Construction method and application of animal model for in-vivo ubiquitination enzyme screening

Abstract

The invention discloses a construction method and application of an animal model for in-vivo ubiquitination enzyme screening, and belongs to the technical field of animal model construction. The traditional in-vitro screening of ubiquitination modification enzyme genes has the defects of unreal simulation environment, poor repeatability, narrow application range and the like, 150 SgRNAs are designed to construct library plasmids, loxP TC9 sequences and filling sequences are introduced to prepare transgenic Sanyang mice, and the transgenic Sanyang mice are used for in-vivo screening after being induced by tamoxifen. The mouse model constructed by the invention can realize gene screening in 29 tissues and organs, the success rate of target verification is more than 80%, the coefficient of variation is lower than 10%, the model can be stably passed, and the screening cost and technical threshold are greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing and applying an animal model for screening ubiquitinizing enzymes in vivo. Background Technology

[0002] The ubiquitin-proteasome pathway is a core intracellular protein degradation regulatory system that precisely regulates key life activities such as gene transcription, immune responses, and the cell cycle through ubiquitination of substrate proteins and proteasome hydrolysis. Among these, ubiquitination enzymes, as the core regulatory molecules of this pathway, are closely related to pathological processes such as tumorigenesis and immune escape due to functional abnormalities, making them an important research subject for the development of targets for tumor immunotherapy.

[0003] Currently, the screening of ubiquitination-modifying enzyme genes mainly relies on in vitro cell models. Gene knockout / knockdown is achieved through lentivirus-mediated CRISPR / Cas9 systems or RNA interference technology, followed by functional testing to screen for potential targets. For example, current techniques for screening ubiquitination-modifying enzymes related to T-cell tumor infiltration require isolating primary T cells from mouse spleens, activating them with CD3 / CD28 antibodies, co-transducing Cas9 and SgRNA libraries via lentivirus, reinfusing the transduced T cells into tumor-bearing mice, and finally identifying key genes by analyzing SgRNA abundance through high-throughput sequencing. However, such in vitro screening techniques have significant drawbacks: The simulated environment is not realistic: In vitro cell culture systems cannot replicate the complex physiological microenvironment in vivo (such as tissue-specific cell-to-cell interactions and immunosuppressive microenvironments), leading to a high false-positive rate when the screened targets are validated in vivo, making it difficult to reflect the true physiological and pathological functions. For example, some ubiquitination-modifying enzymes that exhibit immune-activating functions in in vitro cell models may have their functions inhibited or even reversed in the in vivo tumor microenvironment due to the regulation of factors such as stromal cells and metabolites.

[0004] Poor reproducibility: During in vitro screening, the purity of cell isolation, activation efficiency, and lentivirus transduction efficiency are easily affected by experimental conditions, resulting in significant differences in experimental results between different batches, making it difficult to achieve stable reproducibility.

[0005] Narrow scope of application: Existing technologies rely on viral infection efficiency and can only be used to screen for cell types that are easily infected by viruses, such as primary T cells and some tumor cells. However, they cannot effectively screen for cells with low viral infection efficiency or difficult to culture in vitro, such as bone marrow cells, nerve cells, and fat cells, as well as complex tissues and organs such as spleen, liver, and lungs.

[0006] The process is complex and costly: each screening requires re-isolation of primary cells, packaging of lentiviruses, and optimization of transduction conditions. This not only involves a complicated process that is time-consuming and labor-intensive, but also requires a large amount of reagents and consumables, resulting in high screening costs.

[0007] To address these issues, researchers have attempted to develop in vivo screening tools, but existing technologies still have significant limitations. For example, reported in vivo CRISPR screening technologies are limited to specific cell types (such as primary T cells) and require complex procedures such as cell reinfusion, making it impossible to perform systemic screening of multiple tissues and organs. Furthermore, there is a lack of stable, reusable animal models, requiring the screening system to be rebuilt for each experiment, which makes it difficult to meet the demands for large-scale, high-throughput screening.

[0008] The relevant patent document (publication number CN109706174A) discloses a method for constructing a deubiquitinase UAS-cDNA / ORF plasmid and a transgenic Drosophila library. This method targets 41 Drosophila deubiquitinase genes, constructs UAS-cDNA / ORF plasmids through modular design, and uses the UAS / GAL4 system to achieve gene overexpression in specific tissues for gene function and disease mechanism research. However, the Drosophila used in this technology have a significantly different genetic background and physiological structure from mammals. The Drosophila model cannot simulate the complex tumor immune microenvironment of mammals, making it difficult for the screened targets to be translated into human diseases. Furthermore, it can only perform local screening in a few tissues such as the eyes, wings, and muscles. Summary of the Invention

[0009] The purpose of this invention is to provide: A method for constructing an animal model for screening ubiquitinated enzymes in vivo and its application, and related technologies, to solve the technical problems of traditional in vitro screening of ubiquitinated modified enzyme genes, such as unrealistic simulated environment, poor reproducibility, and narrow applicability, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of standard chemical terminology can be found in the reference "Genetic Engineering", by Wang Xuchu, Science Press.

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as culture medium preparation, strain culture, enzyme digestion and ligation, homologous recombination, etc., shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term “SgRNA (single-guide RNA)” used in this article refers to an RNA molecule approximately 100 nt in length, with its 5′ end (20 nt) complementary to the target DNA and its 3′ end binding to Cas9. SgRNA is responsible for directing the Cas9 nuclease to a specific site in the genome and inducing double-strand breaks (DSBs).

[0016] The term “SgRNA abundance” used in this article refers to the proportion of reads of a specific SgRNA sequence in deep sequencing data after normalization of the total number of reads. It is used to quantify the relative frequency of the SgRNA in a cell population and serves as a proxy indicator for inferring changes in cell fitness after gene knockout.

[0017] The term "Cas9 nuclease" as used in this article refers to an RNA-guided DNA endonuclease derived from Streptococcus that can generate blunt-end double-strand breaks 3 bp upstream of the PAM (NGG) sequence specified by SgRNA, triggering non-homologous end joining (NHEJ) repair and introducing frameshift mutations to achieve gene knockout.

[0018] The term "tamoxifen" as used in this article refers to a small molecule estrogen receptor modulator that, upon binding to the Cre-ERT2 fusion protein, induces its nuclear translocation, thereby triggering loxP recombination. It is commonly used for time-controlled gene editing induction.

[0019] The term “ubiquitination-modifying enzyme genes” used in this article refers to enzyme families (such as E1 activators, E2 conjugates, and E3 ligases) that participate in protein ubiquitination modification, regulate immune cell activation, apoptosis, and tumor immune escape, and are a potential target pool for tumor immunotherapy.

[0020] In a first aspect, the present invention provides a system for screening ubiquitination-modifying enzyme genes in vivo, the system comprising: (a) SgRNA expression cassette: encoding 150 tandemly arranged SgRNA expression units, which target 140 ubiquitination-modifying enzyme genes and 10 control genes, respectively. The SgRNA sequences are shown in SEQ ID NO.1-150. (b) Cas9 nuclease expression element; (c) Elements that can be induced to express Cre recombinase.

[0021] Specifically, the SgRNA expression cassette from 5' to 3' contains, in sequence: a promoter, two unidirectional loxP TC9 recombination sites, a transcription termination sequence located between the loxP sites, and 150 tandemly arranged SgRNA expression units, the loxP sequences of which are shown in Table 1.

[0022] Table 1. loxP sequences

[0023] Secondly, the present invention provides a method for constructing a transgenic mouse model for screening ubiquitination-modifying enzyme genes in vivo, characterized in that the construction method uses any of the above-mentioned systems.

[0024] Specifically, the construction method includes the following steps: S1. Assemble 150 SgRNAs with sequences as shown in SEQ ID NO.1-150 into an SgRNA expression cassette; S2. The SgRNA expression cassette is integrated into the fertilized egg via embryo microinjection, and a stably inherited transgenic animal is obtained; S3. The transgenic animal is crossed with Cas9-expressing animals and UBC-CreERT2-expressing animals to obtain a triple-positive screening model that can simultaneously knock out 150 genes in vivo.

[0025] More specifically, the SgRNA expression cassette is integrated into the mouse chromosome 2 between positions 110316697 and 110316698 via embryo microinjection.

[0026] Thirdly, the present invention provides the application of mouse models obtained by any of the above construction methods in in vivo screening of ubiquitination-modifying enzyme genes and screening of tumor immune targets.

[0027] Specifically, the screening includes the following steps: (1) Inducing the expression of SgRNA in target cells in mouse models; (2) Establishing an in vivo screening environment by inoculating MC38 tumor cells; (3) Collect samples after the screening period ends; (4) Extract genomic DNA from the sample and construct an NGS library; (5) Sequencing analysis of the relative abundance of each SgRNA, and screening based on the relative abundance of each SgRNA.

[0028] More specifically, in step (1), the induction is performed using tamoxifen.

[0029] More specifically, in step (3), the sample includes any one or more of the following: tumor, bone marrow, spleen, small intestine, lung, liver, kidney, colon, muscle, white fat, brown fat, lymph nodes, sex organs, brain, cerebellum, thyroid gland, and thymus.

[0030] More specifically, in step (5), the high abundance of SgRNA indicates that the knockout of the target ubiquitination modifying enzyme gene gives the cell a positive selection advantage, suggesting that the enzyme has a negative regulatory function of tumor immunity or a tumor suppressor-related function; the low abundance indicates that the knockout of the target ubiquitination modifying enzyme gene leads to negative selection or cell lethality, suggesting that the enzyme is an essential gene for the survival, proliferation or positive regulation of tumor immunity of immune cells.

[0031] Preferably, the analysis uses MAGeCK and GSEA bioinformatics tools, and the significance criterion is P<0.05.

[0032] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: This invention provides the first dedicated system and mouse model capable of large-scale in vivo screening of 140 ubiquitination-modifying enzyme genes. It overcomes the limitations of traditional in vitro screening, which cannot simulate the physiological microenvironment in vivo. The screened targets are more closely related to the actual in vivo action state, significantly reducing the false positive rate and greatly improving the success rate of in vivo target validation.

[0033] The system incorporates loxPTC9 recombination sites and 0.5k-10kbp filler sequences into the SgRNA expression cassette, combined with specific chromosomal integration site design, to ensure efficient recombination and uniform expression of the SgRNA library. This solves the technical challenges of interference from multiple SgRNA tandem expression and unstable integration, and the reproducibility and reliability of the screening results are significantly better than existing technologies.

[0034] Transgenic mouse models can be stably passaged and reused without the need to re-prepare cells and viruses for each screening, greatly simplifying the operation process and reducing screening costs. Moreover, they are applicable to a variety of tissues and organs, such as tumors, bone marrow, and spleen, breaking through the limitations of traditional technologies in the screening of cell types and tissues.

[0035] This system and model combine ubiquitination-modifying enzyme gene screening with tumor immune target identification. Through the construction of the MC38 tumor model and NGS high-throughput analysis, it can quickly and accurately identify key genes with tumor immune regulation functions, providing an efficient tool for the development of tumor immunotherapy drugs and accelerating the target translation and application process. Attached Figure Description

[0036] Figure 1 To validate the SgRNA expression cassette integration site.

[0037] Figure 2 The uniformity of 1-50 SgRNA abundance in the 150mer plasmid was determined. The number of 150 SgRNAs in UB-150mer was measured. Uniform abundance indicates that the SgRNAs in the library are complete. When the SgRNA abundance is 0 (or less than 100 times the average abundance), it indicates that the sgRNA is missing and the 150mer transgene is incomplete (data on incomplete SgRNAs are not shown in the figure).

[0038] Figure 3 The uniformity of SgRNA abundance in the 150mer plasmid (51-100) was determined. The number of 150 SgRNAs in UB-150mer was measured. Uniform abundance indicates that the SgRNAs in the library are complete. When the SgRNA abundance is 0 (or less than 100 times the average abundance), it indicates that the SgRNA is missing and the 150mer transgene is incomplete (data on incomplete SgRNAs are not shown in the figure).

[0039] Figure 4 The uniformity of SgRNA abundance in the 150mer plasmid was determined by the number of 150 SgRNAs in the UB-150mer plasmid. Uniform abundance indicates that the SgRNAs in the library are complete. When the SgRNA abundance is 0 (or less than 100 times the average abundance), it indicates that the SgRNA is missing and the 150mer transgene is incomplete (data on incomplete SgRNAs are not shown in the figure).

[0040] Figure 5 For the detection of methylation of the U6 promoter, T in the core region of the U6 promoter indicates no methylation.

[0041] Figure 6 The figure shows the library recombination efficiency after tamoxifen induction. The figure shows the relative abundance of 1-50S gRNA, and the library recombination is relatively uniform (the difference is less than 6-fold).

[0042] Figure 7The figure shows the library recombination efficiency after tamoxifen induction, with the relative abundance of 51-100S gRNAs shown in the figure. The library recombination was relatively uniform (difference less than 6-fold).

[0043] Figure 8 The figure shows the library recombination efficiency after tamoxifen induction. The figure shows the relative abundance of 101-150S gRNA, and the library recombination is relatively uniform (the difference is less than 6-fold).

[0044] Figure 9 Figure 1-50 shows the abundance of SgRNA in a library after tamoxifen induction in three-positive mice. The first bar in the figure shows the relative abundance of Pol2 SgRNA, which is significantly lower than that on the right, indicating that cell death and decreased abundance occurred after Pol2 was knocked out.

[0045] Figure 10 The abundance analysis of SgRNA in the library (51-100) is shown in the figure for tamoxifen-induced sg RNA in triple-positive mice.

[0046] Figure 11 The abundance analysis of SgRNA in the library (101-150) is shown in the figure for tamoxifen-induced sg RNA in triple-positive mice.

[0047] Figure 12 for G2e3, Gbp4, FbXo7, Rnf122, Fbxw5, Trim30d, Anrdcd, Mul1, Phf23, Tes3-ps, Bex3, Mycbp2, Zzef1, Rnf181, Cul4a, Coq10a, Rassf1, Mfsd11, Nlr c3, KIh22, Spsbd, Ubri, Pdcd6, Rnf166, Rnf138, Mkrn2, Prkn, Rnf182, Dda1, Ube2ql1, MsI2, Fbh1, Trim12c, Ccn, Pja2, viylip, Rchy1, Tspy15, U Abundance heatmap of be2w, Fbxo33, Rnf185, Rnf125, Mkrn1, PIpp6, Fancb, Trim34b, Herc6, Cbfb, Pcgf5, RuSC1, Dzip3, Nedd4, Zswim8, Ubac1, Asb12, Ube20, Rnf152, FbxI7, Triml1, Pcmtd1, Ivnsiabp, Asb11, Gent7, Wsb1, Rnf38, Trim43a, Spry2, Mfsd7a, Ube3b, Txnrd3, Rnf167, Trim14, and Rassf5 in 29 tissues.

[0048] Figure 13 for Enox1Gfod2, Mfng, SIc25a43, Riox1, WwP2, KIhl18, NC35, Rnf141, Rlim, Pir, NC29, NC28, NC31, NC32, NC34, NC30, NC36, NC33, Lztr1, Nedd4l, NXn, Park7, Ugt3a1, Rnf220, D7Erd443e, Comtd1, Rnf123, Rnf10, Satl1, KIhl42, Dtx2, Trim41, Ube2cbp, Zc4h2, Trim7, Herc3, Kctd3, Hace1, GV9, Septi Abundance heatmap of n4, PIpp4, Trim52, Gold4, Mccc1, KIhI7, Trim11, Obi1, Kbtbd7, Ash2, Anapc16, Ube2d1, Nat8f1, ApOC4, Acod1, Rbbp6, Med17, Med31, Ube2d3, Rnf113a, Ube2v2, Pinx1, Med27, Fh1, FbxI5, Ube2h, Rnf41, Traio, Hamp2, Med20, Arih1, Anapc11, Anapc2, Gtpbp4, POL2-4, Ube292, Dnajb2 in 29 tissues.

[0049] Figure 14 A gel image for genotyping of three-positive mice. Detailed Implementation

[0050] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0052] Example 1: Construction of plasmids for screening libraries of ubiquitination-modifying enzyme genes in vivo 1. Plasmid construction Construction of 1mer plasmids: 150 target sg sequences were synthesized using forward and reverse primers. Primer annealing was then used to form short sg double-stranded fragments with sticky ends. The sgRNA sequences and corresponding genes are shown in Table 2. The detailed annealing procedure is as follows: 15 μl of F / R-primer was thoroughly mixed with 30 μl of 2× buffer. First, the mixture was kept at 95℃ for 5 min, followed by 70 cycles. In each cycle, the temperature was first maintained at 95℃ for 35 s, then decreased by 1℃. After 70 cycles, the temperature was lowered to 25℃, and the product was stored at 25℃ for later use. The single sg vector was digested with enzymes to generate linear plasmid vectors with sticky ends complementary to the short sg double-stranded fragments. The enzyme used was Esp3I, and the reaction conditions were 37℃ for 40 min. The linear vector and the short sg fragments were mixed at a 1:10 ratio and incubated with T4 ligase at 37℃ for 40 min. The product is then transformed.

[0053] Table 2. sg sequence list

[0054] Using 15mer units as units, ten 15mer plasmids were constructed into a single 150mer plasmid. Specifically, the 150 correctly sequenced 1mer plasmids were divided into ten portions, and each portion of 15 1mer plasmids was ligated using the NEB GoldenGate kit. The reaction conditions were 37℃ for 5 min, 16℃ for 5 min, 30 cycles, and a final 60℃ for 5 min. Transformation was then performed after the reaction. The ten 15mer plasmids and the P101 vector plasmid (containing one sgRNA) were ligated into a 151mer plasmid using the NEB GoldenGate kit. The reaction conditions were the same as above. Electroporation was then performed using Takara E. coli HST08 Premium Electro-Cells after the reaction.

[0055] 2. Plasmid transformation Thaw competent cells, take 10 μl of competent cells, add 1 ng of the target plasmid or 1 μl of recombinant plasmid reaction solution, and gently pipette to mix thoroughly. Then incubate the mixture on ice for 30 min. Perform heat shock treatment, preheating the water bath to 42℃ before transformation. Recover, add 160 μl of antibiotic-free liquid LB medium or SOC liquid medium to the cooled bacterial culture. Preheat the shaker to 37℃ and set the rotation speed to 220 rpm. Place the tube containing the bacterial culture in the shaker and shake for 30-60 min. Spread culture: Pipette 80 μl of the recovered bacterial culture and transfer it to a pre-prepared solid culture dish containing the appropriate antibiotic. Spread the culture evenly using an autoclaved spreader. Pick clones: After overnight culture, add approximately 5 ml of LB liquid medium containing the appropriate antibiotic to each shake tube. The selected bacterial clones were inoculated into a shaker tube, labeled with plasmid information, and then cultured in a shaker at 37°C with a rotation speed of 220 rpm.

[0056] 3. Plasmid extraction (1) Bacterial centrifugation collection: Transfer 5 ml of overnight culture to a suitable centrifuge tube. Balance the centrifuge tube and centrifuge at 4000 rpm for 5-10 min. After centrifugation, carefully pour the supernatant culture medium into the waste container, then invert the centrifuge tube onto a clean paper towel to allow any residual culture medium to flow down and be thoroughly absorbed, reducing the amount of residual culture medium in the tube.

[0057] (2) Bacterial resuscitation: Before the experiment, add the RNase provided in the kit to Buffer P1, mix well, and store at 4°C. Add 250 μl of Buffer P1 to the centrifuge tube containing the bacterial precipitate, and gently pipette or vortex to thoroughly mix the bacterial precipitate with Buffer P1. Resuspend the precipitate completely until there are no visible clumps. Incomplete resuscitation will result in incomplete bacterial lysis, reducing plasmid yield and quality.

[0058] (3) Bacterial lysis: Add 250 μl of Buffer P2 to the resuspended bacterial solution, and gently invert the centrifuge tube to ensure thorough mixing of the bacterial solution and Buffer P2. Avoid vigorous shaking or vortexing, as this can cause bacterial genomic DNA breaks, resulting in genomic fragments being incorporated into the final plasmid product and reducing plasmid purity. After adding Buffer P2, the fully lysed bacterial solution will gradually become viscous and clear. The lysis step needs to be completed as quickly as possible within 5 minutes; otherwise, plasmid damage will occur, reducing the yield. If the solution remains cloudy, the possible causes are too much bacterial solution or too little Buffer P2. Adjust the amount of bacterial solution or Buffer P2 accordingly.

[0059] (4) Neutralization reaction: Add 350 μl of Buffer P3 to the lysis buffer, and gently invert the centrifuge tube to fully neutralize Buffer P3 with Buffer P2. A white flocculent precipitate will appear in the completely neutralized solution. Place the centrifuge tube in a centrifuge and centrifuge at 13,000 rpm for 10 min to collect the flocculent precipitate at the bottom of the centrifuge tube.

[0060] (5) Supernatant Transfer: Place the DNA adsorption column provided with the kit into the collection tube. Use a pipette to transfer the supernatant from step 4 into the adsorption column, being careful not to aspirate the precipitate at the bottom. Then, centrifuge at 13,000 rpm for 30-60 s. After centrifugation, discard the liquid in the collection tube and return the adsorption column to the collection tube. The DNA adsorption column should be conditioned before use to activate its adsorption activity.

[0061] (6) Washing: Add 600 μl of washing buffer W to the DNA adsorption column, centrifuge at 13,000 rpm for 30 s, discard the liquid in the collection tube after centrifugation, and then put the adsorption column back into the collection tube.

[0062] (7) Repeat washing: Follow the operation procedure in step 6 to wash the adsorption column again to ensure that the adsorption column is thoroughly cleaned.

[0063] (8) Drying: Put the adsorption column back into the collection tube and centrifuge at 13,000 rpm for 1 min to completely remove any residual washing solution in the adsorption column.

[0064] (9) DNA elution: Transfer the DNA adsorption column to a sterile 1.5 ml centrifuge tube, add 50 μl of Elution Buffer to the center of the adsorption membrane, cap and let stand at room temperature for 2 min to ensure that the Elution Buffer fully wets the adsorption membrane and that the plasmid is fully dissolved in the Elution Buffer. Then centrifuge at 13,000 rpm for 1 min to elute and collect the DNA.

[0065] (10) Storage: Discard the adsorption column and store the eluted plasmid solution in a -20°C freezer to prevent plasmid DNA degradation.

[0066] 4. Plasmid sequencing

[0067] Example 2: Construction of a Mouse Library The 151mer plasmid constructed in Example 1 was used for microinjection of fertilized eggs by Southern Model Biotechnology Co., Ltd. (hereinafter referred to as Southern Model Biotechnology). Subsequently, the plasmid was mated with Cas9 and UBC-CreERT2 mice to obtain triple-positive mice.

[0068] 1. Genotyping of library mice The primers for PCR were prepared in 20 μl volumes, as shown in Table 3.

[0069] Table 3. Primer configuration sequences for PCR

[0070] The PCR reaction system is shown in Table 4. Table 4. PCR reaction system

[0071] The reaction conditions are shown in Table 5: Table 5. Reaction Condition Parameters

[0072] Mouse genotype identification results are shown in Figure 14 In the gel electrophoresis, the 100bp band corresponds to library positivity; 200bp corresponds to Ubc-Cre positivity; the 300bp band represents the presence of CAG-Cas9 on at least one chromosome; and the 600bp band represents the absence of the CAG-Cas9 transgene on at least one chromosome. Subsequent experiments were conducted using triple-positive mice. Therefore, during the IMAP150 mouse passage, we only retained triple-positive mice and culled mice of other genotypes.

[0073] 2. Identification of the integrity of the mouse library During the breeding of mice carrying libraries, library stability can be challenged by various factors. Common problems include the incorporation of short libraries during the library integration stage and library fragmentation during mouse passage.

[0074] To accurately verify whether a 300-sgRNA library in mice can be stably inherited, we plan to use NGS sequencing to test the library's integrity. In this test, NGS library construction employed a two-round PCR amplification method to obtain the amplicon library for sequencing. The primer information used in the library construction process is as follows: Table 6. Primer sequences for one round of PCR:

[0075] Table 7. Primer sequences for second-round PCR:

[0076] The results showed that the number of 150 SgRNAs was uniform (see...). Figure 2-4 This indicates that the SgRNA in the library is intact and without any deletions.

[0077] 3. Detection of methylation status in the core region of the U6 promoter The following is a detailed procedure for using the Beyotime DNA Bisulfite Conversion Kit (D0068M): (1) Sample preparation: Take 500 ng of mouse genomic DNA (ensuring the volume does not exceed 10 μl) into an eight-tube strip. Make up the volume to 10 μl with ultrapure water, then add 65 μl of the prepared conversion buffer. Gently pipette to mix the solution thoroughly.

[0078] (2) Place the sample in a PCR instrument and react under the following conditions: Table 8. Reaction Condition Parameters

[0079] (3) Magnetic bead sorting: 1) After the PCR reaction is complete, add 150 μl of DNA sorting magnetic beads (hereinafter referred to as magnetic beads) to the reaction solution to recover product fragments. After adding the magnetic beads, gently mix twice using a pipette to ensure that the DNA can fully adhere to the surface of the magnetic beads. This step requires incubation for 5 min.

[0080] 2) Carefully place the eight tubes on the magnetic rack and let them stand for a moment until the magnetic beads are completely attached to the magnets on the magnetic rack. Then carefully discard the supernatant.

[0081] 3) Remove the eight-tube strip from the magnetic rack and add 200 μl of 80% ethanol solution. Gently pipette the magnetic beads to suspend them in the ethanol solution. Then place the eight-tube strip back on the magnetic rack and wait until they are completely attached to the magnet. Discard the supernatant ethanol solution.

[0082] 4) Repeat step 3 to ensure the magnetic beads are thoroughly cleaned.

[0083] 5) Allow the eight-tube strip to air dry at room temperature for about 5 minutes, until the surface of the magnetic beads is dry. The best result is when the surface of the dried magnetic beads is non-reflective and free of cracks.

[0084] 6) After the magnetic beads have dried, remove the eight-tube set from the magnetic rack, add 40 μl of DEPC water to the eight-tube set and mix thoroughly with the magnetic beads, then let it stand for 2 min. After that, place the eight-tube set back on the magnetic rack, and after the magnetic beads have adhered to the tube wall, carefully aspirate the supernatant into a new eight-tube set for subsequent experiments.

[0085] (4) The methylation PCR reaction was performed using a two-round PCR amplification method, and the specific operation was as follows: 1) First round of PCR: Amplification was performed using KOD polymerase (TOYOBO, KMM201). Take 4 μl of the supernatant obtained in step 3 and add it to the reaction system to prepare a total reaction volume of 20 μl.

[0086] 2) Second round PCR: Amplification was performed using Taq polymerase. Take 1 μl of the product from the first round PCR and add it to the reaction system to prepare a total reaction volume of 40 μl.

[0087] (5) The following is the primer information used in the two rounds of PCR reactions: Table 9. Primer sequences used in PCR reactions

[0088] Sequencing analysis: The products obtained from the second round of PCR were sent to Sanger sequencing (hereinafter referred to as Sangon Biotech) for sequencing to obtain sequence methylation information. The results showed no methylation (T) (see...). Figure 5 This indicates that the promoter can normally drive SgRNA expression.

[0089] 4. Identification of library integration sites The specific steps are as follows: (1) Take 100 ng of rat tail genome and place it in a 10 µl PCR reaction system containing 0.5 µl of TaqI endonuclease. Then incubate at 37°C for 1 h. After incubation, place the reaction system at 80°C to inactivate TaqI.

[0090] (2) Accurately take 1 µl from the above-mentioned inactivated product, add 0.4 µl of T4 ligase, and prepare a 10 µl system. Perform the ligation reaction at room temperature for 2 h.

[0091] (3) After the ligation reaction is completed, 1 µl of the ligation product is used for PCR identification of the 5' insertion site. During the first round of PCR amplification, KOD polymerase (TOYOBO, KMM201) is used, and 4 nM dUTP and 2 nM MgCl2 are added to the reaction system. UDG is also added to prevent nonspecific amplification.

[0092] (4) After amplification, take 1 µl of the product and dilute it 100 times with sterile water. Then perform a second round of PCR amplification, using Taq enzyme and adding dUTP.

[0093] (5) After completing two rounds of PCR amplification, the final product is sent to a professional sequencing institution for sequencing. After sequencing, the sequencing results are analyzed using NCBI's Blast tool to determine the specific insertion site of the transgene in the genome.

[0094] The primer sequences for the first and second rounds of PCR are as follows: Table 10. Primer sequences for PCR

[0095] Integration site identification results: The UB-150mer library was inserted at position 110316697 and 1103166 on mouse chromosome 2. The specific primer verification band size was 321 bp. Figure 1 ).

[0096] Example 3: Screening of ubiquitination-modified genes 1. Tamoxifen administration Approximately 14 days after birth, when iMAP-UB mice weighed 6 g or more, tamoxifen was administered via gavage. The specific administration regimen involved gavage on days 1, 3, and 5, with each dose of tamoxifen calculated at 4 μl / g (drug concentration of 20 mg / ml).

[0097] Because tamoxifen is toxic to mice, their weight must be closely monitored throughout the administration period. If a mouse's weight decreases by more than 0.5 g, administration should be stopped immediately, and the mouse's weight trend should be continuously monitored. Once the mouse's weight resumes its normal increase, the remaining dose of tamoxifen should be administered according to the original regimen to ensure the accuracy and effectiveness of the experimental treatment.

[0098] After induction, the efficiency of library recombination was verified, and the results showed that library recombination could occur after oral administration of 75 mg / kg tam (see [link to original text]). Figure 6-8 ); 14 days later, the abundance of the housekeeping gene Pol2 was measured, and a significant decrease was found (see Figure 9-11 The result indicates that cell death occurred after Pol2 knockout, proving that the library can be screened normally.

[0099] 2. Construction of MC38 tumor transplantation model After completing the construction of the IMAP150 mouse model, the next step is to establish the MC38 subcutaneous xenograft model.

[0100] 1) MC38 cell culture Passaged or revived MC38 cells (Nanjing Kebai MC-38 (small / rat colorectal adenocarcinoma cells) CBP60825) were seeded in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin and then cultured in an incubator at 37°C with 5% carbon dioxide. The cell growth status was observed regularly every day.

[0101] Cell passage: Cell passage should be performed when the cells have reached 75%-95% confluence. Carefully aspirate the old culture medium into a waste container using a pipette, and wash the cells twice with preheated PBS (37°C). Add preheated trypsin digestion solution to the culture dish, ensuring the trypsin solution evenly covers the cell layer. Place the culture dish in a 37°C incubator to digest the cells for approximately 1 minute. After 1 minute, observe under a microscope. If most cells are round and some cells begin to detach from the bottom of the culture dish, digestion is complete. Add serum-containing culture medium to the culture dish to stop the trypsin digestion. Then, gently pipette the culture dish to allow all cells to detach from the bottom. Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 1500 g for 3 minutes.

[0102] After centrifugation, carefully aspirate the supernatant culture medium into the waste liquid container, add an appropriate amount of 1×PBS to the remaining cell pellet for resuspending, and use the resuspended cell suspension for subsequent experiments.

[0103] 2) Construction of a mouse MC38 subcutaneous tumor model iMAP-UB mice were subcutaneously inoculated with MC38 xenografts, as detailed below: Seven-week-old iMAP-UB mice that had already received tamoxifen treatment were selected. First, the hair on the upper right forelimb of the mice was carefully removed using a shaver, taking care to avoid damaging the mouse's skin as much as possible to ensure the smooth execution of subsequent injections and reduce the risk of infection.

[0104] Prepare a 1 ml syringe and draw 100 μl of cell suspension (cell concentration 1×10⁻⁶). 7 / ml). After properly securing the mouse, gently and slowly inject the cell suspension subcutaneously into the shaved area of ​​the mouse. During the injection, pay special attention to the injection site to ensure that the cell suspension is injected only into the subcutaneous tissue and prevent accidental injection into the muscle, so as not to affect the normal growth of the tumor or cause other unpredictable results.

[0105] After injection, the growth of the mouse tumors needs to be monitored regularly. The size of the tumors should be recorded in detail, using tools such as calipers for precise measurement. At the same time, the health status of the mice should be closely monitored, including their mental state, diet, and activity level.

[0106] When the tumor diameter reached 1.5 cm, the mice were euthanized by cervical dislocation in accordance with experimental procedures in order to conduct subsequent related experiments.

[0107] 3. Flow cytometry detection and sorting: To collect immune cells for subsequent NGS sequencing, flow cytometry is required to sort immune cells from tumor tissue, bone marrow, and spleen. The specific procedure is as follows: 1) Detection of tumor tissue (1) After mice were euthanized by cervical dislocation, tumor blocks were carefully cut from the edge of the tumor tissue on their backs. Subsequently, the connective tissue attached to the tumor blocks was carefully removed to reduce interference from non-tumor cells. Afterward, the processed tumor blocks were washed once in 1×DPBS to remove surface impurities and bloodstains.

[0108] (2) Prepare 5 ml of 1× digestive enzyme solution. Specifically, add 50 μl of Collagenase D (100 mg / ml), 50 μl of Hyaluronidase (10 mg / ml), and 50 μl of Dnase I (5 mg / ml) to 5 ml of RPMI-1640 medium and mix thoroughly.

[0109] (3) Take 1 ml of the prepared 1× digestive enzyme and place it in an EP tube. Put the previously cleaned tumor block into the digestive enzyme in the EP tube and cut the tumor block into small pieces with scissors. After cutting, transfer it to a container containing 5 ml of 1× digestive enzyme and add an appropriate amount of sterile steel beads. Place the container on a 37°C rotary digester and perform digestion for 30 min to fully disperse the tumor tissue into single cells.

[0110] (4) At the same time, antibody preparation can begin. Generally, it is done at a rate of 10... 6 The solution was prepared by adding 0.5 μl of antibody to 100 μl of the solution for each cell.

[0111] (5) Take a 50 ml centrifuge tube and rinse it once with 1×PBS buffer containing 2% FBS (hereinafter referred to as buffer). This is to prevent cells from adhering to the centrifuge tube wall. Filter the digested cell suspension through a 70 μm filter to remove undigested tissue clumps. If there are undigested cells, they can be ground with a grinding stick while adding buffer to promote cell dispersion.

[0112] (6) Adjust the volume of the filtered and ground cell suspension to 50 ml with 1×DPBS, then centrifuge at 300×g for 10 min. After centrifugation, carefully remove the supernatant.

[0113] (7) Add an appropriate amount of buffer (about 5 ml) to the centrifuged cell pellet and gently pipette to resuspend the cells. Filter the resuspended cells again through a 70 μm filter to remove any remaining cell clumps. Then, add 1×DPBS to bring the cell suspension to 50 ml, centrifuge at 300×g for 10 min, and then discard the supernatant into a waste container.

[0114] (8) Resuspend the cells in an appropriate volume of buffer solution according to the needs of subsequent experiments. Add the previously prepared antibody mixture to the cell suspension, gently shake or gently pipette several times to mix it thoroughly, place it on ice and incubate in a dark environment for 15-30 min to allow the antibody to fully bind to the antigen.

[0115] (9) Add 1 ml of 1×PBS to the cell suspension after incubation, then centrifuge at 300×g for 10 min and remove the supernatant. Finally, resuspend the cells in 400 μl of buffer, aspirate the cell suspension into a pre-wetted flow cytometry tube, and wait for the flow cytometry results.

[0116] The staining schemes for sorting immune cells in tumor tissue are shown in Table 11.

[0117] Table 11. Staining protocols for immune cell sorting

[0118] 4. Extraction of genomes from various tissues and organs throughout the body (1) Anatomy of tissues and organs: iMAP-UB mice, euthanized by cervical dislocation, were placed on a dissection table. Using surgical instruments, the mice's internal organs and tissues were meticulously dissected. Strict aseptic techniques were followed throughout the dissection process to ensure the obtained tissues remained uncontaminated. For each organ and tissue, a small piece, approximately the size of a grain of rice, was removed using a sharp scalpel. The removed organs and tissues covered a wide range, including the spleen, small intestine, lungs, stomach, esophagus, liver, kidneys, colon, muscles, white fat, brown fat, lymph nodes, sex organs (testes, epididymis, and prostate in males; ovaries, fallopian tubes, and uterus in females), brain, cerebellum, thyroid gland, bone marrow, and thymus.

[0119] The separated tissue blocks were quickly placed in a preheated metal bath at 90°C to inactivate endogenous nucleases. This step is crucial as it effectively prevents DNA degradation by nucleases, ensuring the integrity of the DNA in subsequent experiments.

[0120] (2) Tissue digestion and lysis: Carefully transfer the heat-treated tissue blocks into the corresponding wells of a 96-well deep-well plate. Accurately add 500 μl of rat tail lysis buffer and 10 μl of proteinase K using a pipette. After addition, thoroughly mix the tissue blocks, lysis buffer, and proteinase K by gentle agitation or repeated pipetting. Place the 96-well plate on a 55°C rotary table for overnight digestion. Ideally, a fully digested solution should be clear, viscous, and homogeneous, without any noticeable lumps. This indicates that the tissue has been adequately digested and intracellular DNA has been effectively released.

[0121] (3) Extraction of genomic DNA from tissues: DNA adsorption using magnetic beads: Accurately pipette 300 µL of digestion solution from a 96-well deep-plate into a 1.5 ml EP tube. Next, add 150 µL of DNA sorting magnetic beads (hereinafter referred to as magnetic beads). Within 5 minutes of adding the magnetic beads, gently pipette twice to ensure the DNA binds sufficiently to the beads. Then, place the EP tube stably on a magnetic rack and let it stand for a few moments until the DNA has completely adhered to the beads. Carefully discard the supernatant.

[0122] Magnetic bead cleaning: Remove the EP tube from the magnetic rack and add 500 μl of 80% ethanol solution. Gently pipette to suspend the magnetic beads in the ethanol solution, cleaning any impurities adhering to their surface. Then, place the EP tube back on the magnetic rack. Once the beads have adhered to the tube wall, discard the supernatant. Repeat this cleaning step once to ensure the beads are thoroughly cleaned.

[0123] Magnetic bead drying and DNA elution: After cleaning, the EP tubes were allowed to air dry at room temperature for about 5 minutes, until the surface of the magnetic beads was completely dry. After the magnetic beads were dry, 80 μl of DEPC water was added to the tubes, and the mixture was allowed to stand for 2 minutes to allow the magnetic beads to be fully suspended in the water, promoting the elution of DNA from the magnetic beads. The EP tubes were then placed back on the magnetic rack, and after the magnetic beads adhered to the tube walls, the supernatant was carefully aspirated and transferred to new eight-tube sets.

[0124] DNA quality testing: The OD values ​​of the DNA solutions transferred to the eight-tube strips were measured using a spectrophotometer. The purity of the DNA was assessed by the ratios of OD260 / OD280 and OD260 / OD230. Simultaneously, the integrity of the DNA was detected by 1% agarose gel electrophoresis. The clarity and size of the electrophoretic bands indicated whether DNA degradation or breakage had occurred.

[0125] 5. NGS Library Construction and Analysis To analyze the abundance of SgRNA at the P0 site after recombination, we performed NGS library construction using PCR. Three rounds of PCR reactions were conducted using previously extracted genomic DNA as a template.

[0126] First-round PCR reaction: A reaction system with a total volume of 80 μl was prepared using 2×Rapid Taq Master Mix (Novizan). To ensure good library complexity and integrity, 1000 ng of DNA template was added to the system.

[0127] Second round PCR reaction: Accurately aspirate 1 μl of the product from the first round PCR, add 2×Rapid Taq MasterMix, and prepare a reaction system with a total volume of 20 μl for amplification.

[0128] Final round of PCR reaction: Take 1 μl of the product from the second round of PCR, add 2×Rapid Taq Master Mix, and prepare a 20 μl reaction system for further amplification.

[0129] The following are the primer information used in each round of PCR reactions, Tables 12-14.

[0130] Table 12 Primer sequences for one round of PCR

[0131] Table 13 Primer sequences for second-round PCR:

[0132] Table 14. Primer sequences for three rounds of PCR

[0133] The NGS analysis followed the standard procedure for Geneplus's amplicon sequencing service: different samples were assigned different barcode sequences, followed by library construction and sequencing. After obtaining the raw sequencing data, FASTP was used for quality control. Once quality control was successful, CutAdapt software was used to separate the samples based on the barcode information. MAGECK software was then used to analyze the abundance of all SgRNAs in each sample, obtaining the proliferation or deletion phenotypes of 140 SgRNAs and 10 NC SgRNAs after knockout in 19 different tissues and cells (see...). Figure 12-13 ), and successfully identified 38 key regulatory genes for tumor immunity.

[0134] Example of effect We screened for the Anapc2 enzyme, and the results showed that knocking out this gene increased cell death. In the previous study "The anaphase-promoting complex / cyclosome subunit APC / C-Cdh1 is required for the G2 / M transition", knocking down ANAPC2 expression through RNA interference (RNAi) or knocking out ANAPC2 using CRISPR / Cas9 technology directly led to mitotic arrest, abnormal chromosome alignment and separation, and ultimately cell death through apoptosis pathways such as Caspase-3.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A system for screening ubiquitination-modifying enzyme genes in vivo, characterized in that, The system includes: (a) SgRNA expression cassette: encoding 150 tandemly arranged SgRNA expression units, which target 140 ubiquitination-modifying enzyme genes and 10 control genes, respectively. The SgRNA sequences are shown in SEQ ID NO.1-150. (b) Cas9 nuclease expression element; (c) Elements that can be induced to express Cre recombinase.

2. The system according to claim 1, characterized in that, The SgRNA expression cassette, from 5' to 3', contains: a promoter, two unidirectional loxP TC9 recombination sites, a transcription termination sequence located between the loxP TC9 sites, and 150 tandemly arranged SgRNA expression units, the loxP sequence being shown in SEQ ID NO.151-152.

3. A method for constructing a transgenic mouse model for in vivo screening of ubiquitination-modifying enzyme genes, characterized in that, The construction method uses the system described in any one of claims 1-2.

4. The construction method according to claim 3, characterized in that, The construction method includes the following steps: S1. Assemble 150 SgRNAs with sequences as shown in SEQ ID NO.1-150 into an SgRNA expression cassette; S2. The SgRNA expression cassette is integrated into the fertilized egg via embryo microinjection, and a stably inherited transgenic animal is obtained; S3. The transgenic animal is crossed with Cas9-expressing animals and UBC-CreERT2-expressing animals to obtain a triple-positive screening model that can simultaneously knock out 150 genes in vivo.

5. The construction method according to claim 4, characterized in that, The SgRNA expression cassette was integrated into the mouse chromosome 2 between positions 110316697 and 110316698 via embryo microinjection.

6. The application of the mouse model obtained by the construction method according to any one of claims 3-5 in in vivo screening of ubiquitination-modifying enzyme genes and screening of tumor immune targets.

7. The application according to claim 6, characterized in that, Includes the following steps: (1) Inducing the expression of SgRNA in target cells in mouse models; (2) Establishing an in vivo screening environment by inoculating MC38 tumor cells; (3) Collect samples after the screening period ends; (4) Extract genomic DNA from the sample and construct an NGS library; (5) Sequencing analysis of the relative abundance of each SgRNA, and screening based on the relative abundance of each SgRNA.

8. The application according to claim 7, characterized in that, In step (1), the induction is performed using tamoxifen.

9. The application according to claim 7, characterized in that, In step (3), the sample includes any one or more of the following: tumor, bone marrow, spleen, small intestine, lung, liver, kidney, colon, muscle, white fat, brown fat, lymph nodes, sex organs, brain, cerebellum, thyroid gland, and thymus.

10. The application according to claim 7, characterized in that, In step (5), the high abundance of SgRNA indicates that the knockout of the target ubiquitination modifying enzyme gene gives the cell a positive selection advantage, suggesting that the enzyme has a negative regulation of proliferation or a tumor suppressor-like function; the low abundance indicates that the knockout of the target ubiquitination modifying enzyme gene leads to negative selection or cell lethality, suggesting that the enzyme is an essential gene for cell survival and / or proliferation.

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