Method for constructing FBN1 gene knockout pig somatic cells

Abstract

The invention discloses a method for constructing FBN1 gene knockout pig somatic cells. The method comprises the following steps: 1, constructing a gene targeting vector; 2, verifying the gene knockout efficiency; and 3, screening out an expression vector with a better action effect, transfecting human and pig cells, and knocking out the FBN1 gene. According to the method, two pairs of FBN1 gene target sequences are selected by performing homologous comparison on multiple species, two pairs of DNA double strands with different target sequences, identical expressed proteins and identical BsmBI cohesive ends are synthesized by utilizing a PAM design principle of gRNA, the double strands are connected with pPDNA330 plasmids to obtain a recombinant vector carrying the FBN1 homologous genes of the multiple species, and the recombinant vector is used for preparing the FBN1 homologous genes of the multiple species. The gene knockout efficiency of the recombinant vector is detected by using a fluorescent protein expression method, the vector with higher knockout efficiency is selected to transfect recipient cells of multiple species, gene knockout and verification are performed, and simple, efficient and accurate gene knockout of the FBN1 genes of the multiple species is completed.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for constructing FBN1 gene knockout porcine somatic cells. Background Technology

[0002] Fibrillin-1 (FBN1) is a cysteine-rich macrocell-binding extracellular matrix glycoprotein encoded by the FBN1 gene. As a structural component of microfibrils, it provides load-bearing mechanical support in elastic and inelastic connective tissues. Therefore, mutations in the FBN1 gene can lead to a variety of genetic disorders, such as Marfan syndrome, an autosomal dominant inherited disorder characterized by abnormalities of the eyes, bones, and cardiovascular system.

[0003] Altering gene structure typically involves gene mutation, which is the addition, deletion, or alteration of base pairs in the DNA molecule, resulting in changes to the gene structure. Gene mutations are often undirected, random, and unknown. To improve the efficiency of gene alteration, current molecular biology practices typically involve extracting the FBN1 gene from a specific organism and then using a homologous substitution target gene based on the genome's coding sequence. This causes the FBN1 gene to be replaced or destroyed, thus achieving targeted gene knockout. Targeted knockout offers high precision and is easily screened using morphology analysis, significantly improving efficiency. However, this method can only knock out a single gene in a single species and is not applicable to multiple species.

[0004] Currently, data on the FBN1 gene are relatively complete, and after extensive experiments both domestically and internationally, several FBN1 gene knockout cell lines have been obtained. The meat production performance of livestock and poultry in my country is relatively low, and there is a lack of high-yielding meat-producing livestock and poultry. Research has been conducted using ZFNs technology and TALEN technology to edit the FBN1 gene at specific sites in livestock and poultry to induce mutations, screen for effective gene-mutated cell lines, and test the mutation efficiency of these two different gene-editing technologies. This research aims to provide materials for accelerated screening and meat quality improvement of poultry and livestock in my country, and for improving production.

[0005] Gene editing utilizes ZFNs and TALEN technologies. However, for targeted editing, these technologies require the synthesis of DNA sequence-specific binding protein modules; that is, DNA cleavage relies on the FokI nuclease domain. This requirement significantly limits the development of these two technologies. CRISPR / Cas9 technology, on the other hand, uses gRNA to guide endonucleases to edit DNA at specific sites, overcoming the limitations of ZFNs and TALEN technologies and representing a more efficient gene-specific editing technology.

[0006] Currently, some studies have begun to use the CRISPR / Cas9 system for gene knockout experiments. Experiments have shown that gene knockout using the CRISPR / Cas9 system has the advantages of higher efficiency and simpler operation. However, a recombinant vector that can be used to knock out FBN1 genes in multiple species has not yet been developed, nor has a detection method that can quickly and accurately find the target sequence been found. For example, PCT patent CN 105142669 A discloses CRISPR-based genome modification and regulation, demonstrating that the CRISPR system can be used for genome modification and regulation, and that the CRISPR system is simpler and more efficient than ZFN and TALEN. However, this patent does not disclose specific restriction enzyme sites and base sequences for FBN1 gene knockout, nor does it disclose methods for detecting and selecting preferred fragments of the FBN1 gene, nor does it disclose the construction of the recombinant plasmid vector used for FBN1 gene knockout in this invention. Chinese patent CN 104232669 A discloses a method for constructing a vector based on the fish CRISPR / Cas9 system using gene knockout, but this technical solution can only target FBN1 gene knockout in fish and cannot target other species, resulting in low universality. Furthermore, this technical solution does not disclose a detection method that can quickly and accurately find the target sequence. Therefore, the present invention needs to solve the following problems: (1) Construct a gene editing method that can target and knock out FBN1 genes in multiple species; (2) Existing technologies have low efficiency and low applicability in gene editing processes, especially in human and pig genome editing. The entire platform still needs to be optimized and conditions explored; (3) Existing technologies do not have a detection method that can quickly and accurately find target sequences, thereby further improving the success rate of gene editing. Summary of the Invention

[0007] In view of the above, it is necessary to provide a simple, efficient, accurate, and applicable method for FBN1 gene knockout in multiple species, especially in humans and pigs, and to further improve the detection efficiency of target sequences, enabling rapid and accurate identification of target sequences and improving the success rate of gene editing.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for constructing FBN1 gene knockout porcine somatic cells, the method comprising the following steps: (1) Construction of gene targeting vector, the construction method is as follows: A. Download the nucleotide sequences of FBN1 genes from multiple species from the gene database and perform homology comparison to select two pairs of target sequences, namely FBN1 target sequence 1: 5'-AGGCACTGGTATTTGGCAGAG-3' and FBN1 target sequence 2: 5'-AAGATATAAGGCCAATTACTGCTC-3'. B. Based on the homology comparison results and the PAM design principle of gRNA, two pairs of DNA double strands with different target sequences, but expressing the same protein and having the same BsmBI sticky ends were synthesized and named FBN1-1 and FBN1-2 respectively. C. Digest the pPDNA330 plasmid with BsmBI enzyme, perform agarose gel electrophoresis on the digestion product, and then perform gel extraction and recovery. D. Use ligase to ligate FBN1-1 and FBN1-2 to the BsmBI-digested pPDNA330 plasmid, respectively, to obtain recombinant plasmid vectors, named pPDNA330-FBN1-1 and pPDNA330-FBN1-2, respectively. (2) Verification of gene knockout efficiency: Screening and comparison of knockout efficiency of recombinant plasmid vectors pPDNA330-FBN1-1 and pPDNA330-FBN1-2, and selecting recombinant plasmid vector with higher FBN1 gene knockout efficiency to transfect recipient cells. (3) Screening out expression vectors with better effects, transfecting human and pig cells to knock out the FBN1 gene, transfecting recipient cells of different species, collecting transfected recipient cells, extracting cell genomes for sequencing, synthesizing detection primers based on genome sequencing results for PCR amplification, ligating the amplification product into the pEASY-T1 vector, picking single-clone bacteria, sequencing, and detecting the FBN1 gene knockout expression results.

[0009] Furthermore, the upstream sequence of FBN1-1 is 5'-ACCGCTCTGCCAAATACCAGTGCCT-3', and the downstream sequence is 5'-AAACAGGCACTGGTATTTGGCAGAG-3'; the upstream sequence of FBN1-2 is 5'-ACCGAAGATATAAGGCCAATTACTGCTC-3', and the downstream sequence is 5'-AAACGAGCAGTAATTGGCCTTATATCTT-3'.

[0010] Furthermore, the BsmBI enzyme digestion system is reacted at a temperature of 50-60℃ for 2.5-3.5 h, and the enzyme digestion system consists of: 2.5-3.5 μg of pPDNA330 plasmid, 1.5-2.5 μL of 10×NEB Buffer3, 0.2-0.8 μL of BsmBI, and water added to a solution volume of 15-25 μL.

[0011] Furthermore, the screening and verification of the knockout efficiency of the recombinant plasmid vectors pPDNA330-FBN1-1 and pPDNA330-FBN1-2 were performed using a fluorescent protein expression method, the specific method of which is as follows: (1) Using RGS-CR as a validation reporter vector: The validation reporter vector RGS-CR was linearized using restriction endonucleases EcoRI and BamHI; (2) Synthesize two DNA double strands with EcoRI and BamHI sticky ends corresponding to FBN1 target sequence 1 and FBN1 target sequence 2, and name them as #FBN1-1 and #FBN1-2 respectively. The upstream sequence of the #FBN1-1 primer is: 5'-AATTCCTCTGCCAAATACCAGTGCCTGGGG-3', and the downstream sequence is: 5'-GATCCCCCAGGCACTGGTATTTGGCAGAGG-3'. The upstream sequence of the #FBN1-2 primer is: 5'-AATTCAAAGATATAAGGCCAATTACTGCTCTGGG-3', and the downstream sequence is: 5'-GATCCCCAGAGCAGTAATTGGCCTTATATCTTTG-3'; (3) Using T4 ligase, the RGS-CR linearized product of step (1) and the two pairs of DNA double strands #FBN1-1 and #FBN1-2 with EcoRI and BamHI sticky ends of step (2) were ligated to obtain recombinant plasmid vectors, which were named RGS-#FBN1-1 and RGS-#FBN1-2, respectively. (4) Passaged HEK-293T cells were placed in 4 culture dishes; (5) Take 4 centrifuge tubes and number them 1, 2, 3 and 4. Add DMEM containing FBS to each tube. (6) Then add pPDNA330-FBN1-1 and RGS-#FBN1-1 to tube 1, pPDNA330 and RGS-#FBN1-1 to tube 2, pPDNA330-FBN1-2 and RGS-#FBN1-2 to tube 3, and pPDNA330 and RGS-#FBN1-2 to tube 4. Finally, add Roche transfection reagent to each tube, mix well, let stand at room temperature, add the mixture to culture dishes, and incubate in an incubator. After the culture is completed, observe the fluorescence expression of the target gene under a fluorescence microscope.

[0012] Furthermore, the restriction endonuclease EcoRI and BamHI digestion system is performed at a temperature of 35-40℃ for 2.5-3.5 h. The digestion system consists of 1.5-2.5 μg of RGS plasmid, 1.5-2.5 μL of 10×NEB Buffer 3, 0.2-0.8 μL of EcoRI, 0.2-0.8 μL of BamHI, and water to a final volume of 15-25 μL.

[0013] Furthermore, the recipient cells are selected from human HeLa cells or porcine fibroblasts.

[0014] Furthermore, the culture conditions for the human HeLa cells and porcine fibroblasts are as follows: the human HeLa cells and porcine fibroblasts are cultured in DMEM medium containing FBS 24 hours before electroporation, and the culture conditions are 35-40℃ and 4%-6% CO2.

[0015] Furthermore, the detection primers are selected from the upstream and downstream sequences of the human or porcine FBN1-2 gene. The human FBN1-2 gene detection primer is named hFBN1, with the upstream sequence being 5'-AATCTGGTACTCAAACTTGGA-3' and the downstream sequence being 5'-ATATTATTTGTTCTTTGCCATT-3'. The porcine FBN1-2 gene detection primer is named: bufFBN1, the upstream sequence of bufFBN1 is 5'-CATAAAGGAAGAATCAAGCCTA-3', and the downstream sequence is 5'-TTCGCCATTAAAATATAGCATA-3'.

[0016] Further, the PCR system consisted of: 10 μL HS (Premix) (Takara), 1 μL DNA template, 0.5 μL each of forward and reverse primers, and water to a final volume of 20 μL. The reaction program was: 95°C pre-denaturation for 5 min, 35 cycles (95°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 30 sec), 72°C further extension for 7 min, and termination at 4°C. The above PCR reaction was... 5 μL of PCR product was directly subjected to 2% agarose gel electrophoresis at 120V for approximately 30 minutes.

[0017] The method described above for targeting and knocking out the FBN1 gene using CRISPR-Cas9 can be used for FBN1 gene knockout in humans or pigs.

[0018] Furthermore, the gene targeting vector sequence of the FBN1 gene knockout pig is SEQ ID NO.1.

[0019] Furthermore, FBN1 gene knockout pigs are prepared by homologous recombination of 5%-100% of the gene targeting vector sequence.

[0020] The present invention has the following beneficial effects: 1. The inventors of this technical solution analyzed the FBN1 gene of multiple species, performed homology comparison, and selected the two pairs of gene sequences with the highest homology as target sequences to knock out the FBN1 gene. This effectively improved the universality of the target sequence, making the recombinant plasmid vector edited using the target sequence applicable to multiple species. Although this invention selects target sequences with high homology from the FBN1 gene of multiple species through homology comparison, not every selected target sequence can be expressed correctly, and expression verification is still required. Moreover, the genome sequences of different species are different. If only homology comparison is used to select target sequences and other editing technologies, such as ZFNs and TALEN editing technologies, are employed, the editing efficiency will be greatly reduced because these two technologies require the synthesis of DNA sequence-specific binding protein modules. During DNA cleavage, the FokI nuclease domain will restrict the process, and the editing efficiency will be greatly reduced. If the upstream and downstream of the homologous FBN1 gene does not have the FokI nuclease domain, gene cleavage cannot be completed, and gene editing cannot be performed. Similarly, to accurately find target sequences with expression effects, detection methods are essential. These detection methods must be simple, efficient, and accurate to improve the accuracy of target sequences.

[0021] 2. This technical solution uses the CRISPR / Cas9 system. CRISPR-Cas9 requires two components: gRNA and the endonuclease Cas9. gRNA includes crRNA and tracrRNA. gRNA binds to the target specificity of crRNA and the scaffolding characteristics of tracrRNA (how to translate scffold) to form a single transcript. When gRNA and Cas9 are expressed in the cell, the genomic target can be modified or permanently interfered with. Using the Cas9 endonuclease only requires providing a specific 20bp fragment of sgRNA (single guide RNA) to determine target specificity. sgRNA is an RNA of approximately 100 nucleotides, composed of crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA). crRNA can form an RNA-DNA complex with the target DNA; this target DNA sequence is called the prespacer sequence. The sgRNA, composed of crRNA and tracrRNA, interacts with Cas9 to form ribonucleoproteins. The approximately 20 bp at the 5' end of sgRNA (corresponding to crRNA) guides Cas9 to bind to the target sequence via RNA-DNA complementary pairing, thereby cleaving the target site and causing a double-strand break (DSB). In cells, DSBs caused by nucleases are repaired via nonhomologous end-joining (NHEJ) in the absence of a repair template. NHEJ can induce random-length insertions or deletions, disrupting the translation reading frame of coding genes. Therefore, this invention uses the CRISPR / Cas9 system for gene editing, which is not limited by restriction endonucleases, allowing for simpler, more efficient, and multi-site gene modification. It achieves the effect of gene editing of the same target sequence in multiple species using only one expression vector, while being simple and efficient.

[0022] 3. This technical solution uses the validation report vector RGS-CR to verify the knockout efficiency of two pairs of target genes. It uses fluorescent protein to connect with the target gene. When the target gene is knocked out by homologous substitution, the expression of fluorescent protein can be initiated and imaged under green light. This method can express the gene editing effect in vitro, overcoming the problem of long in vivo expression cycle. It can quickly and reliably evaluate the gene transfection efficiency, effectively improve the targeted analysis and detection of target sequences, and improve the accuracy of target sequence selection. Attached Figure Description

[0023] Figure 1 Map of FBN1 multi-species homology alignment and target 1 design sites; Figure 2 A map showing the design sites for FBN1 multi-species homology alignment and target 2; Figure 3 Fluorescence expression detection image of FBN1 targeting efficiency; Figure 4 The image shows the sequencing results of the FBN1 gene mutation in HELA cells. Detailed Implementation All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Any feature disclosed in this specification (including any appended claims and abstract) may be replaced by other equivalent or similar features, unless specifically stated otherwise. That is, unless specifically stated otherwise, each feature is merely one example of a series of equivalent or similar features.

[0025] Example: I. Construction of gene targeting vectors: Design and vector construction of gRNA oligonucleotides targeting FBN1 in multiple species. Nucleotide sequences of FBN1 genes from multiple species were downloaded from the NCBI database and homology alignment was performed. Homology alignment results are attached to the instruction manual. Figure 1 Included with instruction manual Figure 2 Based on the PAM design principles of gRNA and the results of homology alignment, two primer pairs, FBN1-1 and FBN1-2, were synthesized. The primer sequences are as follows. The specific steps for vector construction are as follows: 1. Synthesize FBN1 target sequence 1: 5'-AGGCACTGGTATTTGGCAGAG-3', with the upstream sequence 5'-ACCGCTCTGCCAAATACCAGTGCCT-3' and the downstream sequence 5'-AAACAGGCACTGGTATTTGGCAGAG-3'. Target sequence 2: 5'-AAGATATAAGGCCAATTACTGCTC-3', with the upstream sequence 5'-ACCGAAGATATAAGGCCAATTACTGCTC-3' and the downstream sequence 5'-AAACGAGCAGTAATTGGCCTTATATCTTt-3'. After dissolving, take 4.5 μL of each and add 1 μL of 10×LA PCR Buffer. Mix well, heat at 95℃ for 10 min, and then place at room temperature for 3 h to form a DNA double strand with BsmBI sticky ends. 2. Digest pPDNA330 plasmid with enzymes. The enzyme digestion system is 3 μg pPDNA330 plasmid, 2 μL 10×NEB Buffer 3, 0.5 μL BsmBI, and water added to 20 μL. Incubate at 55℃ for 3 h. Perform agarose gel electrophoresis on the digestion product, excise the gel, recover the product, determine the concentration, and store at -20℃ for later use. 3. Ligate the products obtained in steps a and b above. The ligation system is as follows: 1 μL T4 ligase, 2 μL 10×T4 ligase buffer, 30 ng of pPDNA330 vector digested with BsmBI, 5 μL of double-stranded DNA formed from the target sequence and its complementary sequence, and water to a final volume of 20 μL. Ligate overnight at 16°C. Transform the ligation product into competent DH5α cells, expand the culture, extract plasmids, and perform sequencing to obtain the pPDNA330-FBN1-1 and pPDNA330-FBN1-2 vectors.

[0026] II. Verification of gene knockout efficiency 1. Target sequence knockout efficiency verification reporter vector RGS-CR: The RGS vector was linearized using restriction endonucleases EcoRI and BamHI. The digestion system was as follows: 2 μg RGS plasmid, 2 μL 10×BufferK, 0.5 μL EcoRI, 0.5 μL BamHI, water added to 20 μL, incubated at 37℃ for 3 h; the digestion products were subjected to agarose gel electrophoresis, the target fragment was excised and recovered, the concentration was determined, and stored at -20℃ for later use. 2. Two pairs of primers, #FBN1-1 and #FBN1-2, were synthesized to correspond to the two target sequences of FBN1. The upstream primer sequence of #FBN1-1 is 5'-AATTCCTCTGCCAAATACCAGTGCCTGGGG-3', and the downstream primer sequence is 5'-GATCCCCCAGGCACTGGTATTTGGCAGAGG-3'. The upstream primer sequence of #FBN1-2 is 5'-AATTCAAAGATATAAGGCCAATTACTGCTCTGGG-3', and the downstream primer sequence is 5'-GATCCCCAGAGCAGTAATTGGCCTTATATCTTTG-3'. After dissolving the primers to a concentration of 100 μM, the mixture was treated at 95 °C for 15 min, and then allowed to anneal overnight at a natural temperature to obtain two DNA double-stranded annealed products with EcoRI and BamHI sticky ends, respectively.

[0027] 3. T4 cells were used to ligate the linearized RGS product and the DNA double-strand annealing product, which were then transformed into competent cells. Single colonies were picked and cultured, endotoxins were removed, recombinant plasmids were extracted, and the plasmids were verified by sequencing to obtain the recombinant plasmids RGS-#FBN1-1 and RGS-#FBN1-2.

[0028] 4. One day before transfection, HEK-293T cells were passaged in four 35mm culture dishes.

[0029] 5. On the day of transfection, prepare four 1.5 mL centrifuge tubes. Add 100 μL of DMEM containing 10% FBS to each tube, then add the plasmid. Specifically, add 1000 ng of pPDNA330-FBN1-1 and 100 ng of RGS-#FBN1-1 to tube 1; add 1000 ng of pPDNA330 and 200 ng of RGS-#FBN1-1 to tube 2; and add 1000 ng of pPDNA330 to tube 3. 330-FBN1-2, 200 ng of RGS-#FBN1-2; 1000 ng of pPDNA330 and 200 ng of RGS-#FBN1-2 were added to tube 4; finally, 2 μL of Roche transfection reagent was added to each tube, mixed well, and allowed to stand at room temperature for 15 min. The mixture was then added to four 35 mm culture dishes and incubated at 37 °C in a 5% CO2 incubator. Fluorescence expression was observed after 48 h (experimental results are shown in the instruction manual). Figure 3 Fluorescence analysis showed that pPDNA330-FBN1-2 expressed significantly more green fluorescence than the negative control pPDNA330, indicating that pPDNA330-FBN1-2 can effectively induce DSBs and cause mutations in the FBN1 gene.

[0030] Third, we screened out expression vectors with better effects, transfected human and pig cells, and knocked out the FBN1 gene.

[0031] 1. One day before transfection, human HeLa cells were cultured in 35 mm culture dishes in DMEM containing 10% imported fetal porcine serum (FBS) at 37°C and 5% CO2. On the day of transfection, 2 μg of pPDNA330-FBN1-2 plasmid was introduced according to the Life 3000 kit instructions. 48 h after transfection, cells were collected by trypsin digestion and the genomic DNA was extracted.

[0032] 2. Synthesize the human FBN1 gene knockout detection primer hFBN1, with the upstream primer 5'-AATCTGGTACTCAAACTTGGA-3' and the downstream primer 5'-ATATTATTTGTTCTTTGCCATT-3'. Use the genomic DNA of cells transfected with pPDNA330-FBN1-2 plasmid as a template for PCR amplification. The PCR system is: 10 μL HS (Premix)(Takara), 1 μL DNA template, 0.5 μL each of forward and reverse primers, water to 20 μL. Reaction program: 95℃ pre-denaturation for 5 min, 35 cycles (95℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 30 sec), 72℃ further extension for 7 min, 4℃ to terminate the reaction. Take 5 μL of the PCR product from the above PCR reaction and directly perform 2% agarose gel electrophoresis at 120V for about 30 min. Cut the PCR product into the gel, recover, ligate, transform, pick single clones, and sequence. The PCR amplification product is ligated into the pEASY-T1 vector, and the FBN1 gene mutation is detected by picking single bacterial clones and sequencing. The results show that pPDNA330-FBN1-2 can effectively edit the FBN1 gene in human HeLa cells. The experimental results are shown in the instruction manual appendix. Figure 4 .

[0033] In summary, this invention selects two pairs of target sequences with the highest homology controlling the FBN1 gene in different species through homology comparison, constructs recombinant plasmids carrying the target sequences using the CRISPR-Cas9 editing system, and enables precise analysis of knockout experiment results using fluorescent protein expression detection. A recombinant vector capable of targeting and knocking out the FBN1 gene was constructed and selected. Transfection of human HeLa cells and porcine fibroblasts verified that this method is applicable to FBN1 gene knockout in different species, and even multiple species. The method of this invention is characterized by high targeting rate, accuracy, simplicity, efficiency, and universality, effectively improving gene knockout efficiency and accuracy.

[0034] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for constructing FBN1 gene knockout porcine somatic cells, characterized in that, The method for constructing FBN1 gene knockout porcine somatic cells includes the following steps: (1) Construction of gene targeting vector, the construction method is as follows: A. Download the nucleotide sequences of FBN1 genes from multiple species from the gene database and perform homology comparison to select two pairs of target sequences, namely FBN1 target sequence 1: 5'-AGGCACTGGTATTTGGCAGAG-3' and FBN1 target sequence 2: 5'-AAGATATAAGGCCAATTACTGCTC-3'. B. Based on the homology comparison results and the PAM design principle of gRNA, two pairs of DNA double strands with the same BsmBI sticky ends, which are different from the target sequence but express the same protein, were synthesized and named FBN1-1 and FBN1-2, respectively. C. Digest the pPDNA330 plasmid with BsmBI enzyme, perform agarose gel electrophoresis on the digestion product, and then perform gel extraction and recovery. D. Use ligase to ligate FBN1-1 and FBN1-2 to the BsmBI-digested pPDNA330 plasmid, respectively, to obtain recombinant plasmid vectors, named pPDNA330-FBN1-1 and pPDNA330-FBN1-2, respectively. (2) Verification of gene knockout efficiency: The knockout efficiency of vectors pPDNA330-FBN1-1 and pPDNA330-FBN1-2 was screened, verified and compared. The recombinant plasmid vector with higher FBN1 gene knockout efficiency was selected to transfect recipient cells. (3) Screening out expression vectors with better effects, transfecting human and pig cells to knock out the FBN1 gene, transfecting recipient cells of different species, collecting transfected recipient cells, extracting cell genomes for sequencing, synthesizing detection primers based on genome sequencing results for PCR amplification, ligating the amplification product into the pEASY-T1 vector, picking single clones of bacteria, sequencing them, and detecting the FBN1 gene knockout expression results.

2. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 1, characterized in that, The upstream sequence of FBN1-1 is 5'-ACCGCTCTGCCAAATACCAGTGCCT-3', and the downstream sequence is 5'-AAACAGGCACTGGTATTTGGCAGAG-3'; the upstream sequence of FBN1-2 is 5'-ACCGAAGATATAAGGCCAATTACTGCTC-3', and the downstream sequence is 5'-AAACGAGCAGTAATTGGCCTTATATCTT-3'.

3. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 1, characterized in that, The screening and verification of the knockout efficiency of the recombinant plasmid vectors pPDNA330-FBN1-1 and pPDNA330-FBN1-2 were verified by fluorescent protein expression method. The specific method is as follows: (1) Using RGS-CR as a verification reporter vector: the verification reporter vector RGS-CR was linearized using restriction endonucleases EcoR Ⅰ and BamH Ⅰ. (2) Synthesize two DNA double strands with EcoRI and BamHI sticky ends corresponding to FBN1 target sequence 1 and FBN1 target sequence 2, and name them as #FBN1-1 and #FBN1-2 respectively. The upstream sequence of the #FBN1-1 primer is: 5'-AATTCCTCTGCCAAATACCAGTGCCTGGGG-3', and the downstream sequence is: 5'-GATCCCCCAGGCACTGGTATTTGGCAGAGG-3'. The upstream sequence of the #FBN1-2 primer is: 5'-AATTCAAAGATATAAGGCCAATTACTGCTCTGGG-3', and the downstream sequence is: 5'-GATCCCCAGAGCAGTAATTGGCCTTATATCTTTG-3'; (3) The RGS-CR linearized product from step (1) and the two pairs of DNA double strands #FBN1-1 and #FBN1-2 with EcoRI and BamHI sticky ends from step (2) were ligated using T4 ligase to obtain recombinant plasmid vectors, which were named RGS-#FBN1-1 and RGS-#FBN1-2, respectively. (4) Passaged HEK-293T cells were placed in 4 culture dishes; (5) Take 4 centrifuge tubes and number them 1, 2, 3 and 4. Add DMEM containing FBS to each tube. (6) Then add pPDNA330-FBN1-1 and RGS-#FBN1-1 to tube 1, and add pPDNA330 and RGS-#FBN1-1 to tube 2. Add pPDNA330-FBN1-2 and RGS-#FBN1-2 to tubes 1 and 3, and add pPDNA330 and RGS-#FBN1-2 to tube 4. Finally, add Roche transfection reagent to each tube, mix well, let stand at room temperature, add the mixture to culture dishes, and incubate in an incubator. After incubation, observe the fluorescence expression of the target gene under a fluorescence microscope.

4. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 1, characterized in that, The recipient cells are selected from human HeLa cells or porcine fibroblasts.

5. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 1, characterized in that, The detection primers are selected from the upstream and downstream sequences of the human or porcine FBN1-2 gene. The human FBN1-2 gene detection primer is named hFBN1, with the upstream sequence being 5'-AATCTGGTACTCAAACTTGGA-3' and the downstream sequence being 5'-ATATTATTTGTTCTTTGCCATT-3'. The porcine FBN1-2 gene detection primer is named bufFBN1, with the upstream sequence being 5'-CATAAAGGAAGAATCAAGCCTA-3' and the downstream sequence being 5'-TTCGCCATTAAAATATAGCATA-3'.

6. The method for constructing FBN1 gene knockout porcine somatic cells according to any one of claims 1-5, characterized in that... The method of using CRISPR-Cas9 to target and knock out the FBN1 gene can be used for FBN1 gene knockout in humans or pigs.

7. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 1, characterized in that... The gene targeting vector sequence of the FBN1 gene knockout pig is SEQ ID NO.

1.

8. The method for constructing FBN1 gene knockout porcine somatic cells according to claim 7, characterized in that, The gene targeting vector sequence was used to prepare FFBN1 gene knockout pigs by homologous recombination of 5%-100% of the sequence.

Images