A method for constructing a GHR humanized rat model using a CRISPR system and application thereof

Abstract

The application discloses a method for preparing GHR humanized animal cells by using a CRISPR system, which comprises the following steps: designing and synthesizing sgRNA; mixing the sgRNA with a Cas9 protein to form a CRISPR-Cas9 complex; constructing donor DNA containing coding sequences of a human GHR extracellular region and a rat GHR intracellular region; injecting the CRISPR-Cas9 complex and the donor DNA into the cytoplasm or nucleus of a rat fertilized ovum to knockout the coding sequence of the extracellular region of the rat-derived GHR gene and simultaneously insert the coding sequence of the extracellular region of the human-derived GHR gene into the genome, so that the GHR humanized animal cells are obtained. The application further discloses a method for constructing a GHR humanized rat model by using the CRISPR system, and application of the GHR humanized animal cells and the GHR humanized rat model obtained by the above method in drug screening, drug development or efficacy evaluation. The application can more accurately simulate the interaction between the human GHR and growth hormone and other ligands, improves the accuracy and reliability of research, and has the advantages of simple gene editing operation, high editing efficiency, strong specificity, low off-target effect and wide application range.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a humanized GHR rat model using the CRISPR system and its application. Background Technology

[0002] Growth hormone (GH) exerts important physiological functions such as regulating growth, development, and metabolism by binding to the growth hormone receptor (GHR) on the target cell membrane. Mutations or abnormal expression of the GHR gene are closely related to a variety of diseases, such as growth hormone insensitivity syndrome, obesity, and diabetes. Therefore, constructing animal models that accurately mimic human GHR function is of great significance for studying the physiological mechanisms of GHR, the pathogenesis of related diseases, and drug development.

[0003] Currently, GHR knockout rat (GHR KO rat) models have been reported (List EO, Sackmann-Sala L, Berryman DE et al., Endocrine parameters and phenotypes of the growth hormone receptor gene disrupted (GHR KO rat)). - / - ) mouse[J]. Endocr Rev, 2011, 32(3): 356-386.DOI: 10.1210 / er.2010-0009.). Chinese patent application CN118389520A discloses an sgRNA for targeting the mouse GHR gene, a method for gene knockout in mice, and a method for constructing a GHR gene knockout mouse model. It uses Cas9 protein to knock out the fourth exon of the GHR gene, causing a frameshift mutation, thereby knocking out the mouse GHR gene.

[0004] However, these GHR knockout animal models completely eliminate the animal's own GHR gene through gene editing technology. Because the GHR gene is completely missing, the animal cannot express the functional GHR protein, meaning the GHR gene function is completely lost. The growth hormone signaling pathway is completely blocked, resulting in typical phenotypes such as growth retardation (e.g., short stature) and metabolic abnormalities (e.g., increased insulin sensitivity, altered fat accumulation). Furthermore, due to the lack of a functional receptor, they cannot respond to human growth hormone or related drugs.

[0005] Therefore, there is an urgent need for an accurate and reliable humanized animal model of GHR to be effectively applied to the study of the pathogenesis of GHR-related diseases and in the fields of drug development and efficacy evaluation. Summary of the Invention

[0006] This invention aims to address the lack of rat models that can accurately simulate human GHR function in the existing technology, as well as the problems of complex operation, low efficiency, and high off-target effects in constructing humanized animal models using traditional gene editing technology. It provides a method for constructing a GHR-humanized rat model using the CRISPR system.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing GHR humanized animal cells using a CRISPR system, comprising the following steps: (1) Design and synthesize sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 4; (2) Mix sgRNA with Cas9 protein to form a CRISPR-Cas9 complex; (3) Construct donor DNA containing the coding sequences of the human GHR extracellular region and the rat GHR intracellular region, wherein the nucleotide sequence of the human GHR extracellular region is shown in SEQ ID NO: 21 and the nucleotide sequence of the rat GHR intracellular region is shown in SEQ ID NO: 22; (4) The CRISPR-Cas9 complex and donor DNA were injected into the cytoplasm or nucleus of rat fertilized eggs to knock out the extracellular and transmembrane coding sequences of rat-derived GHR protein, while the extracellular and transmembrane coding sequences of human-derived GHR protein were inserted into the genome to obtain the humanized GHR animal cells.

[0009] Preferably, the nucleotide sequence of the donor DNA encodes the amino acid sequence shown in SEQ ID NO: 6.

[0010] Preferably, in step (2), the molar ratio of sgRNA to Cas9 protein is 1:1.

[0011] Preferably, in step (3), the nucleotide sequence of the donor DNA is as shown in SEQ ID NO: 7, and preferably, the donor DNA contains homologous arms at both ends that are homologous to the rat GHR gene sequence to ensure the smooth progress of homologous recombination. Preferably, the donor DNA does not contain an antibiotic resistance gene to reduce interference with the model.

[0012] Preferably, in step (3), the donor DNA further includes a human GHR transmembrane region coding sequence as shown in SEQ ID NO: 23.

[0013] Preferably, in step (4), the extracellular and transmembrane coding sequences of the rat-derived GHR gene are knocked out, while the extracellular and transmembrane coding sequences of the human-derived GHR gene are inserted into the genome.

[0014] Preferably, in step (3), the donor DNA further includes a signal peptide coding sequence as shown in SEQ ID NO: 24.

[0015] Preferably, in step (4), the concentrations of the CRISPR-Cas9 complex and the donor DNA are both 50 ng / μL.

[0016] Preferably, in step (4), approximately 18 hours after inducing ovulation with human chorionic gonadotropin (hCG), the CRISPR-Cas9 complex and donor DNA are injected into the cytoplasm or nucleus of the rat fertilized egg using microinjection technology.

[0017] Preferably, in step (4), during the injection of the CRISPR-Cas9 complex and the donor DNA, a RAD51 activator is injected into the cytoplasm or nucleus of the fertilized egg. The concentration of the RAD51 activator is preferably 10 μM.

[0018] Secondly, the present invention also provides a GHR humanized animal cell, which is constructed by the above method.

[0019] Thirdly, the present invention also provides a method for constructing a humanized GHR rat model using a CRISPR system, comprising: transplanting rat GHR humanized fertilized oocytes prepared by the above method into the oviduct of a pseudopregnant female rat, allowing them to develop into offspring rats; after the offspring rats are born, their genomes are tested, and positive rats that have successfully achieved GHR gene editing are screened out, namely the GHR humanized rat model.

[0020] Preferably, the genome is detected by PCR, sequencing, and Western blotting (WB) methods. In addition, animal morphological observation can also corroborate whether the humanization replacement of rat GHR protein was successful.

[0021] Fourthly, the present invention also provides an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0022] Fifthly, the present invention also provides a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO: 7.

[0023] Sixthly, the present invention also provides the application of GHR humanized animal cells obtained by the above-described method for preparing GHR humanized animal cells or GHR humanized rat models obtained by the above-described method for constructing GHR humanized rat models in drug screening, drug development or efficacy evaluation.

[0024] Preferably, the drug is a treatment for GHR-related diseases.

[0025] This invention uses the CRISPR system to edit the rat GHR gene, retaining the signal peptide and intracellular region coding sequences of the rat GHR, and replacing the extracellular and transmembrane region coding sequences of the rat GHR with the extracellular and transmembrane region coding sequences of the human GHR. The humanized rat model of GHR constructed by the method of this invention can retain the normal physiological function of GHR. In addition, the receptor protein is a human homolog, and its binding ability to GH and signal transduction mode (such as the activation efficiency of the downstream JAK-STAT pathway) are closer to the human physiological state, which can simulate the regulatory mechanism of the human GH-GHR axis.

[0026] This invention employs CRISPR combined with exogenous DNA-mediated HDR humanization. CRISPR-mediated gene knockout (KO) utilizes the CRISPR / Cas system to recognize and cleave a specific sequence in the rat GHR gene, causing a DNA double-strand break (DSB). Simultaneously, exogenous homologous donor DNA containing the human gene sequence is introduced. During cell repair, the exogenous donor DNA serves as a template for precise repair via homology-directed repair (HDR), replacing part or the full length of the rat endogenous GHR gene sequence with the human homologous GHR gene sequence. This preserves gene function, resulting in the expression of human GHR protein in rats, rather than the rat endogenous GHR. The physiological function of GHR is thus preserved through the human gene, achieving "humanized" expression.

[0027] Compared with existing GHR knockout rats, the humanized GHR rats of the present invention have irreplaceable advantages in the following aspects: (1) Improved accuracy and reliability of the model: The signal peptide and intracellular region of rat GHR were preserved, ensuring the normal localization and signal transduction function of GHR in rats; the extracellular region of rat GHR was replaced with that of human GHR, enabling the model to more accurately simulate the interaction between human GHR and growth hormone and other ligands, thus improving the accuracy and reliability of the study. Humanized rats possess complete GH-GHR signaling pathway function and can be used to study the disease mechanisms related to GH overdose (such as acromegaly) or abnormal signaling pathways, as well as the effects of GH on lifespan, cognition, and other functions. In contrast, GHR knockout rats, due to the complete absence of the pathway, can only be used to study the pathological state of complete loss of GHR function, and their application scope is relatively limited.

[0028] (2) Providing a precise drug screening and evaluation platform: Humanized models are the most direct and reliable preclinical models for evaluating the pharmacological effects and toxicities of therapeutic drugs (such as agonists, antagonists, and antibodies) targeting human GHR. In contrast, GHR knockout rats cannot be used to test any drugs targeting human GHR because they do not express human targets. Humanized models can accurately predict the potency, pharmacokinetics, and potential toxicity of drugs in humans, greatly reducing the risks of clinical development.

[0029] (3) It has superior translational medicine value: Due to the expression of human targets, the experimental data obtained on this model (such as drug efficacy and changes in biomarkers) are more correlated with human clinical trials, and the translational success rate is significantly improved. In contrast, the mechanistic findings on growth and metabolism obtained in GHR knockout rats, although similar to the pathology of human GHR dysfunction diseases (such as Laron syndrome) due to the complete absence of signaling pathways, cannot be used to directly evaluate interventions targeting human receptors.

[0030] (4) It can be used as a highly compatible research tool: This model can be combined with human GH injection to test the effect of exogenous GH, making it an ideal model for evaluating the bioactivity of human growth hormone preparations. In contrast, GHR knockout rats do not respond to any GH and cannot be used for this type of study.

[0031] (5) Simple operation and high efficiency: Gene editing using the CRISPR system is relatively simple to operate and highly efficient, and can quickly obtain positive rat models.

[0032] (6) High specificity and low off-target effect: By carefully designing sgRNA, the specificity of the CRISPR system can be improved, the off-target effect can be reduced, and the interference to other genes in the rat genome can be reduced.

[0033] (7) Wide range of applications: This humanized rat model of GHR can be widely used in the study of the pathogenesis of GHR-related diseases, drug screening and efficacy evaluation. Attached Figure Description

[0034] Figure 1 A flowchart of the method for preparing humanized GHR rats according to the present invention is shown.

[0035] Figure 2 A schematic diagram is shown showing the editing of the rat GHR gene using the CRISPR system to obtain GHR humanized rats.

[0036] Figure 3 The amino acid sequence alignments of humans and rats are shown, along with the sites of replaced amino acid regions.

[0037] Figure 4 The quality control results of the synthesized sgRNA sample are shown.

[0038] Figure 5 The DNA cleavage efficiency of sgRNA is shown.

[0039] Figure 6 The enzyme digestion identification results of the recombinant vector are shown.

[0040] Figure 7 The phenotypes of wild-type rats and GHR humanized rats are shown in comparison.

[0041] Figure 8 The results of human GHR protein expression in wild-type rats and GHR humanized rats are shown. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0043] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0044] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0045] Unless otherwise defined, 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 pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0047] This invention uses the CRISPR system to edit the rat GHR gene, retaining the signal peptide and intracellular region coding sequences of the rat GHR, and replacing the extracellular and transmembrane region coding sequences of the rat GHR with the extracellular and transmembrane region coding sequences of the human GHR. Example 1: Design and Synthesis of sgRNA Five specific sgRNAs were designed based on the sequences at both ends of the extracellular region of the rat GHR gene (GenBank GeneID:25235).

[0048] The design of sgRNA must follow the design principles of the CRISPR system to ensure high specificity and cleavage efficiency. The designed sgRNA is synthesized using gene synthesis technology.

[0049] Since the sgRNA sequence directly determines the location of DNA double-strand breaks, and the sgRNA itself affects the efficiency of CRISPR-mediated DNA double-strand breaks, as well as potential off-target efficiency and off-target locations, in order to obtain the best gene editing efficiency and avoid unnecessary off-target effects, this invention designs and screens candidate sgRNAs with high specificity and cleavage efficiency.

[0050] The sequences of each sgRNA are shown in Table 1 below.

[0051] Table 1. sgRNA sequences

[0052] The designed sgRNA sequence was handed over to a gene synthesis company (Suzhou Genewiz Biotechnology Co., Ltd.) for synthesis.

[0053] Figure 4 The sgRNA gel electrophoresis results are shown, indicating that the synthesized sgRNA is of good quality and has not been degraded.

[0054] To assess the cleavage activity of the sgRNA to be used, in vitro T7E1 assays were performed.

[0055] T7E1 is an endonuclease that recognizes and cleaves non-complementary base pairs in double-stranded DNA, commonly used to detect mutations generated by gene editing technologies such as CRISPR-Cas9. This experiment is an in vitro molecular-level assay. To evaluate the sgRNA-mediated cleavage efficiency of target DNA, gel electrophoresis was performed. Figure 4 The grayscale values ​​of the middle and lower strips are used to assess cutting efficiency.

[0056] The T7E1 enzyme recognizes and cleaves mismatch sites in the DNA double helix caused by editing mutations (such as insertions or deletions). After digestion, the products show bands of different sizes on gel electrophoresis: Uncut band (a): Intact PCR product.

[0057] Post-cleavage bands (b, c): smaller fragments produced by cleavage by the T7E1 enzyme.

[0058] By analyzing the grayscale values ​​of these bands, the editing efficiency can be calculated. The grayscale value reflects the DNA content in the band.

[0059] The experimental procedure includes: extracting edited cellular DNA, PCR amplification of the fragment containing the target site, purification of the product, denaturation and annealing to form heteroduplexes, T7E1 digestion, and finally analysis of the bands by electrophoresis.

[0060] The cleavage activity of the sgRNA to be tested was determined by analyzing the grayscale of the electrophoretic bands.

[0061] The formula for calculating grayscale analysis is as follows: Editing efficiency can be calculated using f(cut) = (b+c) / (a+b+c), where a is the grayscale value of the uncut strip, and b and c are the grayscale values ​​of the two cut strips. The final Indel efficiency (%) = 100 (1-√(1-f(cut)))%.

[0062] Based on the T7E1 experimental grayscale data ( Figure 5 The DNA cleavage efficiency of each sgRNA was evaluated. The results are shown in Table 2 below.

[0063] Table 2. DNA cleavage efficiency of sgRNA

[0064] The in vitro editing efficiency of sgRNA-4 was 40.1%, which was better than other candidate sgRNAs. Therefore, sgRNA-4 was selected for the next step of the experiment.

[0065] Example 2: Construction of donor DNA Based on the human GHR gene (GenBank GeneID:2690, NM_017094.3 → NP_058790.1 Using the rat GHR gene (GenBank GeneID:25235) as a template, NM_000163.5 → NP_000154.1 Using ) as a template, donor DNA is constructed.

[0066] Based on the sequence design, further designs were created, such as... Figure 3 The image shows the amino acid sequence alignments of humans and rats, as well as the sites of replaced amino acid regions. The vector contains the left homologous arm, the human GHR extracellular region gene fragment, the human GHR transmembrane region, the rat GHR intracellular region gene fragment, the rat 3'UTR, the polyA transcription termination element, and the right homologous arm.

[0067] The rat-derived sequence corresponds to the NCBI accession number NM_017094.3 (growth hormone receptor isoform 1 precursor), and the human-derived sequence corresponds to the NCBI accession number NM_000163.5 (growth hormone receptor isoform 1 precursor).

[0068] The vector was constructed as follows: three homologous recombination fragments were designed (LA: left arm, KI: knock in, RA: right arm). The 5' homologous arm corresponds to the LA fragment (SEQ ID NO: 8), the human KDR gene fragment and other elements for humanization insertion correspond to the KI fragment (SEQ ID NO: 10), and the 3' homologous arm corresponds to the RA fragment (SEQ ID NO: 9).

[0069] The above sequence was synthesized into the pUC plasmid vector (Genwiz, pUC-GW-AMP vector) by Genewiz.

[0070] The amino acid sequence encoded by the donor DNA includes: rat signal peptide (1-18aa) - human extracellular region (19-264aa) - human transmembrane region (265-288aa) - rat intracellular region (289-637aa).

[0071] The amino acid sequence encoded by the human-rat chimeric donor DNA is shown in SEQ ID NO: 6.

[0072] The nucleotide sequence of the donor DNA is shown in SEQ ID NO: 7. The donor DNA includes the coding sequences for the linked signal peptide (SEQ ID NO: 24), the human extracellular region (SEQ ID NO: 21), the human transmembrane region (SEQ ID NO: 23), and the rat intracellular region (SEQ ID NO: 22).

[0073] The sequence was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0074] Enzyme digestion was performed on the vector obtained from Genewiz, and the sizes of the digested fragments were 4655, 3354, and 2013 bp, respectively, which were consistent with the expected sizes.

[0075] Example 3: Microinjection of fertilized eggs 1. Superovulation: Female rats aged 6-8 weeks were selected ( Rattus norvegicusPregnant mare serum gonadotropin (PMSG) 0.3 U / μL was injected intraperitoneally, followed by human chorionic gonadotropin (hCG) 0.3 U / μL 48 hours later, and then rats were placed in the same cage as male rats.

[0076] 2. Collection of fertilized eggs: The following morning, the female mice were examined for vaginal plugs. The mice with vaginal plugs were euthanized, the oviducts were removed, and the fertilized eggs were flushed out under a stereomicroscope.

[0077] The synthesized sgRNA-4 was combined with Cas9 protein (GenScript, GenCRISPR). TM Mix Cas9 v1.2 (product number Z03702-100) in a 1:1 molar ratio and incubate at 37°C for 10 minutes to form a Cas9-sgRNA complex.

[0078] 3. Microinjection: Under micromanipulation, the Cas9-sgRNA complex and donor DNA (both at a concentration of 50 ng / μL, 2 pL) were injected into the cytoplasm of the fertilized egg.

[0079] To increase the efficiency of in situ replacement of the rat GHR gene, this invention innovatively injects RAD51 activator (RS1, CAS No.: 312756-74-4; Sigma-Aldrich, catalog No. R9782-5MG) into the cytoplasm of fertilized eggs. The activator is added to the above-mentioned microinjection mixture composed of Cas9-sgRNA complex and donor DNA at a concentration of 0 or 10 μM according to the grouping. The injection volume per fertilized egg is approximately 2 pL.

[0080] The positive rate results are as follows:

[0081] The role of RAD51 activators is to stabilize RAD51 nucleoprotein filaments and prolong their lifespan. RS-1, as a RAD51 activator, can bind to the RAD51 protein itself, altering its conformation and promoting its binding to single-stranded DNA, forming a tighter and more stable complex. Theoretically, more stable nucleoprotein filaments mean more time for homologous template searching and a reduced risk of being displaced from the DNA by other proteins (such as anti-recombinant proteins), thereby improving the success rate of homologous pairing and strand invasion. However, there are currently no publicly available reports on the effective use of RS-1 as a RAD51 activator in increasing the positive rate in samples injected into rat fertilized eggs. This invention is the first to discover that by optimizing the fertilized egg manipulation method to prepare animal models, the production efficiency of positive mice can be universally improved under various conditions.

[0082] 4. Embryo transfer: The injected fertilized eggs were then transferred into the oviducts of pseudopregnant female mice in estrus, with 15-20 fertilized eggs transferred to each female mouse.

[0083] Figure 7 Medium: SD wild-type male mice around 10 weeks old, approximately 260g (GHR) wt / wt GHR humanized rats, male, approximately 10 weeks old, about 210g (GRH) H / H Due to the humanization of GHR, the binding efficiency between mouse GH and humanized GHR is reduced. Reduced protein homology due to species differences and structural changes in the protein also lead to decreased biological function, which is a normal phenotype in humanized models. Morphological observation confirms the successful model construction. The theoretical phenotypes include reduced body weight, shorter tail, coarser and duller fur, and relatively short and weak limbs.

[0084] The humanized GHR rat constructed in this invention can be used for drug development and biological research related to diseases that are associated with abnormal GH and GHR signaling axes.

[0085] Example 4: Identification of Nucleic Acid Levels in GHR Humanized Rats After the offspring rats were born, a small amount of tail tip tissue was cut off, genomic DNA was extracted, primers were identified, PCR reaction was performed, and the PCR products were identified by Sanger sequencing to screen out positive rats that had successfully undergone GHR gene editing.

[0086] The identification primers are as follows:

[0087] The PCR reaction system is as follows:

[0088] PCR reaction program: 98℃, 10 seconds; 55℃, 15 seconds; 72℃, 5 seconds / kb; repeat the above steps 35 times.

[0089] Sequencing was provided by Genewiz. The PCR product sequence was compared with the LA, KI, and RA sequences. If all sequences matched, the rat was identified as a GHR humanized positive rat.

[0090] Example 5: Identification of protein levels in GHR humanized rats Protein level identification was performed on offspring rats that tested positive for sequencing. Approximately 1 cm of tail tip tissue was harvested from the positive rats with correct sequencing, and proteins were extracted and identified using Western blotting. Positive rats that successfully underwent GHR gene editing were screened at the protein level.

[0091] The expression of human GHR protein in wild-type SD rats and GHR humanized rats was detected using anti-human GHR specific antibody (GHR Antibody (MAB 5): sc-69880).

[0092] The steps for the Western blotting experiment are as follows: (1) Sample preparation Cell / tissue lysis: Lyse on ice for 30 minutes using RIPA lysis buffer (containing protease inhibitor); centrifuge (12,000 rpm, 15 minutes, 4°C) and collect the supernatant.

[0093] Protein quantification: The concentration was determined by the BCA method and adjusted to a consistent level (2 μg / μL).

[0094] (2) SDS-PAGE electrophoresis Gel preparation: separating gel (10-12% acrylamide, pH 8.8); stacking gel (5% acrylamide, pH 6.8).

[0095] Sample loading: 20-30 μg protein + 5× Loading Buffer per well.

[0096] The pre-colored marker is used as a reference.

[0097] Electrophoresis conditions: 80V (stacking gel) → 120V (separating gel), 1.5 hours (bromophenol blue to the bottom of the gel).

[0098] (3) Transfer membrane Wet transfer method: constant current 200 mA, 90 minutes (PVDF membrane needs to be activated with methanol).

[0099] Transfer buffer: Tris-Glycine + 20% methanol.

[0100] Verify transfer efficiency: Ponceau S staining or marker transfer.

[0101] (4) Blocking and antibody incubation Sealing: 5% skim milk / TBST, room temperature for 1 hour.

[0102] Primary antibody incubation: dilution ratio (1:1000), overnight at 4°C.

[0103] Secondary antibody incubation: HRP-labeled secondary antibody (1:5000), 1 hour at room temperature.

[0104] (5) Development and analysis ECL luminescence: Add luminescent solution (Pierce ECL) and image using a chemiluminescence analyzer.

[0105] Internal reference calibration: Commonly used internal reference: β-actin (40-45 kDa).

[0106] Figure 8 Western blot (WB) results showed that in GHR humanized rats (GHR... H / H In the samples, a clear band with a molecular weight of approximately 130 kDa was detected using the anti-human GHR specific antibody, consistent with the theoretical molecular weight of human GHR protein, indicating that humanized rats successfully expressed human GHR protein. In contrast, no specific band was detected in wild-type (WT) rat samples using the same anti-human GHR antibody, indicating that the endogenous rat GHR protein in wild-type rats cannot cross-react with the anti-human GHR antibody, thus verifying the species specificity of the antibody.

[0107] Meanwhile, using β-actin as an internal control (molecular weight approximately 42 kDa), stable and consistent bands were detected in both humanized GHR rats and wild-type rats, indicating that the loading amount of the two groups of samples was balanced, and there were no significant deviations in the protein extraction and transfer processes, making the experimental system stable and reliable.

[0108] The above results confirm at the protein level that human GHR protein was successfully expressed in the humanized rat model of GHR, and that this expression is species-specific, providing a key basis for subsequent functional studies and drug screening based on human GHR.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing GHR humanized animal cells using a CRISPR system, characterized in that, Includes the following steps: (1) Design and synthesize sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 4; (2) The sgRNA is mixed with Cas9 protein to form a CRISPR-Cas9 complex; (3) Construct donor DNA containing the coding sequences of the human GHR extracellular region and the rat GHR intracellular region, wherein the nucleotide sequence of the human GHR extracellular region is shown in SEQ ID NO: 21 and the nucleotide sequence of the rat GHR intracellular region is shown in SEQ ID NO: 22; (4) The CRISPR-Cas9 complex and the donor DNA are injected into the cytoplasm or nucleus of rat fertilized eggs to knock out the extracellular coding sequence of the rat-derived GHR gene, and the extracellular coding sequence of the human-derived GHR gene is inserted into the genome to obtain the humanized GHR animal cells.

2. The method according to claim 1, characterized in that, In step (2), the molar ratio of sgRNA to Cas9 protein is 1:

1.

3. The method according to claim 1, characterized in that, In step (3), the nucleotide sequence of the donor DNA is as shown in SEQ ID NO: 7, and preferably the donor DNA contains homologous arms at both ends that are homologous to the rat GHR gene sequence.

4. The method according to claim 1, characterized in that, In step (4), the concentrations of both the CRISPR-Cas9 complex and the donor DNA are 50 ng / μL.

5. The method according to claim 1, characterized in that, The nucleotide sequence of the donor DNA encodes the amino acid sequence shown in SEQ ID NO:

6.

6. The method according to claim 1, characterized in that, In step (4), during the injection of the CRISPR-Cas9 complex and the donor DNA, RAD51 activator is injected into the cytoplasm or nucleus of the fertilized egg.

7. A GHR humanized animal cell, prepared by the method according to any one of claims 1 to 6.

8. A method for constructing a humanized GHR rat model using a CRISPR system, characterized in that, include: The GHR humanized animal cells prepared by any one of claims 1 to 6 are transplanted into the oviduct of pseudopregnant female mice to develop into offspring rats. After the offspring rats are born, their genomes are tested, and positive rats that have successfully achieved GHR gene editing are screened out, which are the GHR humanized rat models.

9. The application of GHR humanized animal cells obtained by the method according to any one of claims 1 to 6 or GHR humanized rat models obtained by the method according to claim 8 in drug screening, drug development or efficacy evaluation.

10. An sgRNA, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 4.

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