High-efficiency enriched anti-blue ear and high-fecundity edited pig donor cells as well as preparation method and application thereof

Abstract

The invention discloses a method for efficiently enriching anti-blue ear and porcine reproductive capacity edited pig donor cells. The method comprises the following steps: introducing CRISPR-Cas9 plasmids targeting a BMP15 gene, CRISPR-Cas9 plasmids targeting a CD163 gene and alternative reporter plasmids edited by double genes into the pig donor cells; culturing the porcine donor cells to enable gene editing and reporter gene expression to occur; detecting and sorting cells with positive report gene expression so as to obtain enriched double-gene editing donor cells; the alternative reporter plasmid for double-gene editing comprises the following elements which are connected in sequence: a recognition sequence of a BMP15 gene editing target, a transcription terminator sequence, a recognition sequence of a CD163 gene editing target and a fluorescent reporter gene sequence. According to the invention, the alternative reporter plasmid for BMP15 and CD163 double-gene editing is constructed, and the editing condition of target sites of the BMP15 and CD163 genes of a cell genome can be converted into a detectable red fluorescence phenotype, so that the enrichment efficiency of anti-blue ear high-fecundity edited pig donor cells is improved, and the double-gene editing efficiency of the cells is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering biotechnology, specifically relating to a method for efficiently enriching high-fertility edited pig donor cells resistant to porcine reproductive and respiratory syndrome (PRRS), its preparation method, and its application. Background Technology

[0002] In the process of preparing dual-gene-edited pig models for xenotransplantation or biomedical research, obtaining high-quality, precisely edited donor cells is a crucial first step. Currently, the mainstream technical approach in this field uses primary porcine cells (such as fetal fibroblasts) as starting material, and achieves simultaneous editing of two gene loci by transfecting plasmid vectors containing the CRISPR-Cas9 system and two specific guide RNAs (gRNAs). Specifically, primary porcine cells are typically isolated and cultured first. Once the cells are stable, two plasmids, one carrying a Cas9 protein expression cassette and the other a specific gRNA sequence, are introduced into the cells using techniques such as liposome encapsulation transfection or electroporation transfection. After transfection, the cells are cultured in an incubator for a certain period to allow the CRISPR-Cas9 system to fully express and complete gene cutting and editing. Subsequently, these processed cells are collected as donor cell nuclei for subsequent somatic cell nuclear transfer (SCNT), with the ultimate goal of obtaining genotype-defined dual-gene-edited pigs.

[0003] However, this seemingly clear experimental approach still faces a series of technical bottlenecks in practice, often resulting in unsatisfactory efficiency in obtaining ideal donor cells. The most prominent problem is the generally low proportion of cells with simultaneous dual-gene editing. This is mainly due to the following factors: First, cell transfection efficiency is inherently difficult to achieve 100%. Whether using liposomes or electroporation, a certain percentage of cells fail to successfully introduce the exogenous plasmid, and these cells naturally cannot undergo any editing. Second, even if the plasmid successfully enters the cell, the cleavage activity of the two gRNAs often differs, potentially leading to one gene site being efficiently edited while the other site is edited with low efficiency, thus reducing the probability of dual-editing events. Furthermore, the cell's own DNA repair mechanisms (such as non-homologous end joining or homologous recombination repair) are random, potentially producing unexpected editing results. Simultaneously, primary cells have limited proliferation capacity and are highly sensitive to external transfection stimuli; their survival status also directly affects the editing effect. These factors combine to make it extremely difficult and costly to screen cell clones from a cell population for the desired modifications in both genes (such as biceleste knockout), which is one of the main obstacles to the large-scale application of this technology.

[0004] Therefore, developing more efficient co-transfection strategies, optimizing gRNA design, and improving cell screening methods remain areas that urgently need breakthroughs in this field. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a method for efficiently enriching highly prolific edited porcine donor cells resistant to porcine reproductive and respiratory syndrome (PRRS).

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for efficiently enriching highly prolific edited porcine donor cells resistant to porcine reproductive and respiratory syndrome (PRRS), comprising: S1, Target BMP15 CRISPR-Cas9 plasmids for genes, targeting CD163 The CRISPR-Cas9 plasmid of the gene and the alternative reporter plasmid for dual gene editing were introduced into porcine donor cells; S2. Culture the porcine donor cells to induce gene editing and reporter gene expression; S3. Detect and sort cells that express reporter genes positively to obtain enriched dual-gene editing donor cells; The alternative reporter plasmid for dual gene editing includes the following elements linked in sequence: BMP15 Identification sequences of gene editing targets 、 Transcription terminator sequence, CD163 Identification sequences of gene editing targets and fluorescent reporter gene sequences.

[0008] Preferably, the BMP15 The gene editing target identification sequence is shown in SEQ ID NO: 1. Preferably, the transcription termination sequence is shown in SEQ ID NO: 2. Preferably, the... CD163 The gene editing target identification sequence is shown in SEQ ID NO: 3. Preferably, the fluorescent reporter gene is the DsRed reporter gene.

[0009] Preferably, the DsRed reporter gene sequence is shown in SEQ ID NO: 4. Preferably, the sequence of the dual-gene-edited alternative reporter plasmid is shown in SEQ ID NO: 5.

[0010] Preferably, the target BMP15 The CRISPR-Cas9 plasmid of the gene contains a target BMP15 The sgRNA of the gene has the nucleotide sequence shown in SEQ ID NO: 6.

[0011] Preferably, the target CD163 The CRISPR-Cas9 plasmid of the gene contains a target CD163 The sgRNA of the gene has the nucleotide sequence shown in SEQ ID NO: 7.

[0012] Preferably, the target BMP15 The nucleotide sequence of the CRISPR-Cas9 plasmid of the gene is shown in SEQ ID NO: 8. Preferably, the targeted... CD163 The nucleotide sequence of the CRISPR-Cas9 plasmid of the gene is shown in SEQ ID NO: 9.

[0013] Preferably, in step S1, the plasmid is introduced by transfection.

[0014] Preferably, in step S3, cells expressing fluorescent proteins are sorted by flow cytometry.

[0015] Preferably, the porcine donor cells are porcine testicular cells.

[0016] In a second aspect, the present invention provides an alternative reporter plasmid for dual gene editing, comprising the following elements connected in sequence: BMP15 Identification sequences of gene editing targets 、 Transcription terminator sequence, CD163 Identification sequences of gene editing targets and DsRed reporter gene sequences.

[0017] Thirdly, the present invention provides a kit for enriching dual-gene-edited cells, comprising the dual-gene-editing alternative reporter plasmids described in the present invention.

[0018] Fourthly, this invention provides the application of the aforementioned dual-gene editing alternative reporter plasmid and kit in enriching high-fertility edited pig donor cells resistant to porcine reproductive and respiratory syndrome (PRRS).

[0019] Fifthly, the present invention provides alternative reporter plasmids and kits for dual gene editing for use in breeding gene-edited pigs that are resistant to porcine reproductive and respiratory syndrome (PRRS) and have high fertility.

[0020] Current methods for producing pig donor cells with dual gene editing typically involve transfecting primary cells with CRISPR-Cas9 plasmids. This involves transfecting two CRISPR-Cas9 plasmids into primary pig cells via plasmid transfection or electroporation, and then collecting the cells as donor cells for somatic cell nuclear transfer. However, because the efficiency of genome editing is influenced to some extent by factors such as CRISPR-Cas9 plasmid transfection efficiency and expression levels, as well as gRNA activity, the proportion of donor cells obtained using traditional methods that simultaneously undergo dual gene editing is usually relatively low.

[0021] This technology, based on the principle of targeted deletion of transcription terminator sequences, has developed alternatives. BMP15 , CD163 An alternative report system for dual gene editing. This invention constructs... BMP15, CD163Alternative reporter plasmids for dual-gene editing, capable of processing cellular genomes BMP15, CD163 The editing of gene target sites is converted into a detectable red fluorescent phenotype, thereby improving the enrichment efficiency of donor cells from highly fertile porcine reproductive and respiratory syndrome (PRRS) pigs. The innovation of this alternative reporter system for dual gene editing lies in the reporter plasmid... BMP15, CD163 There is a transcription terminator sequence between the target gene sites, blocking the transcription of the downstream DsRed protein sequence. Only when both genes are bound and cleaved by the Cas9 protein can the intermediate transcription terminator sequence be targeted and deleted, restoring the expression of the downstream DsRed fluorescent protein. Experiments in porcine testicular (Swine Testis, ST) cells have demonstrated this. BMP15, CD163 The dual-gene editing alternative reporter plasmid successfully restored DsRed fluorescent protein expression after editing. Extranuclear alternative reporter plasmids can predict intranuclear genome editing; when both target gene sites of the reporter plasmid are cleaved, the intermediate terminator sequence is removed, and downstream DsRed fluorescent protein is successfully expressed, intranuclear genome editing is also highly probable. Flow cytometry sorting using the red fluorescence phenotype can significantly improve… BMP15, CD163 The enrichment efficiency of dual-gene edited cells effectively improves the dual-gene editing efficiency of cells.

[0022] This invention is the first to use targeted deletion of transcription terminator sequences to restore the expression of downstream DsRed fluorescent reporter genes to enrich dual-gene-edited cells. The corresponding advantage is that, compared with the traditional frameshift mutation blocking strategy, which can only enrich single-gene-edited cells, this technology can effectively enrich dual-gene-edited cells, providing a new strategy for the comprehensive genetic improvement of multiple traits in animals. Attached Figure Description

[0023] Figure 1 show BMP15 and CD163 Gene sgRNA design and in vitro enzyme digestion verification.

[0024] Figure 2 This diagram illustrates the principle of an alternative reporter plasmid for dual gene editing.

[0025] Figure 3 This demonstrates the transfection efficiency of the alternative reporter plasmid for dual gene editing.

[0026] Figure 4 This demonstrates the enrichment effect of dual-gene editing cells using alternative reporter plasmids. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] I. Experimental Methods 1. BMP15, CD163 Gene target site selection and expression vector construction according to BMP15 (Gene ID: 448811) and CD163 Based on the gene sequence (Gene ID: 397031) and the basic design principles of sgRNA in the CRISPR-Cas9 system, and using software analysis (http: / / crispr.mit.edu / ), a target pig was designed. BMP15 and CD163 The specific sequences of the gene's sgRNA are shown in Table 1.

[0031] Table 1. Targets BMP15 and CD163 sgRNA sequence information of the gene

[0032] After synthesizing sgRNA, its activity was demonstrated by in vitro enzyme digestion experiments.

[0033] Construct a Cas9 expression vector, ligate it with T4 ligase via BbsI digestion, and then target the Cas9 expression vector. BMP15 The sgRNA (SEQ ID NO: 6) of the gene was ligated into the pX460 empty vector plasmid to obtain the targeted gene. BMP15 CRISPR-Cas9 plasmid of the gene ( BMP15 Edit plasmid (SEQ ID NO: 8); target CD163 The sgRNA (SEQ ID NO: 7) of the gene was ligated into the pX458 empty vector plasmid to obtain the targeted gene. CD163 CRISPR-Cas9 plasmid of the gene ( CD163 The recombinant expression vector was successfully constructed using a bacterial culture sequencing method (SEQ ID NO: 9). The plasmid was edited.

[0034] Construction of an alternative reporter plasmid for dual gene editing: The DsRed reporter gene sequence (SEQ ID NO: 4) was cloned into the vector pEGFP-N1 (Kanamycin) via 5' AgeI and 3' NotI to construct the plasmid pDsRed-N1. Using recombination methods, the... CD163 Gene sequence, transcription terminator (SEQ ID NO: 3) BMP15 The gene sequence was cloned into the vector pDsRed-N1 (Kanamycin) using 5'NheI and 3'AgeI to construct the plasmid PDsRed- CD163 + BMP15 (Double cut point), i.e., two genes ( CD163 + BMP15 An alternative reporter plasmid edited by [the author], the nucleotide sequence of which is shown in SEQ ID NO: 5.

[0035] The construction and verification of the above-mentioned carriers were outsourced to commercial companies.

[0036] 2. ST cell culture and transfection ST cells (porcine testicular cells) were cultured in DMEM high-glucose medium containing 10% v / v fetal bovine serum in a 37°C, 5% CO2 incubator. Cells were washed twice with PBS solution and the medium was replaced after 24 h. Cells were cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 4 μg / mL in 6-well cell culture plates. Once the cell confluence in the 6-well plates approached 90%, transfection was prepared. During transfection, each well was treated with 4 μg of [a specific drug / method]. BMP15 Editing plasmid, 4 μg CD163 The formula for the edited plasmid and 2 μg of the alternative reporter plasmid for double gene editing was added for transfection, while a no-reporter plasmid group (i.e., transfection only) was set up. BMP15 Editing plasmids and CD163 The plasmid was edited, and positive and blank control groups for each fluorescence were also included. Electroporation transfection parameters were: 1400 V, 10 ms, 3 pulses.

[0037] After transfection, the culture medium was prepared in antibiotic-free DMEM high-glucose medium at 37°C and 5% CO2. After 12 h of transfection, the medium was replaced with penicillin-streptomycin dual-antibiotic DMEM high-glucose medium.

[0038] 3. Fluorescence microscopy imaging Cell fluorescence was observed using an inverted fluorescence microscope at 24 h, 48 h, and 72 h after electroporation transfection to observe the expression level of DsRed fluorescent protein in cell editing plasmids and alternative reporter plasmids.

[0039] 4. Flow cytometry analysis and sorting Adherent ST cells were first digested with trypsin. After digestion was terminated, the cells were resuspended in PBS to form single cells, filtered through a 50 μm nylon membrane into flow cytometry tubes, and analyzed for fluorescence ratio and intensity using excitation light at wavelengths of 561-579 nm in a Beckman MoFlo Astrios EQs flow cytometer. Red fluorescent positive cells were then sorted and collected into 96-well plates. The sorted cells were continuously cultured and passaged, and the genome was extracted.

[0040] 5. Sanger sequencing and TIDE analysis Using Taq PCR StarMix (GenStar), the sgRNA-recognized sites were amplified separately. BMP15 and CD163 The gene fragment was successfully amplified using agarose gel electrophoresis. The PCR products were then subjected to Sanger sequencing and TIDE analysis, which indicates that the alternative reporter system successfully enriched the dual gene-edited cells.

[0041] II. Experimental Results 1. BMP15, CD163 Gene sgRNA design and in vitro enzyme digestion verification This technology was designed specifically for pigs. BMP15, CD163 sgRNA of genes ( Figure 1 (A) The activity of sgRNA was verified by in vitro enzyme digestion.

[0042] PCR amplification was performed using the primers shown in Table 2. BMP15, CD163 Gene fragments were subjected to in vitro enzyme digestion experiments to demonstrate the targeting of... BMP15, CD163 All genetically engineered sgRNAs exhibited high knockout efficiency. Figure 1 (B in the middle).

[0043] Table 2. BMP15 and CD163 Gene primers

[0044] 2. Design of Alternative Dual Gene Editing Reporter Plasmids In the target gene of the alternative reporter plasmid for dual gene editing ( BMP15, CD163 A transcription terminator sequence is introduced between the sites, thereby blocking the transcription of the downstream DsRed fluorescent protein coding sequence and preventing its expression. Only when BMP15 and CD163 When the target sites are bound and cleaved by the Cas9 protein, the intermediate transcription terminator sequence can be targeted and deleted, thereby restoring the expression of the downstream DsRed fluorescent reporter gene. Therefore, dual-gene-edited cells have a red fluorescent phenotype and can be enriched by flow cytometry. Figure 2 (A in the middle).

[0045] Because the editing status of surrogate reporter plasmids is correlated with the editing status of the nuclear genome, this technique can efficiently enrich dual-gene-edited positive cells in the genome. Compared to traditional frameshift mutation blocking strategies, which can only enrich single-gene-edited cells, this technique can effectively enrich dual-gene-edited cells. Figure 2 (B in the middle).

[0046] 3. Validation of the effect of alternative dual gene editing reporter plasmids Fluorescence and flow cytometry data after transfection with the empty DsRed plasmid and the double-gene-edited alternative reporter plasmid showed that the expression of DsRed protein in the alternative reporter plasmid was successfully blocked by the upstream-designed transcription terminator sequence, preliminarily indicating that the design of the transcription terminator was reasonable. Figure 3 (A in the middle).

[0047] co-transfection of ST cells BMP15 , CD163 After the editing plasmid was combined with a double-gene-editing alternative reporter plasmid, the cells showed obvious red fluorescence. This indicates that the reporter plasmid... BMP15 , CD163 When all target sites are cleaved, the transcription terminator sequence in the alternative reporter plasmid is targeted and deleted, and DsRed protein expression is no longer blocked, allowing for normal transcription and translation. Figure 3 (B in the middle).

[0048] This demonstrates that the design of the alternative reporter plasmid is correct and can reflect the response of the Cas9 protein to... BMP15 , CD163 The identification and cleavage of the target site of the gene. Furthermore, in the case of three plasmids (… BMP15 Editing plasmids, CD163 Within 48-72 hours after co-transformation of the edited plasmid and the dual-gene editing alternative reporter plasmid, the red fluorescence produced by expression of the alternative reporter plasmid was the strongest. This may be because the targeted deletion of the transcription terminator sequence on the alternative reporter plasmid occurs later than the expression of the edited plasmid, thus the peak red fluorescence intensity lags behind the peak fluorescence intensity of the edited plasmid itself.

[0049] 4. Detection of enrichment efficiency of dual gene-edited cells using alternative report systems. Genomes were extracted from porcine cells obtained by conventional electroporation and porcine cells sorted using an alternative reporter system with dual gene editing. Taq PCR StarMix (GenStar) was used to amplify genomes containing sites recognized by sgRNAs. BMP15 and CD163 Gene fragments (primer sequences are shown in Table 2), and PCR products were subjected to Sanger sequencing. Figure 4 (A in the middle).

[0050] TIDE analysis of sequencing results confirmed that alternative reporter systems for dual gene editing can effectively improve... BMP15 and CD163 The efficiency of editing two genes simultaneously ( Figure 4 (B in the middle).

[0051] Traditional electroporation methods BMP15 The gene editing efficiency was 6.7%. CD163 The gene editing efficiency was 6.3%, while the flow cytometry analysis showed that the enriched cells after using an alternative reporter system for dual gene editing had a higher efficiency. BMP15 The gene editing efficiency was 36.4%. CD163 The gene editing efficiency was 80.2%, showing a significant increase. This result indicates that the alternative reporter system can successfully enrich dual-gene-edited cells, effectively improving the dual-gene-editing efficiency of cells.

[0052] 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 efficiently enriching highly prolific edited porcine donor cells resistant to porcine reproductive and respiratory syndrome (PRRS), characterized in that, Including the following steps: S1, Target BMP15 CRISPR-Cas9 plasmids for genes, targeting CD163 The CRISPR-Cas9 plasmid of the gene and the alternative reporter plasmid for dual gene editing were introduced into porcine donor cells; S2. Culture the porcine donor cells to induce gene editing and reporter gene expression; S3. Detect and sort cells that express reporter genes positively to obtain enriched dual-gene editing donor cells; The alternative reporter plasmid for dual gene editing includes the following elements linked in sequence: BMP15 Identification sequences of gene editing targets 、 Transcription terminator sequence, CD163 Identification sequences of gene editing targets and DsRed reporter gene sequences.

2. The method according to claim 1, characterized in that, The BMP15 The gene editing target identification sequence is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that, The transcription terminator sequence is shown in SEQ ID NO:

2.

4. The method according to claim 1, characterized in that, The CD163 The gene editing target identification sequence is shown in SEQ ID NO:

3.

5. The method according to claim 1, characterized in that, The DsRed reporter gene sequence is shown in SEQ ID NO:

4.

6. The method according to claim 1, characterized in that, The target BMP15 The CRISPR-Cas9 plasmid of the gene contains a target BMP15 The sgRNA of the gene has the nucleotide sequence shown in SEQ ID NO:

6.

7. The method according to claim 1, characterized in that, The target CD163 The CRISPR-Cas9 plasmid of the gene contains a target BMP15 The sgRNA of the gene has the nucleotide sequence shown in SEQ ID NO:

7.

8. An alternative reporter plasmid for dual gene editing, characterized in that, Includes the following elements connected in sequence: BMP15 Identification sequences of gene editing targets 、 Transcription terminator sequence, CD163 Identification sequences of gene editing targets and DsRed reporter gene sequences.

9. A kit for enriching dual-gene-edited cells, characterized in that, This includes alternative reporter plasmids for dual gene editing as described in claim 8.

10. The application of the dual-gene editing alternative reporter plasmid as described in claim 8 and the kit as described in claim 9 in the enrichment of high-fertility edited porcine donor cells resistant to porcine reproductive and respiratory syndrome (PRRS).

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