sgRNA targeting porcine PLIN1 gene and its application

Abstract

The present invention relates to the field of biotechnology, and provides a sgRNA targeting the porcine PLIN1 gene and its application. The sgRNA action site is located on the second and fourth exons of the porcine PLIN1 gene. The present invention also provides a method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line using a CRISPR / CAS9 system, which is based on the porcine PLIN1 gene sequence, designs and synthesizes the sgRNA targeting the porcine PLIN1 gene, then constructs a CRISPR / Cas9 targeting vector containing the sgRNA, transfers it into porcine fetal fibroblasts, obtains the porcine fetal fibroblasts with the PLIN1 gene knocked out, and the cells can be used for the preparation of PLIN1 gene-edited pigs, which is not only of great significance for pig breeding, but also has important reference value for revealing the occurrence mechanism of human hereditary lipodystrophy and seeking treatment methods.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an sgRNA targeting a porcine PLIN1 gene and an application thereof. Background Art

[0002] Lipid droplet coating protein (also known as perilipin, adiponectin, perilipin1, PLIN1) is a type of phosphorylated protein wrapped around lipid particles in adipocytes and steroidogenic cells, which has a bidirectional regulatory effect on fat decomposition. In the basal (fed) state, PLIN1 is coated on the surface of lipid droplets, acting as a neutral lipid protective barrier and inhibiting lipolysis. In fasting or exercise conditions, PLIN1 can be highly phosphorylated by protein kinase A, which facilitates lipase to approach the surface of lipid droplets and promotes lipolysis.

[0003] Pigs occupy a dominant position in my country's meat production and consumption structure and are an important source of animal protein for Chinese residents. Improving lean meat rate and reducing fat content are important goals of livestock and poultry breeding. Studies have shown that PLIN1 gene polymorphism has a certain effect on fat deposition in Qinchuan cattle and is related to the average daily weight gain, lean meat rate and backfat thickness of pigs. It can be used as a candidate gene to improve the lean meat rate or meat quality of livestock.

[0004] Seeking and creating disease models of human genetic malnutrition has become a research hotspot in the medical field. People with mutations that lack the function of the PLIN1 gene show symptoms of hereditary lipodystrophy with abnormal distribution and metabolism of adipose tissue, including thyroid hypermetabolism, hyperproteinemia, hyperlipidemia, hyperinsulinemia, insulin resistance, etc. Common model animals, PLIN1 knockout mice, also have similar characteristics. Under the same food consumption conditions, they have 30% less fat than wild-type mice, increased fat decomposition, and increased plasma leptin, blood lipids, and blood sugar levels. However, compared with mice, pigs' metabolism, disease occurrence and other processes are more similar to those of humans, and can serve as an ideal human disease model.

[0005] Therefore, seeking a sequence that can efficiently and specifically identify the pig PLIN1 gene and using the Cas9 enzyme to efficiently cut the site will lay a solid foundation for the preparation of PLIN1 gene-edited pigs. This is not only of great significance for pig breeding, but also has important reference value for revealing the occurrence mechanism of human hereditary lipodystrophy and seeking treatment methods. Summary of the invention

[0006] The purpose of the present invention is to provide a sgRNA targeting the porcine PLIN1 gene and an application thereof.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a sgRNA targeting the porcine PLIN1 gene, wherein the sgRNA action site is located on the second and fourth exons of the porcine PLIN1 gene, and the DNA sequence of the sgRNA action site is selected from at least one of the sequences shown in SEQ ID NO: 1-3.

[0008] Preferably, the DNA sequence of the sgRNA action site is as shown in SEQ ID NO:2.

[0009] In a second aspect, the present invention provides a porcine PLIN1 gene targeting vector, which is a sgRNA expression vector based on the CRISPR / Cas9 system, wherein the DNA sequence of the sgRNA action site is as described above.

[0010] Furthermore, the targeting vector contains a U6 promoter that can drive the expression of sgRNA and a gene encoding the expression of Cas9 protein, for example, a PX458 vector.

[0011] In a third aspect, the present invention provides use of the targeting vector in preparing a pig cell line with PLIN1 gene knockout.

[0012] Furthermore, the porcine cell line comprises a porcine fetal fibroblast cell line.

[0013] In a fourth aspect, the present invention provides a method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line using the CRISPR / CAS9 system, which comprises designing and synthesizing an sgRNA targeting the porcine PLIN1 gene based on the porcine PLIN1 gene sequence, then constructing a CRISPR / Cas9 targeting vector containing the sgRNA, transferring the sgRNA into porcine fetal fibroblasts, and screening target-positive cell clones, i.e., porcine fetal fibroblasts with the PLIN1 gene knocked out; wherein the DNA sequence of the sgRNA action site is as described above.

[0014] Furthermore, the sequences of specific PCR primers used to identify target positive cell clones are shown in SEQ ID NOs: 7-8.

[0015] In a fifth aspect, the present invention provides any of the following uses of the porcine fetal fibroblast cell line prepared according to the above method:

[0016] 1) Used to study the function of PLIN1 protein;

[0017] 2) Used to construct PLIN1 knockout pigs;

[0018] 3) Used as a cell model or drug screening model to study human hereditary lipodystrophy.

[0019] In a sixth aspect, the present invention provides the use of a PLIN1 gene knockout pig constructed using a porcine fetal fibroblast cell line prepared according to the above method as an animal model or drug screening model for studying human hereditary lipodystrophy and its accompanying metabolic diseases such as thyroid hypermetabolism, hyperproteinemia, hyperlipidemia, hyperinsulinemia, insulin resistance, etc.

[0020] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] The present invention provides a method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line by using a CRISPR / CAS9 system. The method comprises the following steps: designing and synthesizing an sgRNA targeting the porcine PLIN1 gene according to the porcine PLIN1 gene sequence, then constructing a CRISPR / Cas9 targeting vector containing the sgRNA, and transferring the sgRNA into porcine fetal fibroblasts to obtain porcine fetal fibroblasts with the PLIN1 gene knocked out. The cells can be used for the preparation of PLIN1 gene-edited pigs, which is not only of great significance for pig breeding, but also of great reference value for revealing the occurrence mechanism of human hereditary lipodystrophy and seeking a treatment method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the specific recognition region of the sgRNA designed for the second exon and the fourth and fifth exons of the porcine PLIN1 gene in the present invention.

[0023] Figure 2 This is a schematic diagram of the PLIN1 gene editing site and sgRNA sequence information in a preferred embodiment of the present invention.

[0024] Figure 3 A sequencing map is constructed for the CRISPR / Cas9 targeting vector in a preferred embodiment of the present invention.

[0025] Figure 4 This is a fluorescence image of PFF cells transfected with CRISPR / Cas9 targeting vector in a preferred embodiment of the present invention.

[0026] Figure 5 This is a diagram showing the cutting efficiency of the PX458-sg2 reconstructed vector in a preferred embodiment of the present invention.

[0027] Figure 6 This is a sequencing comparison diagram of monoclonal cells in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0028] The present invention aims to provide a sgRNA that specifically recognizes the pig PLIN1 site, its encoding DNA and application. An expression vector is constructed using a short DNA fragment encoding the sgRNA sequence, and the expression vector can express that the sgRNA sequence can specifically recognize the pig PLIN1 gene site and perform site-specific cutting, laying the foundation for the preparation of gene-edited pigs.

[0029] The present invention adopts the following technical solution:

[0030] The present invention targets the second exon and the fourth and fifth exons of the porcine PLIN1 gene ( Figure 1 ), 6 sgRNAs specifically targeting the porcine PLIN1 gene were designed based on CRISPR / Cas9 technology (Table 1).

[0031] Furthermore, six sgRNAs were constructed into CRISPR / Cas9 targeting vectors, named PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5 and PX458-sg6. The six CRISPR / Cas9 targeting vectors were used to transfect porcine fetal fibroblasts (PEF) for cutting efficiency detection. It was found that sg1, sg2 and sg3 had cutting activity on the porcine PLIN1 gene, that is, they had cutting activity on the second and fourth exons of the PLIN1 gene, and their corresponding nucleotide sequences were shown in SEQ ID NO:1-3, respectively, and sg2 had the highest cutting efficiency of 38%.

[0032] Furthermore, the primers used for genotype identification are as follows (SEQ ID NO: 7-8):

[0033] PLIN1-F: 5′-ACCTCCCTAGTCTGGGTTCC-3′

[0034] PLIN1-R: 5′-TGACTGGTAGCTCTCTGGCT-3′

[0035] The amplified wild-type target fragment has a size of 809 bp.

[0036] Furthermore, the px458-sg2 vector containing the sgRNA2 sequence was transfected into PEF cells, and combined with cell flow sorting technology, a monoclonal cell line of porcine PLIN1 gene editing was obtained.

[0037] The present invention provides a sgRNA that specifically recognizes the porcine PLIN1 site, its encoding DNA and application. By using one or more sgRNA CRISPR / Cas9 targeting vectors to transfect porcine fetal fibroblasts, a monoclonal cell line of porcine PLIN1 gene editing can be obtained. Somatic cell nuclear transplantation and other technologies can be used to prepare porcine PLIN1 gene-edited pigs, thereby creating new pig breeding materials or establishing a disease model of human hereditary lipodystrophy.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] Example 1 Design of sgRNA sequence and vector construction for specific recognition of porcine PLIN1 gene

[0040] Based on the porcine PLIN1 gene sequence in NCBI (Gene ID: 654411 or ENSSSCG00000001844), the second exon, the fourth exon and the fifth exon were respectively identified. Figure 1 ), two sgRNAs were designed for each, and they were named sg1 (SEQ ID NO: 1), sg2 (SEQ ID NO: 2), sg3 (SEQ ID NO: 3), sg4 (SEQ ID NO: 4), sg5 (SEQ ID NO: 5) and sg6 (SEQID NO: 6). The sequence information and specific recognition sites are shown in Table 1. Figure 2 As shown:

[0041] Table 1 sgRNA sequences

[0042]

[0043] Note: The underlined part is the PAM sequence.

[0044] Figure 2 Two pairs of sgRNAs were designed for the second, fourth, and fifth exons of the porcine PLIN1 gene, respectively.

[0045] The designed sgRNA is added with restriction sites to synthesize oligonucleotide chains (Oligo), and the sequence information is shown in Table 2:

[0046] Table 2 Oligo chain synthesis sequence

[0047]

[0048] Note: The underlined part is the restriction site sequence.

[0049] The commercial CRISPR / Cas9 vector PX458-GFP (Addgene, USA) was cut with BbsⅠ enzyme, and gel recovery was performed. The linearized vector was connected to the 6 annealed oligo double strands through the sticky ends, followed by transformation, plating, picking single colonies, and shaking bacteria for propagation. The obtained bacterial solution was sequenced and it was found that all 6 sgRNAs had been successfully connected to the vector PX458-GFP and could be used for subsequent experiments. The 6 reconstructed vectors were named PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5 and PX458-sg6.

[0050] Universal primers were used to sequence six vectors, namely PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5 and PX458-sg6. Figure 3 The sequencing results showed that all six sgRNAs were successfully constructed into the CRISPR / Cas9 targeting vector PX458.

[0051] Example 2 Screening of efficient sgRNA targeting porcine PLIN1 locus

[0052] PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5 and PX458-sg6 were transfected into porcine PEF cells. After 48 hours, some cells were observed to emit green light under a fluorescence microscope, indicating that the cells were successfully transfected ( Figure 4 ). Flow cytometry was then used to enrich the luminescent cells and extract DNA for analysis of sgRNA cutting efficiency. The primers used for genotyping are as follows:

[0053] PLIN1-F: 5′-ACCTCCCTAGTCTGGGTTCC-3′

[0054] PLIN1-R: 5′-TGACTGGTAGCTCTCTGGCT-3′

[0055] The results showed that PX458-sg2 had the highest cutting efficiency, reaching 38% ( Figure 5 ), followed by PX458-sg3 and PX458-sg1, with 13% and 12%, respectively, and no cutting efficiency was detected for the other three vectors.

[0056] Example 3 Screening and verification of PLIN1 gene-edited PEF cell lines

[0057] 5 µg of px458-sg2 vector was transfected into PEF cells. After 48 hours, the luminescence was observed under a fluorescence microscope, and the cells were sorted using a flow cytometer. The positive single cells after successful sorting continued to be cultured in a cell culture incubator, and the culture medium was changed every 3 days. When the cells grew to 80%, they were transferred to a 48-well plate and continued to be expanded. The culture medium was changed every 3 days. When the cells grew to 80% in the 48-well plate, some cells were taken to extract genomic DNA for genotype identification, and the remaining cells continued to be expanded.

[0058] Through PCR amplification and sequencing, a total of 100 monoclonal cells were obtained in this screening, of which 4 were PEF cells with homozygous PLIN1 gene fragment deletion ( Figure 6 ), accounting for 4% of the total number of monoclones, missing 31, 75, 13 and 135 bases respectively; 20 were heterozygous cells, accounting for 20% of the total number of monoclones. The 4 PEF cells with homozygous PLIN1 gene fragment deletions were subsequently used to prepare PLIN1 gene-edited pigs by somatic cell cloning.

[0059] In summary, the sgRNA that specifically recognizes the porcine PLIN1 gene provided by the present invention can efficiently achieve the knockout of the porcine PLIN1 gene by means of the CRISPR / Cas9 gene editing system and the dual fluorescence enrichment method. These cells can be used to study the function of the porcine PLIN1 gene, and can also be combined with somatic cell cloning and other methods to prepare PLIN1 gene-edited pigs, providing new breeding materials for the pig industry and a lipodystrophy disease model for humans.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An sgRNA targeting the porcine PLIN1 gene, characterized in that: The action site of the sgRNA is located on the second exon of the porcine PLIN1 gene, and the DNA sequence of the sgRNA is shown in SEQ ID NO:

2.

2. A porcine PLIN1 gene targeting vector, characterized in that: The targeting vector is a sgRNA expression vector based on the CRISPR / Cas9 system, and the targeting vector contains the sgRNA according to claim 1.

3. The targeting carrier according to claim 2, characterized in that: The targeting vector contains a U6 promoter that can drive the expression of sgRNA and a gene encoding the Cas9 protein.

4. Use of the targeting vector according to claim 3 in preparing a pig cell line with PLIN1 gene knockout; The porcine cell lines include porcine fetal fibroblast cell lines.

5. A method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line using the CRISPR / Cas9 system, characterized in that: According to the porcine PLIN1 gene sequence, sgRNA targeting the porcine PLIN1 gene is designed and synthesized, and then a CRISPR / Cas9 targeting vector containing the sgRNA is constructed and transferred into porcine fetal fibroblasts, and target-positive cell clones are screened, namely, porcine fetal fibroblasts with PLIN1 gene knockout; wherein the DNA sequence of the sgRNA is shown in SEQ ID NO:2.

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