SgRNA for targeted knockout of ASIP gene and application thereof

Abstract

The invention discloses sgRNA for targeted knockout of an ASIP gene and application of the sgRNA, belongs to the technical field of genetic engineering, and provides the sgRNA for targeted knockout of the ASIP gene, the sgRNA comprises as1-sgRNA and as2-sgRNA, the nucleotide sequence of the as1-sgRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the as2-sgRNA is as shown in SEQ ID NO.3. The invention further discloses a preparation method of the sgRNA for targeted knockout of the ASIP gene and application of the sgRNA for targeted knockout of the ASIP gene. The sgRNA for targeted knockout of the ASIP gene is applied to preparation of a duck-derived cell strain with ASIP gene mutation. According to the invention, the sgRNA of the intron region of the specific targeting duck ASIP gene is adopted, and the CRISPR / Cas9 technology is utilized to construct the duck fibroblast strain with the ASIP gene knocked out, so that a foundation is laid for deeply researching an expression regulation mechanism of the ASIP gene, and the genetic mechanism of duck feather color variation can be further finely analyzed.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an sgRNA for targeted knockout of an ASIP gene and an application thereof. Background Art

[0002] Ducks are an important poultry species with a wide range of breeding scales and important economic value worldwide. As an important economic poultry, ducks have extremely high commercial value in terms of meat and eggs. Their important economic traits such as coat color, growth performance, and fat deposition have always been the focus of research in the field of poultry genetic breeding. Studies have shown that these traits are often finely regulated by many genes, among which the agouti signaling protein (ASIP) gene plays a key role.

[0003] The agouti signaling protein (ASIP) gene is located on duck chromosome 21 and is a key gene that affects feather color. Previous studies have found that the causal variation of duck Agouti feather color is located in the upstream transposon insertion of the ASIP gene, and the transposon insertion causes the ASIP gene transcript to undergo alternative splicing, affecting the gene expression level, but there are few reports on its regulatory mechanism of affecting gene expression.

[0004] As a cutting-edge genome site-specific editing technology, CRISPR / Cas9 plays a key role in modern molecular biology research. It mainly relies on crRNA and tracrRNA to combine into sgRNA, mediating the recognition and cutting of the target sequence by the Cas9 protein to cause DNA double-strand breaks, and there is a self-repair mechanism for DNA damage in the cell, which uses the repair mechanism to increase or decrease the number of bases and change the original sequence, thereby performing gene editing. Therefore, the present invention uses the CRISPR / Cas9 system targeting the ASIP gene to construct duck embryonic fibroblasts with a transposon insertion mutation upstream of the duck ASIP gene, indicating that transposon insertion is a key factor affecting the expression level of the ASIP gene. Summary of the invention

[0005] To solve the above technical problems, the present invention proposes a sgRNA for targeted knockout of the ASIP gene and its application, and constructs a duck ASIP gene mutated fibroblast cell line using the CRISPR / Cas9 gene editing system containing the sgRNA, laying a foundation for in-depth research on the regulatory mechanism of structural variation of the ASIP gene.

[0006] To achieve the above object, the present invention provides an sgRNA for targeted knockout of the ASIP gene, wherein the sgRNA comprises as1-sgRNA and as2-sgRNA, the nucleotide sequence of the as1-sgRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the as2-sgRNA is shown in SEQ ID NO.3.

[0007] The present invention also provides the use of the sgRNA for targeted knockout of the ASIP gene in preparing a duck-derived cell line with ASIP gene mutation.

[0008] Preferably, the duck-derived cell line is duck embryonic fibroblasts in the logarithmic growth phase.

[0009] The present invention also provides a vector for targeted knockout of the ASIP gene, wherein the vector is an sgRNA expression vector based on the CRISPR / Cas9 system, and the vector contains the sgRNA.

[0010] The present invention also provides a CRISPR / Cas9 gene editing system for targeted knockout of the ASIP gene, wherein the CRISPR / Cas9 gene editing system comprises the coding sequence of the sgRNA and the Cas protein.

[0011] The present invention also provides the use of the vector for targeted knockout of the ASIP gene or the CRISPR / Cas9 gene editing system for targeted knockout of the ASIP gene in preparing a duck-derived cell line with ASIP gene mutation.

[0012] The present invention also provides a method for knocking out the ASIP gene in a mutant duck-derived cell line, comprising the following steps:

[0013] (1) According to the sgRNA targeting the ASIP gene knockout, two reverse complementary nucleotide chains are designed and mixed and annealed to form a double strand;

[0014] (2) connecting the double-stranded DNA obtained in step (1) with the pX330 vector to obtain a recombinant vector;

[0015] (3) transfecting duck-derived cells with the recombinant vector obtained in step (2), and obtaining duck-derived cell lines with duck ASIP gene mutations through cultivation, screening, and identification;

[0016] The duck-derived cells in step (3) are duck embryonic fibroblasts in the logarithmic growth phase.

[0017] The present invention also provides a mutant duck-derived cell strain with ASIP gene knocked out, which is prepared by utilizing the method for the mutant duck-derived cell strain with ASIP gene knocked out.

[0018] The present invention also provides application of the ASIP gene knockout mutant duck-derived cell strain in duck breeding.

[0019] The present invention also provides the use of the ASIP gene knockout mutant duck-derived cell strain in breeding ducks with different feather colors.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention provides a sgRNA that specifically targets the intron region of the duck ASIP gene, and can use CRISPR / Cas9 technology to construct a duck fibroblast cell line with ASIP gene knockout, laying a foundation for in-depth research on the expression and regulation mechanism of the ASIP gene, which is conducive to further refining the analysis of the genetic mechanism of duck feather color variation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The pX330-as1-sgRNA plasmid and the pX330-as2-sgRNA plasmid sequencing maps are shown in Figure 1, where A is the pX330-as1-sgRNA plasmid sequencing map and B is the pX330-as2-sgRNA plasmid sequencing map;

[0024] Figure 2 This is the electrophoresis diagram of PCR products of DD genotype monoclonal cells. 2 in the figure represents the control ID genotype cells, 1 and 3 to 7 in the figure represent positive single cell clones with DD genotype, M in the figure represents DL2000 DNA marker, and the sizes of maker bands from top to bottom in the figure are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively;

[0025] Figure 3 The difference in ASIP gene expression between DD genotype duck fibroblast cell line and wild ID genotype duck fibroblast cell line. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] All primers used in the present invention were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0032] Example 1

[0033] 1. sgRNA target selection and oligonucleotide chain design and synthesis

[0034] Research has confirmed that there is a PAV (presence / absence variants, gain and loss variants) in the upstream region of the ASIP gene, which is a causal mutation of duck feather color. It has been proved that this mutation site is the causal mutation of the ASIP gene, a key gene affecting duck feather color traits. The genotype of the cells used is ID type, and the expression level of the ID type is higher than that of the DD type. After knockout, the I allele is not expressed, resulting in a decrease in the gene relative to the wild type, causing changes in feather color.

[0035] The transposon sequence of the ASIP gene was found on the NCBI website (NCBI accession number: JACEUL010000017). According to the CRISPR / Cas targeting principle, the sgRNA sequence targeting the ASIP gene transposon mutation site was selected with a high score (as shown in Table 1) using the CRISPR online design tool (http: / / crispr.mit.deu / ). The restriction site sequence was added to the 5' end of the sense strand and the antisense strand to complement the sticky end formed after BbsI digestion.

[0036] Table 1 Nucleotide sequences of sgRNA target sites

[0037]

[0038]

[0039] 2. Construction of pX330-sgRNA knockout vector

[0040] 1. The pX330 vector was linearized with endonuclease BbsI and digested at 37°C for 4h. The gel was cut and identified by electrophoresis using 1% agarose gel and recovered according to the instructions of the gel recovery kit (OMEGA kit, Henan Dongge Biotechnology Co., Ltd.). The sgRNA oligonucleotide primer powder was diluted to a concentration of 100 μmol / L with sterile enzyme-free water, and 5 μL of as1-sgRNA was mixed with 5 μL of as1-sgRNA antisense strand, and 5 μL of as2-sgRNA was mixed with 5 μL of as2-sgRNA antisense strand, respectively, at 37°C for 30min; 95°C for 5min; naturally cooled to room temperature, and annealed to form double-stranded as1-sgRNA and double-stranded as2-sgRNA.

[0041] 2. The double-stranded as1-sgRNA and double-stranded as2-sgRNA were connected to the pX330 vector respectively. The connection system was: 1μL T4 ligase, 1μL 10×Ligase, 6μL double-stranded as1-sgRNA or 6μL double-stranded as2-sgRNA, 2μL linearized pX330 vector, and connected at 37℃ for 1h to obtain the connection products pX330-as1-sgRNA and pX330-as2-sgRNA.

[0042] 3. Transform the ligation products pX330-as1-sgRNA and pX330-as2-sgRNA into DH5α competent cells, evenly spread on ampicillin-resistant solid LB plates, and invert and culture at 37°C overnight. Pick a single clone for shaking culture, and select positive clones to be sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0043] The results are as follows Figure 1Middle A and Figure 1 As shown in B, the as1-sgRNA and as2-sgRNA sequences were correctly inserted into the pX330 plasmid, and the position, direction and sequence of the inserted sequence were consistent with expectations, indicating that the pX330-as1-sgRNA and pX330-as2-sgRNA expression vectors were successfully constructed.

[0044] 3. Duck embryonic fibroblast culture and knockout vector transfection

[0045] 1. Duck embryonic fibroblasts in logarithmic growth phase (Synopharm (Shanghai) Biotechnology Co., Ltd.) were inoculated into 60 mm cell culture dishes and routinely cultured using DMEM-F12 medium (Thermo Fisher Scientific) containing 10% serum. The cell density before transfection was 80%.

[0046] 2. The recombinant vector and lentiviral expression vector PLKO.1 (donated from the School of Basic Medical Sciences, Zhengzhou University) were mixed in a volume ratio of pX330-sgRNA (pX330-as1-sgRNA and pX330-as2-sgRNA were 2:2): PLKO.1 was 4:1. The mixed lentiviral expression system was co-transfected into the duck embryonic fibroblasts cultured in step 1 according to the instructions of Lipofectamine@3000 transfection reagent (Thermo Fisher Scientific). At the same time, duck embryonic fibroblasts transfected with only pX330 were set as a control group.

[0047] 4. Monoclonal screening of ASIP gene mutant cell lines

[0048] 1. After 24 hours of transfection, replace with DMEM-F12 medium containing 10% serum and a final concentration of 6 μg / mL puromycin and continue culturing for 24 hours to screen for successfully transfected duck embryonic fibroblasts. When all cells in the control group die, replace with DMEM-F12 medium containing 10% serum for subsequent positive monoclonal cell strain screening.

[0049] 2. Take the surviving cells from the knockout group and inoculate them in a 96-well plate using the limiting dilution method. The specific steps are as follows: Digest the cells with trypsin and count them. Use the gradient dilution method to dilute them to 1 cell per 100 μL of culture medium. Spread one cell dilution per well in a 96-well plate. After the cells form monoclonal colonies, spread them evenly in a 24-well plate after digestion. Expand the cells in the 24-well plate and use a portion of them to extract genomic DNA for typing identification (such as Figure 2 After the positive cell clones with DD genotype were grown to confluence, they were inoculated into 6-well plates for culture.

[0050] 5. Identification of ASIP gene mutant cell lines

[0051] 1. The cells in the 6-well plate were expanded and cultured. One part was used to extract genomic DNA for typing identification to obtain positive cell clones with DD genotype; the other part was used to extract RNA and detect the relative level of ASIP gene mRNA by RT-qPCR. Figure 3 As shown, there was a significant difference in the mRNA level of ASIP gene in ASIP gene mutant cells (DD) and normal cells (ID), and the ASIP gene expression level in ID genotype cells was significantly higher than that in DD genotype cells, indicating that the ASIP gene mutant duck fibroblast cell line was successfully constructed.

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A sgRNA for targeting and knocking out the ASIP gene, characterized in that: The sgRNA comprises as1-sgRNA and as2-sgRNA, the nucleotide sequence of the as1-sgRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the as2-sgRNA is shown in SEQ ID NO.

3.

2. Use of the sgRNA for targeted knockout of the ASIP gene as claimed in claim 1 in preparing a duck-derived cell line with ASIP gene mutation.

3. The application according to claim 2, characterized in that: The duck-derived cell strain is duck embryonic fibroblasts in the logarithmic growth phase.

4. A vector for targeted knockout of ASIP gene, characterized in that: The vector is a sgRNA expression vector based on the CRISPR / Cas9 system, and the vector contains the sgRNA according to claim 1.

5. A CRISPR / Cas9 gene editing system for targeted knockout of ASIP gene, characterized in that: The CRISPR / Cas9 gene editing system comprises the coding sequences of the sgRNA and Cas protein described in claim 1.

6. Use of the vector for targeted knockout of ASIP gene as claimed in claim 4 or the CRISPR / Cas9 gene editing system for targeted knockout of ASIP gene as claimed in claim 5 in preparing a duck-derived cell line with ASIP gene mutation.

7. A method for knocking out the ASIP gene in a mutant duck cell line, characterized in that: The following steps are involved: (1) According to the sgRNA targeting ASIP gene knockout according to claim 1, two reverse complementary nucleotide chains are designed and mixed and annealed to form a double strand; (2) connecting the double-stranded DNA obtained in step (1) with the pX330 vector to obtain a recombinant vector; (3) transfecting duck-derived cells with the recombinant vector obtained in step (2), and obtaining duck-derived cell lines with duck ASIP gene mutations through cultivation, screening, and identification; The duck-derived cells in step (3) are duck embryonic fibroblasts in the logarithmic growth phase.

8. A mutant duck cell line with knockout of ASIP gene prepared by the method for knocking out of ASIP gene mutant duck cell line according to claim 7.

9. Use of the ASIP gene knockout mutant duck-derived cell line as claimed in claim 8 in duck breeding.

10. Use of the mutant duck-derived cell line with ASIP gene knockout as claimed in claim 8 in breeding ducks with different feather colors.

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