Pair of sgRNA specifically targeting pig PRMT3 gene and application of sgRNA

Abstract

The invention provides a pair of sgRNAs specifically targeting pig PRMT3 gene and application thereof, the sgRNAs comprise sgRNA1 and sgRNA2, the sense strand of the DNA sequence of the sgRNA1 is as shown in SEQ ID NO: 2, and the DNA sequence of the corresponding complementary strand of the sgRNA1 is as shown in SEQ ID NO: 1; a sense strand of a DNA sequence of the sgRNA 2 is as shown in SEQ ID NO: 4, a DNA sequence of a corresponding complementary strand of the sgRNA 2 is as shown in SEQ ID NO: 3, and the sgRNA can effectively edit a PRMT3 gene sequence, is used for preparing a porcine cell line of PRMT3KO, is expected to improve efficiency in virus separation of PEDV (porcine epidemic diarrhea virus) and vaccine production, has a clearer understanding of a pathogenic mechanism of coronavirus, and can be used for preparing disease-resistant pigs for resisting PRV and PDCoV at the same time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and is a pair of sgRNAs specifically targeting the porcine PRMT3 gene and a cell line with porcine PRMT3 gene sequence knocked out obtained by using the pair of sgRNAs. Background Art

[0002] Pigs are one of the important livestock breeds in my country, and they play an important role in agricultural economics, medical disease models and molecular genetics research. Gene-edited pigs have important application value in biological research, breeding of new agricultural varieties and biomedical research. The emergence of the CRISPR / Cas9 system has greatly improved the efficiency of pig gene editing.

[0003] Protein arginine methyltransferase (PRMT) is an enzyme that catalyzes the asymmetric dimethylation of arginine residues in a monomethylated or dimethylated manner. PRMT3 is a type I PRMT that mainly catalyzes the asymmetric dimethylation of arginine residues, through participating in processes such as cell metabolism and gene regulation. The expression of PRMT3 is regulated by viral infection, and PRMT3 negatively regulates the antiviral innate immune signaling pathway, inhibiting the production of type I interferon and the expression of antiviral genes.

[0004] Therefore, gene editing of the porcine PRMT3 gene, changing or modifying the PRMT3 gene coding sequence, and avoiding viral infection have important application value for the study of pig gene function, the preparation and breeding of new gene-edited pigs. CRISPR / cas9-mediated genome engineering has been widely and efficiently used in various species, and can achieve conditional gene knockout, gene knock-in, gene replacement, point mutation, etc., providing an attractive strategy for the study of gene function. Cas9 targeted DNA cutting is mediated by a guide RNA chain, namely sgRNA (single guide RNA). Therefore, whether sgRNA can specifically and accurately target the target gene site is a prerequisite for whether CRISPR-Cas9 can perform its function.

[0005] Therefore, designing sgRNA with specificity and precise targeting of target gene sites is a key factor in CRISPR-Cas9 technology. Summary of the invention

[0006] Based on the existing research foundation and technical platform of porcine PRMT3 gene regulation, the present invention utilizes CRISPR / Cas9-mediated gene knockout technology and uses LLC-PK1 porcine kidney cell line as the research object to explore the effect of porcine PRMT3 gene on PRV, PDCoV and PEDV infection, which is applied to the prevention and control of porcine PRMT3 gene on PRV, PDCoV and PEDV as well as the study of disease resistance targets, and can provide scientific research value and application value in other research fields.

[0007] The present invention provides a pair of sgRNAs specifically targeting the porcine PRMT3 gene, which are designed for the target sites upstream and downstream of the first exon of the porcine PRMT3 gene, and have high targeting efficiency and knockout efficiency. Based on the pair of sgRNAs, the porcine PRMT3 gene sequence can be deleted, and the targeting efficiency and knockout efficiency are high, and porcine PRMT3 gene knockout cells can be prepared. The knockout is performed based on the CRISPR / Cas9 system, and the sgRNA sequence includes sgRNA1 and sgRNA2. The target sequence recognized by sgRNA1 is shown in SEQ ID NO.1, and the target sequence recognized by sgRNA2 is shown in SEQ ID NO.3. After the porcine PRMT3 gene is knocked out, its expression is inhibited; the sense strand of the DNA sequence of the sgRNA1 is shown in SEQ ID NO: 2, and the DNA sequence of its corresponding complementary strand is shown in SEQ ID NO: 1; the sense strand of the DNA sequence of the sgRNA2 is shown in SEQ ID NO: 4, and the DNA sequence of its corresponding complementary strand is shown in SEQ ID NO: 3.

[0008] The present invention also provides a knockout vector, which can inhibit the expression of the pig PRMT3 gene, and is obtained by connecting the double-stranded DNA of the above-mentioned sgRNA 1 and sgRNA2 with a vector. The vector is a PX330 vector. The preparation method of the knockout vector of the CRISPR-Cas9 system comprises: synthesizing the positive strand of the DNA sequence of the sgRNA1 such as SEQ ID NO: 2, and the DNA sequence of its corresponding complementary strand such as SEQ ID NO: 1; the positive strand of the DNA sequence of the sgRNA2 such as SEQ ID NO: 4, and the DNA sequence of its corresponding complementary strand such as SEQ ID NO: 3, and then annealing the single-stranded DNA sequence to synthesize double-stranded DNA, and connecting it to the Bbs I restriction site of the PX459 vector backbone to form a complete targeting plasmid.

[0009] The present invention also provides the use of the above sgRNA sequence in preparing a cell line in which the expression of the porcine PRMT3 gene is inhibited; the cell line is the LLC-PK1 porcine kidney cell line. The method for constructing the LLC-PK1 porcine kidney cell line is as follows: a CRISPR / Cas9 gene knockout vector containing the sgRNA specifically targeting the porcine PRMT3 gene is introduced into the cell to knock out the porcine PRMT3 gene. The introduction of the CRISPR / Cas9 gene knockout vector into the cell can be achieved by conventional biological methods such as electrotransfection.

[0010] The present invention also provides the use of the above-mentioned sgRNA or knockout vector or cell line in the preparation of a product having at least one of the following functions: 1. specific identification of the porcine PRMT3 gene (specific identification of the porcine PRMT3 gene exon); 2. knockout of the porcine PRMT3 gene (specific identification of the porcine PRMT3 gene exon); 3. gene editing of the porcine PRMT3 gene; 4. preventing pigs from being infected with PDCoV and PRV viruses; 5. breeding or preparing gene-edited pigs; 6. PEDV virus isolation and vaccine production.

[0011] In some optional embodiments, the product is a kit.

[0012] In some optional embodiments, the kit comprises the above-mentioned sgRNA and a reagent for storing the sgRNA, and a Cas9 protein molecule.

[0013] The present invention also provides a non-disease diagnosis and treatment method, by introducing the CRISPR / Cas9 system containing the above-mentioned sgRNA that recognizes the pig PRMT3 gene into pig-derived biological materials (specifically, in vitro pig somatic cells) to achieve gene editing of the pig PRMT3 gene, and the expression of the pig PRMT3 gene is significantly inhibited.

[0014] The present invention also provides the use of the above-mentioned sgRNA sequence in the preparation of drugs for preventing and controlling PDCoV and PEDV with the porcine PRMT3 gene as a target.

[0015] The beneficial effects of the present invention include at least: The present invention is based on a pair of sgRNAs in the pig group that can specifically recognize the pig PRMT3 gene sequence, and uses CRISPR / Cas9 technology to successfully knock out the pig PRMT3 gene in the LLC-PK1 pig kidney cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the WB identification diagram of large fragment knockout of sgRNA1 and sgRNA2.

[0017] Figure 2 This is the effect of porcine PRMT3 gene knockout on PEDV proliferation (n=3, **P<0.001, ***P<0.0001).

[0018] Figure 3 The effect of porcine PRMT3 gene knockout on PRV proliferation (n=3, *P<0.05).

[0019] Figure 4 This is the effect of pig PRMT3 gene knockout on PDCOV proliferation (n=3, **P<0.001, ****P<0.00001). DETAILED DESCRIPTION

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The present invention downloads the nucleotide sequence of the porcine PRMT3 gene (Gene ID: 100521429, NC_010444.4 Reference Sscrofa11.1 Primary Assembly, Range 38884311-39032944 complement) from the NCBI database, analyzes the porcine PRMT3 gene sequence, and according to the design principle of CRISPR / Cas9 recognition of target sites, screens the target sites of porcine PRMT3 and designs and screens sgRNAs for the target sites, and finally obtains sgRNA1 and sgRNA2 with the highest knockout efficiency, as shown in SEQ ID NO.2 and SEQ ID NO.4, and constructs a CRISPR / Cas9 knockout vector containing the sgRNA, thereby achieving efficient knockout of the porcine PRMT3 gene.

[0024] SEQ ID NO: 1: 5'-CGCTGTCTGACAGCTCGGGC-3' SEQ ID NO: 2: 5'-GCCCGAGCTGTCAGACAGCG-3' SEQ ID NO: 3: 5'-TCTGAGCATCAGTTTAATAT-3' SEQ ID NO: 4: 5'-ATATTAAACTGATGCTCAGA-3' Example 1: Knockout of the porcine PRMT3 gene in the LLC-PK1 porcine kidney cell line using sgRNA and Cas9.

[0025] A pair of sgRNAs were designed for the upstream and downstream targets of the first exon, and the sgRNA sequences were synthesized in the form of primers. The sgRNA annealing products were connected to the linearized pX330 vector after enzyme digestion, namely the Cas9 / gRNA vector. The gene editing vector PX330 mainly contains sgRNA driven by the U6 promoter, spCas9 gene driven by the CBh promoter, and necessary resistance genes for Escherichia coli amplification. Other regulatory sequences include enhancers and nuclear localization sequences, etc., and do not contain marker genes and reporter genes. After transcription, sgRNA forms a nucleic acid-protein complex with Cas9 protein, and Cas9 plays a role at a specific site by relying on the specific binding of sgRNA to the genomic target sequence.

[0026] The targeting vector was transferred into LLC-PK1 cell line by electroporation. After obtaining monoclonal clones, the knockout results were analyzed by WB. WB analysis showed that the upstream and downstream sgRNA targeting vectors can destroy the porcine PRMT3 gene. The WB images of the large fragment knockout of sgRNA1 and sgRNA2 are shown in the figure below. Figure 1 shown.

[0027] qPCR detection of the effect of pig PRMT3 knockout on PEDV replication Figure 2 shown.

[0028] Wash the cells with 2 mL PBS, discard the PBS, and add 2 mL 0.5% trypsin to digest the cells. After digestion is complete, add 3 mL of fresh culture medium containing 5% FBS to resuspend the cells. 5 The cells were inoculated in a 6-well plate at a density of 10 cells / well and cultured with 2 mL of cell culture medium containing 5% FBS. After the cells adhered to the wall, the cell culture medium was removed and DMEM containing the virus was added. After culturing in a cell culture incubator for 2 hours, fresh culture medium containing 5% FBS was replaced and cultured for 48 hours. The infection dose of the virus was MOI = 0.01. The cells were collected at the corresponding time points for RNA extraction and qPCR detection.

[0029] qPCR evaluation of the ability of PRMT3 KO to restrict PRV proliferation Figure 3 shown.

[0030] Wash the cells with 2mL PBS, discard the PBS, and add 2mL 0.5% trypsin to digest the cells; after digestion, add 3mL fresh culture medium containing 5% FBS to resuspend the cells, inoculate them in a 6-well plate at a density of 3*105 cells / well, and culture them with 2mL cell culture medium containing 5% FBS. After the cells adhere to the wall, remove the cell culture medium, add DMEM containing the virus, culture in the cell culture incubator for 2h, and then replace with fresh culture medium containing 5% FBS and continue to culture for 48h, where the infection dose of the virus is MOI=0.001. Collect 200μl of cell supernatant at the corresponding time point for viral genome extraction and qPCR detection.

[0031] qPCR evaluation of the ability of PRMT3 KO to restrict PD-CoV proliferation Figure 4 shown.

[0032] Wash the cells with 2mL PBS, discard the PBS, and add 2mL 0.5% trypsin to digest the cells; after digestion, add 3mL fresh culture medium containing 5% FBS to resuspend the cells, inoculate them in a 6-well plate at a density of 3*105 cells / well, and culture them with 2mL cell culture medium containing 5% FBS. After the cells adhere to the wall, remove the cell culture medium, add DMEM containing the virus, culture in the cell culture incubator for 2h, and then replace with fresh culture medium containing 5% FBS and continue to culture for 48h, where the infection dose of the virus is MOI=0.01. Collect cells at the corresponding time points for RNA extraction and qPCR detection.

[0033] The results showed that the cell line could promote the proliferation of PEDV in cells to a certain extent, and limit the proliferation of PRV and PDCOV to a certain extent, which is of great significance for isolating and culturing PEDV attenuated strains or wild strains and improving the preparation of PEDV attenuated live vaccines to a certain extent in industry, for the screening of drug targets against porcine acute epidemic diarrhea virus and for the genetic breeding of disease-resistant pigs against PRV and PDCOV.

[0034] It can be understood in the present invention that the Cas9 nucleic acid molecule also includes functional elements that act together with the mRNA or DNA encoding Cas9, and examples of other functional elements include but are not limited to promoters, terminators, enhancers and marker genes, as well as DNA molecule parts used as vectors; the Cas9 molecule can be a conventional Cas9 molecule or a Cas9 molecule modified by molecular biology, as long as it has the shearing function of the Cas9 molecule, the present invention does not limit this. The method for gene editing of the eIF4G1 gene in the pig genome provided by the present invention adopts the above-mentioned sgRNA or the DNA molecule encoding the above-mentioned sgRNA, and combines the CRISPR / Cas9 system, the knockout efficiency is high and the specificity is strong, and the off-target rate is lower when performing sequence knockout.

[0035] In the present invention, the system is a plasmid expressing a DNA molecule encoding sgRNA and a cas9 protein, specifically pX330-eIF4G1-sgRNA1, pX330-eIF4G1-sgRNA2 and / or pX330-eIF4G1-sgRNA3.

[0036] The gene editing of pigs described in the present invention can be performed only on the PRMT3 gene in the pig genome, or multiple genes including the PRMT3 gene in the pig can be edited. It should be noted that the application provided by the present invention is used for non-therapeutic research on pig gene function, including research on site-specific integration of exogenous genes at the pig PRMT3 site. The sgRNA and its encoding molecule, gene editing method or kit provided by the present invention are applied to pig gene editing, with high knockout efficiency and low off-target rate.

[0037] The kit described in the present invention can be used to perform gene editing on only the PRMT3 gene in the pig genome, or can be used to perform gene editing on multiple genes in the pig including the PRMT3 gene.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pair of sgRNAs specifically targeting the porcine PRMT3 gene, wherein the sgRNA sequences include sgRNA1 and sgRNA2, wherein the sense strand of the DNA sequence of the sgRNA1 is such as SEQ ID NO: 2, and the DNA sequence of its corresponding complementary strand is such as SEQ ID NO: 1; the sense strand of the DNA sequence of the sgRNA2 is such as SEQ ID NO: 4, and the DNA sequence of its corresponding complementary strand is such as SEQ ID NO:

3.

2. A knockout vector, characterized in that: The expression of the pig PRMT3 gene is inhibited by connecting the double-stranded DNA of sgRNA1 and sgRNA 2 as described in claim 1 to a vector.

3. The knockout vector according to claim 3, characterized in that: The vector is a PX330 vector.

4. A cell line, characterized in that: Contains the knockout vector as claimed in claim 2.

5. Use of the sgRNA according to claim 1 or the knockout vector according to claim 2 or the cell line according to claim 4 in preparing a product having at least one of the following functions, characterized in that:

1. Specific identification of porcine PRMT3 gene; Second, knock out the porcine PRMT3 gene; 3. Gene editing of the porcine PRMT3 gene; 4. Prevent pigs from being infected with PDCoV and PRV viruses; 5. Breeding or preparing gene-edited pigs; 6. PEDV virus isolation and vaccine production.

6. The product according to claim 5, characterized in that: The product is a test kit.

7. A gene editing method for purposes other than disease diagnosis and treatment, characterized in that: When the CRISPR / Cas9 system for recognizing the porcine PRMT3 gene as described in claim 1 is introduced into porcine somatic cells in vitro, the expression of the porcine PRMT3 gene is significantly inhibited.

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