Sgrna targeting porcine PLIN1 gene and use thereof

Abstract

An sgRNA targeting a porcine PLIN1 gene and use thereof. The sgRNA action site is located on the second exon and the fourth exon of the porcine PLIN1 gene. Provided is a method for knocking out a PLIN1 gene in a porcine fetal fibroblast cell line by using a CRISPR / CAS9 system. The method comprises designing and synthesizing an sgRNA targeting a porcine PLIN1 gene according to a porcine PLIN1 gene sequence, then constructing a CRISPR / Cas9 targeting vector containing the sgRNA, and transferring same into a porcine fetal fibroblast cell to obtain a PLIN1 gene-knocked-out porcine fetal fibroblast cell. The cell can be used for the preparation of a PLIN1 gene-edited porcine, and is not only of great significance for porcine breeding, but also of great reference value for revealing the occurrence mechanism of human hereditary lipodystrophy and seeking a treatment method.

Description

sgRNA targeting the porcine PLIN1 gene and its applications

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Application No. CN202411414056.1, filed on October 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to an sgRNA targeting the porcine PLIN1 gene and its applications. Background Technology

[0004] Lipid droplet coating proteins (also known as perilipin, adiponectin, perilipin1, PLIN1) are phosphorylated proteins that coat lipid particles within adipocytes and steroid-producing cells, playing a bidirectional regulatory role in lipolysis. Under basal (feeding) conditions, PLIN1 coats the surface of lipid droplets, acting as a neutral lipid protective barrier and inhibiting lipolysis. Under fasting or exercise conditions, PLIN1 can be highly phosphorylated by protein kinase A, facilitating lipase access to the lipid droplet surface and promoting lipolysis.

[0005] 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 percentage and reducing fat content is an important goal in livestock and poultry breeding. Studies have shown that the PLIN1 gene polymorphism has a certain impact on fat deposition in Qinchuan cattle, and is related to the average daily weight gain, lean meat percentage, and backfat thickness of pigs. It can be considered as a candidate gene for improving lean meat percentage or meat quality in livestock.

[0006] The search for and creation of disease models of human hereditary malnutrition has become a research hotspot in the medical field. Individuals with PLIN1 gene loss-of-function mutations exhibit hereditary lipodystrophy symptoms characterized by abnormal adipose tissue distribution and metabolism, including thyroid hypermetabolism, hyperproteinemia, hyperlipidemia, hyperinsulinemia, and insulin resistance. PLIN1 knockout mice, a common model organism, also exhibit similar characteristics, showing a 30% reduction in fat compared to wild-type mice under the same food consumption conditions, with increased lipolysis and elevated plasma levels of leptin, blood lipids, and blood glucose. However, compared to mice, pigs' metabolism and disease development are more similar to humans, making them a potentially ideal model for human diseases.

[0007] Therefore, finding a sequence that can efficiently and specifically recognize the porcine PLIN1 gene and using the Cas9 enzyme to efficiently cleave this 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 of great reference value for revealing the pathogenesis of human hereditary lipodystrophy and seeking treatment methods. Summary of the Invention

[0008] The purpose of this invention is to provide an sgRNA targeting the porcine PLIN1 gene and its application.

[0009] To achieve the objective of this invention, in a first aspect, this invention provides an 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.

[0010] Preferably, the DNA sequence of the sgRNA action site is shown in SEQ ID NO:2.

[0011] Secondly, the present invention provides a porcine PLIN1 gene targeting vector, wherein the targeting vector is an sgRNA expression vector based on the CRISPR / Cas9 system, wherein the DNA sequence of the sgRNA action site is as described above.

[0012] Furthermore, the targeting vector contains a U6 promoter capable of initiating sgRNA expression and a gene encoding the Cas9 protein. For example, the PX458 vector.

[0013] Thirdly, the present invention provides the application of the targeting vector in the preparation of PLIN1 gene knockout pig cell lines.

[0014] Furthermore, the porcine cell line includes a porcine fetal fibroblast cell line.

[0015] Fourthly, this invention provides a method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line using a CRISPR / Cas9 system. The method involves designing and synthesizing an sgRNA targeting the porcine PLIN1 gene based on the porcine PLIN1 gene sequence, constructing a CRISPR / Cas9 targeting vector containing the sgRNA, transfecting it into porcine fetal fibroblasts, and screening for positive target cell clones, which are then PLIN1 gene knockout porcine fetal fibroblasts. The DNA sequence of the sgRNA action site is as described above.

[0016] Furthermore, the sequences of the specific PCR primers used to identify target-positive cell clones are shown in SEQ ID NO:7-8.

[0017] Fifthly, the present invention provides any of the following applications of the porcine fetal fibroblast cell line prepared according to the above method:

[0018] 1) Used to study the function of the PLIN1 protein;

[0019] 2) Used to construct PLIN1 gene knockout pigs;

[0020] 3) Used as a cell model or drug screening model for studying human hereditary lipodystrophy.

[0021] Sixthly, the present invention provides the application of PLIN1 gene knockout pigs 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 associated metabolic diseases such as thyroid hypermetabolism, hyperproteinemia, hyperlipidemia, hyperinsulinemia, and insulin resistance.

[0022] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0023] This invention provides a method for knocking out the PLIN1 gene in a porcine fetal fibroblast cell line using the CRISPR / Cas9 system. The method involves designing and synthesizing an sgRNA targeting the porcine PLIN1 gene based on the porcine PLIN1 gene sequence, constructing a CRISPR / Cas9 targeting vector containing the sgRNA, and then transfecting it into porcine fetal fibroblasts to obtain PLIN1 gene knockout porcine fetal fibroblasts. These cells can be used to prepare PLIN1 gene-edited pigs, which is not only of great significance for pig breeding but also provides important reference value for elucidating the pathogenesis of human hereditary lipodystrophy and seeking treatment methods. Attached Figure Description

[0024] Figure 1 shows the specific recognition regions of the sgRNA designed for the second, fourth, and fifth exons of the porcine PLIN1 gene in this invention.

[0025] Figure 2 is a schematic diagram of the PLIN1 gene editing site and sgRNA sequence information in a preferred embodiment of the present invention.

[0026] Figure 3 is a sequencing diagram of the CRISPR / Cas9 targeting vector construction in a preferred embodiment of the present invention.

[0027] Figure 4 shows the fluorescence of PFF cells transfected with the CRISPR / Cas9 targeting vector in a preferred embodiment of the present invention.

[0028] Figure 5 shows the cutting efficiency of the PX458-sg2 reconstruction carrier in a preferred embodiment of the present invention.

[0029] Figure 6 is a sequencing comparison diagram of monoclonal cells in a preferred embodiment of the present invention. Detailed Implementation

[0030] This invention aims to provide an sgRNA that specifically recognizes the porcine PLIN1 gene site, its encoding DNA, and its applications. An expression vector is constructed using a short DNA fragment encoding the sgRNA sequence. This expression vector expresses the sgRNA sequence, enabling it to specifically recognize and cleave the porcine PLIN1 gene site, laying the foundation for the creation of gene-edited pigs.

[0031] The present invention adopts the following technical solution:

[0032] This invention designs six sgRNAs (Table 1) specifically targeting the porcine PLIN1 gene based on CRISPR / Cas9 technology, targeting the second, fourth, and fifth exons of the porcine PLIN1 gene (Figure 1).

[0033] Furthermore, the six sgRNAs were constructed into CRISPR / Cas9 targeting vectors, named PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5, and PX458-sg6, respectively. Porcine fetal fibroblasts (PEFs) were transfected with these six CRISPR / Cas9 targeting vectors, and their cleavage efficiency was assessed. It was found that sg1, sg2, and sg3 exhibited cleavage activity against the porcine PLIN1 gene, specifically against the second and fourth exons of the PLIN1 gene. Their corresponding nucleotide sequences are shown in SEQ ID NO:1-3, with sg2 showing the highest cleavage efficiency at 38%.

[0034] Furthermore, the primers used for genotyping are as follows (SEQ ID NO:7-8):

[0035] PLIN1-F: 5′-ACCTCCCTAGTCTGGGTTCC-3′

[0036] PLIN1-R: 5′-TGACTGGTAGCTCTCTGGCT-3′

[0037] The amplified wild-type target fragment is 809 bp in size.

[0038] Furthermore, the px458-sg2 vector containing the sgRNA2 sequence was transfected into PEF cells, and a single-clonal cell line with porcine PLIN1 gene editing was obtained by combining cell flow cytometry sorting technology.

[0039] This invention provides an sgRNA that specifically recognizes the porcine PLIN1 site, its encoding DNA, and its applications. Using a CRISPR / Cas9 targeting vector containing one or more of these sgRNAs, porcine fetal fibroblasts can be transfected to obtain a porcine PLIN1 gene-edited monoclonal cell line. Somatic cell nuclear transfer and other techniques can be used to prepare porcine PLIN1 gene-edited pigs, thereby creating new breeding materials for pigs or establishing a disease model of human hereditary lipodystrophy.

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0041] Example 1: Design and construction of sgRNA sequence specifically recognizing porcine PLIN1 gene

[0042] Based on the porcine PLIN1 gene sequence (Gene ID: 654411 or ENSSCG00000001844) in NCBI, two sgRNAs were designed for each of its second, fourth, and fifth exons (Figure 1), 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 (SEQ ID NO:6), respectively. The sequence information and specific recognition sites are shown in Table 1 and Figure 2.

[0043] Table 1 sgRNA sequences

[0044] Note: The underlined part is the PAM sequence.

[0045] Figure 2 shows two pairs of sgRNAs designed for the second, fourth, and fifth exons of the porcine PLIN1 gene.

[0046] The designed sgRNA was modified by adding restriction enzyme sites to synthesize oligonucleotide chains (Oligo), and the sequence information is shown in Table 2:

[0047] Table 2 Oligo chain synthesis sequence

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

[0049] The commercially available CRISPR / Cas9 vector PX458-GFP (Addgene, USA) was digested with BBSI and recovered via gel electrophoresis. The linearized vector was then ligated to six annealed Oligo double-stranded molecules via their sticky ends. Transformation, plating, single colony picking, and culture propagation by shaking were then performed. Sequencing of the resulting bacterial cultures revealed that all six sgRNAs had been successfully ligated to the PX458-GFP vector and were ready for subsequent experiments. The six reconstructed vectors were named PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5, and PX458-sg6.

[0050] Six vectors, PX458-sg1, PX458-sg2, PX458-sg3, PX458-sg4, PX458-sg5, and PX458-sg6, were sequenced using universal primer pairs. Figure 3 shows the sequencing results, confirming that all six sgRNAs were successfully constructed into the CRISPR / Cas9 targeting vector PX458.

[0051] Example 2: Screening of highly efficient sgRNAs targeting the porcine PLIN1 site

[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 glow green under a fluorescence microscope, indicating successful transfection (Figure 4). Flow cytometry was then used to enrich the glowing cells, and DNA was extracted for sgRNA cleavage efficiency analysis. The primers used for genotyping were 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, at 13% and 12% respectively. No cutting efficiency was detected for the other three vectors.

[0056] Example 3: Screening and Validation of PLIN1 Gene-Edited PEF Cell Lines

[0057] 5 μg of the px458-sg2 vector was transfected into PEF cells. After 48 h, the luminescence was observed under a fluorescence microscope, and the cells were sorted using a flow cytometer. The successfully sorted positive single cells were cultured in a cell culture incubator, with the culture medium changed every 3 days. When the cells reached 80% confluence, they were transferred to 48-well plates for further expansion. The culture medium was changed every 3 days. When the cells reached 80% confluence in the 48-well plates, a portion of the cells were harvested to extract genomic DNA for genotyping. The remaining cells were then further expanded.

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

[0059] In summary, the sgRNA specifically recognizing the porcine PLIN1 gene provided by this invention can efficiently knock out the porcine PLIN1 gene using the CRISPR / Cas9 gene editing system and dual-fluorescence enrichment method. These cells can be used for research on 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 model of lipodystrophy in humans.

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

[0061] The PLIN1 gene knockout porcine fetal fibroblasts prepared by this invention can be used to prepare PLIN1 gene-edited pigs, which is not only of great significance for pig breeding, but also of great reference value for revealing the pathogenesis of human hereditary lipodystrophy and seeking treatment methods.

Claims

1. An sgRNA targeting the porcine PLIN1 gene, characterized in that, The target 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 an sgRNA expression vector based on the CRISPR / Cas9 system, and the targeting vector contains the sgRNA as described in claim 1.

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

4. The application of the targeting vector according to claim 3 in the preparation of PLIN1 gene knockout porcine cell lines; The porcine cell lines include porcine fetal fibroblast cell lines.

5. A method for knocking out the PLIN1 gene in porcine fetal fibroblast cell lines using the CRISPR / Cas9 system, characterized in that... Based on the porcine PLIN1 gene sequence, an sgRNA targeting the porcine PLIN1 gene was designed and synthesized. Then, a CRISPR / Cas9 targeting vector containing the sgRNA was constructed, which was then transformed into porcine fetal fibroblasts. Positive cell clones that hit the target were screened, which are porcine fetal fibroblasts with PLIN1 gene knockout. The DNA sequence of the sgRNA is shown in SEQ ID NO:2.

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