse in the later stage, so that rapid and reversible specific degradation and removal of p300 protein in the mouse living body are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a mouse model for specifically degrading p300 protein and a construction method thereof. Background Art

[0002] Proteins are the direct executors of various biological processes within cells, and the dynamic regulation of their functions is crucial for understanding cellular physiological and pathological mechanisms. However, traditional molecular regulation technologies primarily focus on the genetic level, indirectly influencing protein expression through methods such as gene knockout or RNA interference. This approach has several limitations, such as the irreversibility of gene knockout, which prevents dynamic regulation of protein function. While RNA interference can reduce protein expression levels to a certain extent, its effect is relatively slow and has off-target effects, potentially affecting the expression of other non-target genes. Therefore, research on technologies that can directly target and regulate protein levels is particularly important.

[0003] The p300 protein is a multifunctional transcriptional coactivator that belongs to the KAT3 histone acetyltransferase family and was originally identified in the study of gene expression regulation in eukaryotes. p300 is widely involved in core biological processes such as chromatin remodeling, transcriptional activation, DNA damage repair, and cell fate determination through protein interaction networks mediated by histone acetyltransferase activity and domains. In the field of cancer, abnormal activation of p300 is closely related to a variety of malignant tumors such as prostate cancer, breast cancer, and leukemia. For example, it drives the androgen receptor AR signaling pathway to promote prostate cancer progression; or maintains tumor cell survival by activating oncogenic transcriptional programs such as c-Myc. In addition, p300 can mediate the excessive activation of inflammatory factors such as NF-κB in inflammatory diseases, and aggravate neurofibrillary tangles caused by Tau protein acetylation in neurodegenerative diseases, which is closely related to the development of Alzheimer's disease. In the mouse genome Ep300 Located on chromosome 15, it is approximately 87 kb in length. The start codon ATG is located in exon 1, and the stop codon TGA is located in exon 31.

[0004] Despite the increasing importance of p300 as a therapeutic target, existing interventions still face significant challenges. Existing small molecule inhibitors, such as C646 and CCS1477, mainly act by targeting the bromodomain BRD or histone acetyltransferase HAT domain of p300, but these domains are highly conserved in the cyclic AMP response element binding protein CBP, making it difficult for inhibitors to distinguish between the two and unable to degrade the protein itself. PROTAC technology recruits target proteins and E3 ligases simultaneously through bifunctional molecules, but its design relies on the selectivity of target protein ligands. Existing PROTAC degraders targeting p300, such as XYD129, XYD190, XYD198, QC-182, etc., all show cross-degradation of CBP. Gene editing technologies such as CRISPR-Cas9 knockout of p300 can easily cause embryonic lethality or severe developmental defects, while RNA interference technology is limited by its transient nature and delivery efficiency.

[0005] In view of the above limitations, there is an urgent need to develop new technologies that can dynamically and reversibly regulate p300 protein levels. Summary of the Invention

[0006] In order to achieve rapid and reversible degradation and clearance of p300 protein in vivo, the present invention provides a method for constructing a mouse model for specific degradation of p300 protein. The technical solution adopted in the present invention is: The present invention provides a method for constructing a mouse model for specifically degrading p300 protein, comprising the following steps: The Donor vector backbone was connected with 5' homology arms, 3×GGGGS, FKBP1A Gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homology arm were used to obtain Donor recombinant vector; The nucleotide sequence of the 5' homology arm is shown in SEQ.ID.NO.2; the nucleotide sequence of the 3×GGGGS is shown in SEQ.ID.NO.3; FKBP1A The gene synonymous mutation sequence is shown in SEQ.ID.NO.4; the nucleotide sequence of the P2A is shown in SEQ.ID.NO.5; the nucleotide sequence of the green fluorescent protein EGFP sequence is shown in SEQ.ID.NO.6; and the nucleotide sequence of the 3' homology arm is shown in SEQ.ID.NO.7; Based on the mouse chromosome 15 Ep300 gRNA was designed for exon 31 of the gene, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1; The gRNA, Cas9 protein and the Donor recombinant vector are mixed and injected into mouse fertilized eggs to make the FKBP1A Gene synonymous mutation sequence and green fluorescent protein EGFP sequence were knocked into the mouse genome; The injected fertilized eggs are cultured in vitro and then transplanted into surrogate mice to continue developing until pups are born, obtaining F0 generation mice. The F0 generation mice are hybridized with wild-type mice, or the offspring obtained by hybridization are further bred to obtain mice that specifically degrade p300 protein.

[0007] Preferably, the Donor recombinant vector is constructed according to the following steps: Using the BAC plasmid as a template, a fragment containing the 5' homology arm from the mouse genome was amplified to obtain fragment-1. The sequence of fragment 1 is shown in SEQ.ID.NO.2; Using the BAC plasmid as a template, a fragment containing the 3' homology arm from the mouse genome was amplified to obtain fragment-3. The sequence of fragment 3 is shown in SEQ.ID.NO.7. The 3×GGGGS, FKBP1A The gene synonymous mutation sequence, P2A and green fluorescent protein EGFP sequence were connected in sequence to obtain fragment-2; The fragment-1, fragment-2, fragment-3 and the Donor vector backbone are connected to obtain the Donor recombinant vector. The nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.12.

[0008] Preferably, the primer sequences for amplifying the fragment-1 are shown as SEQ.ID.NO.8 and SEQ.ID.NO.9; The primer sequences for amplifying the fragment-2 are shown in SEQ.ID.NO.10 and SEQ.ID.NO.11.

[0009] Preferably, when the fragment-1, fragment-2, fragment-3 and the Donor vector backbone are connected, the 20 μL connection system contains: 88.6 ng Donor vector backbone, 39.2 ng fragment-1, 23.1 ng fragment-2, 23.9 ng fragment-3, 10 μL NEBuilder HiFi DNA Assembly Master Mix.

[0010] Preferably, after the gRNA and the Cas9 protein are mixed and incubated at 25° C., the Donor recombinant vector is added, mixed evenly, and co-injected into mouse fertilized eggs.

[0011] Preferably, the concentration of the gRNA is 90 pmol / μL~110 pmol / μL, the concentration of the Cas9 protein is 18 μM~22 μM, and the concentration of the Donor recombinant vector is 12 ng / μL~18 ng / μL; The volume ratio of the gRNA to the Cas9 protein is 3-5:1, and the total volume of the mixture of the gRNA and Cas9 protein and the Donor recombinant vector is 20 μL.

[0012] Preferably, the concentration of the gRNA is 100 pmol / μL, the concentration of the Cas9 protein is 20 μM, and the concentration of the Donor recombinant vector is 15 ng / μL; The volume ratio of the gRNA to the Cas9 protein is 4:1.

[0013] The present invention also provides a mouse model capable of rapidly degrading p300 protein, wherein the mouse model is obtained by the construction method.

[0014] Preferably, dTAG is administered to the mice that specifically degrade p300 protein to achieve rapid and reversible degradation and clearance of p300 protein in the mice.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a mouse model that specifically degrades p300 protein. The present invention is based on CRISPR / Cas9-mediated gene editing technology. Ep300 The human FKBP1A protein coding sequence, P2A and green fluorescent protein EGFP sequence were knocked into the gene, marked as FKPB-P2A-EGFP tag sequence, and introduced FKBP1A Synonymous mutations are made to avoid interference from endogenous splicing sites, avoid off-target effects, etc., thereby establishing a mouse model for rapid and specific degradation of p300 protein, namely the mouse p300-FKPB-P2A-EGFP knock-in model. The EGFP fluorescent protein plays an indicator role, and the successful knock-in of the target sequence can be judged by observing the fluorescence. The addition of the P2A self-cleavage polypeptide between the FKBP and EGFP sequences has little effect on the protein function, and can ensure that the target protein still maintains its natural function after shearing. Therefore, EGFP will not interfere with the structure of the p300-FKBP fusion protein, ensuring the smooth binding of the dTAG ligand to FKBP. The mouse p300-FKPB-P2A-EGFP knock-in model can stably and efficiently express the p300 protein fused with the FKPB tag protein, referred to as the p300-FKPB fusion protein. In addition, the modeling process of the mouse p300-FKPB-P2A-EGFP knock-in model in the present invention has the advantage of a short operating cycle.

[0016] The present invention is achieved by Ep300, FKBP1A A fusion protein linker 3×GGGGS sequence was added between the synonymous mutation sequences to promote the expression of p300-FKPB fusion protein.

[0017] The present invention achieves rapid and reversible specific degradation and clearance of p300 protein in mice by administering dTAG to the knock-in mice obtained in the later stage.

[0018] The present invention targets F0 generation positive mice constructed using CRISPR / Cas9 technology. Due to the different genetic lineages resulting from the cutting efficiency and possible non-homologous repair, the F0 generation positive mice are mated with wild-type mice to obtain F1 generation positive mice with stable genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of a transgenic mouse model expressing an FKPB tag constructed using CRISPR / Cas9 technology in an embodiment of the present invention.

[0020] Figure 2 This is the plasmid map of the Donor vector used for homologous recombination repair.

[0021] Figure 3 This is the breeding plan for F0 generation mice.

[0022] Figure 4 Diagram of the PCR identification strategy for F0 and F1 generation mouse genotypes.

[0023] Figure 5 Electrophoresis diagram for PCR identification of F1 generation mouse genotypes, A: 5'HA homologous recombination-positive genome; B: 3'HA homologous recombination-positive genome.

[0024] Figure 6 This is the sequencing map of the knock-in sequence of F1 generation mice.

[0025] Figure 7 These are electrophoresis images of PCR identification of F2 generation mouse genotypes. A: identification results of the 506 bp band; B: identification results of the 1611 bp and 459 bp bands.

[0026] Figure 8 This is a Western blot analysis of the expressed p300-FKBPP fusion protein. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0028] The inventive concept of the present invention is as follows: The dTAG protein degradation system offers the possibility to target any protein within the cell in a rapid, inducible, and reversible manner.

[0029] The dTAG system is a targeted protein degradation technology based on genetically engineered tags and bifunctional small molecules. Using CRISPR-Cas9 or lentiviral technology, the target protein is fused to an artificially engineered FKBP tag to create a chimeric protein. The heterobifunctional small molecule degrader, dTAG, binds to the FKBP tag at one end and recruits an E3 ubiquitin ligase, such as CRBN or VHL, at the other end to form a ternary complex, inducing ubiquitination and proteasomal degradation of the target protein. dTAG's regulation of target protein abundance is rapid, reversible, and tunable, providing a rapid, dose-dependent effect on total cellular protein abundance, precise temporal control, and reversible effects after the degrader is washed away. This avoids the limitations of methods like RNAi or CRISPR-Cas9, which suffer from limited dynamic control, uncertainty about effect size, and often complete irreversibility.

[0030] A specific sgRNA is designed for the C-terminus of the target protein gene, and a donor plasmid containing an FKBP tag sequence is constructed. This plasmid contains homology arms to promote homologous recombination. The sgRNA and Cas9 protein are co-transfected with the donor plasmid into mouse embryonic stem cells (mESCs), where the FKBP tag is inserted into the C-terminus of the target gene through homologous recombination. The edited embryonic stem cells are injected into mouse embryos to generate chimeric mice, which are then bred to obtain transgenic mice carrying the FKBP tag. The advantage of this method is that it can achieve specific modification at the genomic level, ensuring that the fusion of the FKBP tag and the target protein occurs within the cell's native gene expression framework, thereby more closely resembling protein expression and function under physiological conditions.

[0031] The dTAG system can rapidly, adjustably, and reversibly degrade target proteins, and has been widely used in various cell lines and mouse models to study target protein functions. The present invention successfully constructed p300-FKBP-P2A-EGFP knock-in mice, which is crucial for using the dTAG system to study the specific functions of p300 proteins, discover and verify new targets, and achieve specific target protein degradation with high spatiotemporal resolution in living models. This strategy not only avoids dependence on the inherent ligands of the target, but also can regulate protein function through the dosage of degraders, providing a potential breakthrough for analyzing the specific transient effects of p300 in diseases and developing precision therapies. Compared with drug inhibitors and degraders, dTAG small molecule degraders have obvious advantages, including simpler production processes, more convenient clinical applications, and overcoming drug resistance to inhibitors; the degree of protein knockdown can be adjusted by changing the dosage; the ability to take effect faster; and the ability to specifically degrade p300 protein alone.

[0032] In summary, the construction of p300-FKPB fusion protein-expressing mice is particularly critical for the rapid, reversible and specific degradation and clearance of p300 protein in mice.

[0033] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0034] Example 1 A mouse model for specific degradation of p300 protein and a method for constructing the same are as follows: The present invention uses CRISPR / Cas9-mediated gene editing technology to establish a p300-FKPB-P2A-EGFP knock-in model in C57BL / 6J mice.

[0035] 1. Targeting human genes FKBP1A , designed a mouse transgenic strategy and used CRISPR / Cas9 technology to FKBP1A The CDS sequence was knocked into mouse chromosome 15 Ep300 In exon 31 of the gene, Ep300 The gene is a gene encoding p300 protein. Figure 1 As shown, Ep300 The reference sequence of the gene in NCBI is: NM_177821.6. The specific steps include:

[0036] 1.1. Design gRNA based on CRISPR / Cas9 system, Ep300 The sequences of the gRNA action sites specific to exon 31 of the genes are shown in Table 1 .

[0037] Table 1 gRNA action sites Note: In Table 1, the italic bases in the sequences are PAM.

[0038] 1.2. According to the principle of homologous recombination repair and combined with the knock-in target sequence, the Donor vector is designed. The Donor vector contains 5' homology arm, 3×GGGGS, FKBP1A The main components of the gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homology arm are shown in Table 2, and the nucleotide sequences of the corresponding elements are shown in Table 3. The target sequence can also be called the knock-in sequence; FKBP1A Gene synonymous mutation sequences are marked as Mutant Human FKBP1A CDS-p.F37V.

[0039] 5' homology arm, 3×GGGGS, FKBP1A The sequence information of the gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homology arm are shown in SEQ.ID.NO.2 to SEQ.ID.NO.7 respectively.

[0040] Table 2 Main components of Donor vector Table 3 Partial nucleotide sequence of Donor vector Note: In Table 3, the underlined bases are introduced synonymous mutations. The sequence before mutation is TTT, and the sequence after mutation is GTT.

[0041] 1.3, using In-Fusion technology, the above 5' homology arms, 3×GGGGS, Mutant Human FKBP1A CDS-p.F37V, P2A, EGFP and 3' homology arms were assembled into Donor vector, i.e. 5' homology arms, fusion protein linker 3×GGGGS, FKBP1A Gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homology arm. The specific process is as follows:

[0042] (1) Using high-fidelity Taq DNA polymerase and the BAC RP23-16K17 plasmid as a template, fragments 1 and 3 containing homologous sequences from the mouse genome were amplified. The sequences of the amplification primers are shown in Table 4, the amplification system is shown in Table 5, and the amplification procedure is shown in Table 6.

[0043] Fragment-1: a fragment containing the 5' homology arm, namely SEQ.ID.NO.2; Fragment-3: a fragment containing the 3' homology arm, namely SEQ.ID.NO.7.

[0044] A 3×GGGGS-Mutant Human FKBP1A CDS-P2A-EGFP sequence was synthesized and designated as fragment-2, wherein the FKBP1A CDS does not contain the ATG start codon.

[0045] Table 4 Nucleotide sequences of amplification primers Table 5 PCR system for amplifying fragments 1 to 3 Table 6 PCR program for amplifying fragments 1 to 3 (2) Using a seamless cloning kit, ligate the PCR amplified fragment with the linearized plasmid according to the reaction system shown in Table 7. The reaction was performed using a PCR instrument and incubated at 50°C for 60 min; then placed on ice for transformation.

[0046] The linearized plasmid is 4430bp, customized internally by Saiye Bio, with the internal product number Cat: VB139; specifically, it is a linearized plasmid obtained by modifying the commercial plasmid addgene#50005 to provide the vector backbone.

[0047] Table 7 In-Fusion System (3) The In-Fusion ligation product was transformed into DH5-alpha competent cells, and then the competent cells were added to LB liquid culture medium and cultured on a shaker at 37°C for 30 minutes. The resulting culture was spread on LB solid culture medium containing ampicillin and cultured overnight. After ampicillin resistance screening, the monoclonal clones grown on the LB solid culture medium were picked and cultured in LB liquid culture medium. After that, the bacterial liquid was subjected to PCR identification. After confirming that the ligation was successful, the bacterial liquid was sent for sequencing. According to the sequencing results, the monoclonal clones with successful ligation and correct sequence were selected for shaking, and the bacterial liquid was collected for plasmid extraction to obtain the Donor recombinant vector. Its plasmid map is shown as follows: Figure 2 shown.

[0048] The nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.12, 1bp to 4749bp can be found in sequence 12 in the computer-readable vector of the nucleotide or amino acid sequence table, and 4750bp to 8731bp can be found in sequence 13.

[0049] 1.4. Preparation of injection complex.

[0050] Solution in Tube 1: Add 0.8 μL of 100 pmol / μL gRNA to RNase-free water, then add 0.2 μL of 20 μM Cas9 protein, mix thoroughly, and incubate in a 25°C metal bath for 10 minutes to obtain solution in Tube 1. The gRNA is the sequence shown in SEQ.ID.NO.1.

[0051] Solution in tube 2: Donor recombinant vector with a final concentration of 15 ng / μL.

[0052] The solution in tube 1 and the solution in tube 2 were mixed to obtain an injection complex, and the total volume of the injection complex was 20 μL.

[0053] 1.5. Microinjection and embryo transfer.

[0054] After superovulation of female C57BL / 6J mice, fertilized C57BL / 6J eggs were obtained through in vitro fertilization. The resulting injection complex was then injected into the fertilized C57BL / 6J eggs using a 0.1 μm glass microinjection needle. The microinjected fertilized eggs were then transferred into the oviducts of surrogate C57BL / 6J mice via embryo transfer. F0 generation mice were born, and their genotypes were identified by PCR.

[0055] The specific operation of the above embryo transplantation is as follows: the fertilized eggs after microinjection are cultured in vitro to morulas and quality tested; a small incision is made in the oviduct capsule of the surrogate mother mouse, and after the direction of the oviduct fimbria is clearly seen, the transplant tube is inserted 3mm into the oviduct fimbria from the opening, and the embryos with good development quality are blown into the magnum, and the transplant tube is pulled out after a short pause.

[0056] 1.6. The positive F0 generation mice were mated with wild-type C57BL / 6J mice to obtain F1 generation mice. The genotypes of the mice were identified by PCR. If positive mice were born, that is, F1 generation heterozygotes, it means that the knock-in sequence has been integrated into the germ cells. Figure 3 and Figure 4 shown.

[0057] 1.7. The F1 generation heterozygous mice were self-crossed, and the resulting F2 generation mice were genotyped to obtain p300-FKPB-P2A-EGFP homozygous knock-in mice, that is, mice expressing the p300-FKBP fusion protein.

[0058] 1.8. By subsequently administering dTAG to the knock-in mice, rapid and reversible specific degradation and clearance of p300 protein in the mice can be achieved.

[0059] 2. PCR identification results of mouse genotype.

[0060] 2.1. DNA extraction

[0061] Select newborn mice from 3 weeks of age, cut off approximately 0.3 cm of their tails, and place them in a 1.5 mL EP tube containing 50 μL of lysis buffer and 1 μL of proteinase K. Centrifuge until the tail is at the bottom of the tube and incubate in a 55°C metal bath overnight. The next day, inactivate the proteinase K by incubating in a 95°C metal bath for 5 minutes. Vortex the tail for 15 seconds to completely lyse the tail, and centrifuge at 12,000 rpm for 5 minutes. The supernatant can be used directly as a template for genotyping or stored at -20°C until needed.

[0062] 2.2. PCR identification results of F1 generation mouse genotype.

[0063] DNA was extracted from the mouse tail tip and its genotype was identified by PCR amplification.

[0064] according to Figure 4 The PCR identification strategy in

[15] was used. Using primers F2 and R2, a 3.4 kb fragment should be amplified in genomes positive for 5' HA homologous recombination, while no band should be observed in genomes negative for 5' HA homologous recombination. Using primers F1 and R1, a 2.5 kb fragment should be amplified in genomes positive for 3' HA homologous recombination, while no band should be observed in genomes negative for 3' HA homologous recombination. The amplification system is shown in Table 8, and the amplification procedure is shown in Table 9. Specific primers used for PCR identification are shown in Table 10.

[0065] Table 8 PCR system for identifying positive genomes of 5'HA and 3'HA homologous recombination Table 9 PCR program for identifying 5'HA and 3'HA homologous recombination positive genomes Table 10 PCR amplification primers for identifying 5'HA and 3'HA homologous recombination-positive genomes Identification results such as Figure 5 As shown, mice No. 17, 20, 21, 22, and 23 are F1 generation mice that are positive for homologous recombination in both arms.

[0066] Note: If the DNA sample is not very pure or if there is insufficient PCR extension time, long PCR products may not be amplified. Figure 4 For other PCR identification strategies, using primers F3 and R4, the target allele should be amplified as a 506 bp fragment. The sequences of primers F3 and R4 are shown in Table 12.

[0067] 2.3. Sequencing results of knock-in sequences of F1 generation mice.

[0068] PCR amplification was performed using primers F3 and R3, and the product was sequenced.

[0069] The sequencing results are as follows Figure 6 The results showed that the knock-in site of the positive F1 generation No. 17 mouse was correct and the knock-in sequence was correct without mutation. The specific primers used for PCR amplification are shown in Table 11.

[0070] Table 11 PCR amplification primers for identification of F1 generation mice 2.4. PCR identification results of F2 generation mouse genotypes.

[0071] DNA was extracted from the tail tip of mice and genotyped by PCR amplification. Figure 4 The PCR identification strategy in the present invention uses primers F4 and R5 as well as F3 and R4 for PCR. The genotypes of F2 generation mice are divided into three categories based on the length of the PCR fragment: homozygous, heterozygous, and wild type. The PCR fragment lengths corresponding to different genotypes are different. The homozygous has two bands of 1611bp and 506bp; the heterozygous has three bands of 1611bp, 459bp, and 506bp; and the wild type has one band of 459bp. The specific primers used for PCR identification are shown in Table 12. Homozygous is referred to as Ho, heterozygous is referred to as He, and wild type is referred to as Wt.

[0072] See the results Figure 7 , P34~P36, P38 and P39 are homozygous; P30~P33 and P37 are heterozygous, and P40 is Wt.

[0073] Table 12 PCR amplification primers for genotyping of F2 mice 3. Evaluation of the targeting strategy for transient knockout of p300.

[0074] The expression of p300 protein in F2 generation mouse tissue was detected by Western blot. The present invention takes muscle tissue as an example.

[0075] Identification results such as Figure 8 As shown, after dTAG administration to F2 heterozygous mice, the p300-FKPB fusion protein was degraded, and the target band was larger than the p300 protein in WT mice, while the p300 protein in the muscle tissue of mice not administered dTAG was normally expressed.

[0076] The above results show that the present invention uses In-Fusion technology to construct Donor vectors, and injects Donor vectors, gRNA and Cas9 mRNA into mouse fertilized eggs to transform human FKBP1A The gene sequence was knocked into the mouse genome using CRISPR / Cas9-mediated gene editing technology and used as a Ep300 The FKPB tag sequence expressed by gene fusion was then transferred into embryos and the resulting positive F0 generation mice were bred to obtain parents that could stably and efficiently express the p300-FKPB fusion protein. This enabled the use of dTAG protein rapid degradation system model mice to achieve rapid, complete, and reversible p300 protein-specific degradation induced by small molecule degraders, providing new ideas and approaches to solving the problem that traditional gene knockout animal models cannot explore the direct regulatory mechanism of the protein of interest.

[0077] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0078] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model that specifically degrades p300 protein, characterized in that: The following steps are involved: The Donor vector backbone was connected with 5' homology arms, 3×GGGGS, FKBP1A Gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homology arm were used to obtain Donor recombinant vector; The nucleotide sequence of the 5' homology arm is shown in SEQ.ID.NO.2; the nucleotide sequence of the 3×GGGGS is shown in SEQ.ID.NO.3; FKBP1A The gene synonymous mutation sequence is shown in SEQ.ID.NO.4; the nucleotide sequence of the P2A is shown in SEQ.ID.NO.5; the nucleotide sequence of the green fluorescent protein EGFP sequence is shown in SEQ.ID.NO.6; and the nucleotide sequence of the 3' homology arm is shown in SEQ.ID.NO.7; Based on the mouse chromosome 15 Ep300 gRNA was designed for exon 31 of the gene, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1; The gRNA, Cas9 protein and the Donor recombinant vector are mixed and injected into mouse fertilized eggs to make the FKBP1A Gene synonymous mutation sequence and green fluorescent protein EGFP sequence were knocked into the mouse genome; The injected fertilized eggs are cultured in vitro and then transplanted into surrogate mice to continue developing until pups are born, obtaining F0 generation mice. The F0 generation mice are hybridized with wild-type mice, or the offspring obtained by hybridization are further bred to obtain mice that specifically degrade p300 protein.

2. The construction method according to claim 1, characterized in that The Donor recombinant vector is constructed according to the following steps: Using the BAC plasmid as a template, a fragment containing the 5' homology arm from the mouse genome was amplified to obtain fragment-1. The sequence of fragment 1 is shown in SEQ.ID.NO.2; Using the BAC plasmid as a template, a fragment containing the 3' homology arm from the mouse genome was amplified to obtain fragment-3. The sequence of fragment 3 is shown in SEQ.ID.NO.

7. The 3×GGGGS, FKBP1A The gene synonymous mutation sequence, P2A and green fluorescent protein EGFP sequence were connected in sequence to obtain fragment-2; The fragment-1, fragment-2, fragment-3 and the Donor vector backbone are connected to obtain the Donor recombinant vector. The nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.

12.

3. The construction method according to claim 2, characterized in that The primer sequences for amplifying the fragment-1 are shown in SEQ.ID.NO.8 and SEQ.ID.NO.9; The primer sequences for amplifying the fragment-2 are shown in SEQ.ID.NO.10 and SEQ.ID.NO.

11.

4. The construction method according to claim 2, characterized in that When ligating the fragment-1, fragment-2, fragment-3 and the Donor vector backbone, the 20 μL ligation system contains: 6 ng Donor vector backbone, 39.2 ng fragment-1, 23.1 ng fragment-2, 23.9 ng fragment-3, 10 μL NEBuilder HiFi DNA Assembly Master Mix.

5. The construction method according to claim 1, characterized in that After the gRNA and the Cas9 protein were mixed and incubated at 25° C., the Donor recombinant vector was added, mixed evenly, and injected into mouse fertilized eggs.

6. The construction method according to claim 5, characterized in that: The concentration of the gRNA is 90 pmol / μL~110 pmol / μL, the concentration of the Cas9 protein is 18 μM~22 μM, and the concentration of the Donor recombinant vector is 12 ng / μL~18 ng / μL; The volume ratio of the gRNA to the Cas9 protein is 3-5:1, and the total volume of the mixture of the gRNA and Cas9 protein and the Donor recombinant vector is 20 μL.

7. The construction method according to claim 5, characterized in that: The concentration of the gRNA is 100 pmol / μL, the concentration of the Cas9 protein is 20 μM, and the concentration of the Donor recombinant vector is 15 ng / μL; The volume ratio of the gRNA to the Cas9 protein is 4:

1.

8. A mouse model for rapid degradation of p300 protein, characterized in that: The mouse model is obtained by the construction method described in claim 1.

9. The mouse model according to claim 8, characterized in that The dTAG is administered to the mice that specifically degrade the p300 protein, thereby achieving rapid and reversible degradation and clearance of the p300 protein in the mice.

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