Reagent and method for editing pig CD71 gene

Abstract

The invention discloses a reagent and a method for editing a pig CD71 gene, and belongs to the technical field of biology. The technical problem to be solved by the invention is how to edit the pig CD71 gene. The reagent for editing the pig CD71 gene disclosed by the invention contains sgRNA, and the target sequence of the sgRNA is SEQ ID No.5 or SEQ ID No.2 in a sequence table. The sgRNA disclosed by the invention can specifically recognize a third exon region of the pig CD71 gene, the pig CD71 gene can be edited by utilizing a CRISPR / Cas9 system, and the cutting efficiency of the pig CD71 gene can reach 44.14%. The method can be applied to gene function research, disease-resistant breeding and the like of pig cells or individuals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to reagents and methods for editing the porcine CD71 gene. Background Art

[0002] Pigs are important agricultural and economic animals, which have great scientific value in aspects such as improvement of agricultural production traits, animal models of human diseases, and research on xenotransplantation. With the emergence of gene editing technologies such as ZFN, TALEN, and CRISPR / Cas9, the production traits of pigs (such as meat production, meat quality, disease resistance, etc.) have been greatly improved, promoting the process of genetic improvement of pigs.

[0003] CD71 is the transferrin receptor (TFRC), also known as transferrin receptor protein 1 (TfRl), which is a type II transmembrane protein and the main iron uptake protein on the cell surface; it helps cells uptake iron and promotes DNA synthesis by binding to transferrin (Tf), and plays an important role in the processes of cell maturation, proliferation, and differentiation. Except for highly differentiated cells, CD71 is expressed on the surface of various cells, such as immature red blood cells, placental tissues, and rapidly dividing cells; however, the expression levels of CD71 in different tissues and cells also vary. Therefore, editing the CD71 gene is of great significance for in-depth study of its function. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to edit the porcine CD71 gene.

[0005] To solve the above technical problem, the present invention first provides a reagent for editing the porcine CD71 gene, the reagent contains sgRNA, and the targeting sequence of the sgRNA is SEQ ID No.5 or SEQ ID No.2 in the sequence listing.

[0006] In the above reagent, the sequence of the sgRNA can be SEQ ID No.10 or SEQ ID No.7 in the sequence listing.

[0007] The above reagent can be composed of Cas9 protein and the sgRNA.

[0008] The present invention also provides a recombinant vector for editing the porcine CD71 gene, which is a vector containing a coding gene of Cas9 protein and a DNA molecule of the sgRNA.

[0009] The above recombinant vector may be a vector obtained by inserting the target sequence fragment of the sgRNA (i.e., the DNA fragment shown in SEQ ID No.5 or SEQ ID No.2) upstream of the gRNA backbone of the pX458 vector.

[0010] The present invention also provides a method for editing the porcine CD71 gene, the method comprising: introducing the recombinant vector into porcine cells to achieve editing of the porcine CD71 gene.

[0011] The porcine cells may be porcine fetal fibroblasts or porcine ileal epithelial cells.

[0012] Cells with the CD71 gene edited by using the method for editing the porcine CD71 gene also fall within the protection scope of the present invention.

[0013] The use of the reagent for editing the porcine CD71 gene in the editing of the porcine CD71 gene, or in the preparation of porcine CD71 gene editing products, also falls within the protection scope of the present invention.

[0014] The use of the recombinant vector in the editing of the porcine CD71 gene, or in the preparation of porcine CD71 gene editing products, also falls within the protection scope of the present invention.

[0015] The sgRNA for editing the porcine CD71 gene of the present invention can specifically recognize the third exon region of the porcine CD71 gene, and the CRISPR / Cas9 system can be used to edit the porcine CD71 gene. It is found through experiments that the sgRNA of the present invention in combination with the CRISPR / Cas9 system can efficiently perform gene editing operations on the porcine CD71 gene, and the cleavage efficiency of the porcine CD71 gene can reach 44.14%. The present invention can be applied to aspects such as gene function research and disease-resistant breeding of porcine cells or individuals.

[0016] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Sequencing results of some sgRNA vectors.

[0018] Figure 2This is an electrophoresis diagram for detecting the activity of sgRNA. Note: M represents 1000 bp DNA Marker; 1-3 represent the digestion products of cells transfected with pX458-CD71-sgRNA1; 4-6 represent the digestion products of cells transfected with pX458-CD71-sgRNA2; 7-9 represent the digestion products of cells transfected with pX458-CD71-sgRNA3; 10-12 represent the digestion products of cells transfected with pX458-CD71-sgRNA4; 13-15 represent the digestion products of cells transfected with pX458-CD71-sgRNA5; WT (wide type) represents the digestion products of wild cells; H2O represents the negative control.

[0019] Figure 3 This is a fluorescence diagram of porcine ileal epithelial cells transfected. Note: NC (negative control) represents non-transfected negative control cells. sgRNA1 and sgRNA4 respectively represent GFP-positive cells after 48 h of transfection with the plasmid pX458-CD71-sgRNA1 vector and the plasmid pX458-CD71-sgRNA4 vector.

[0020] Figure 4 This is the flow sorting of positive cells.

[0021] Figure 5 This is the sequencing result of partial edited porcine ileal epithelial cells obtained by sgRNA1.

[0022] Figure 6 This is the sequencing result of partial edited porcine ileal epithelial cells obtained by sgRNA4.

[0023] Figure 7 This is the sequencing result of partial gene-edited porcine fetal fibroblasts. Specific implementation manners

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. In the following examples, quantitative tests are all set with at least three repeated experiments, and the results are averaged.

[0025] Example 1. Construction of a set of sgRNA expression plasmids targeting the CD71 gene 1. Selection of sgRNA target sites Based on the third exon and adjacent partial intron sequence (SEQ ID No.1) of the porcine CD71 gene (Gene ID: 397062, update date: August 17, 2024), sgRNA target sites for gene knockout were designed, with the PAM sequence being NGG. The target sequences of each sgRNA are shown in Table 1 below.

[0026] Table 1 Target sequences of sgRNA

[0027] 2. Construction of sgRNA expression vectors Oligonucleotide (Oligo) DNA sequences were designed according to the target sequences of the 5 sgRNAs described in Table 1 and sent to Beijing Sangon Biotech Co., Ltd. for synthesis. The specific sequences are shown in Table 2 below.

[0028] Table 2 Oligo DNA sequences of sgRNA

[0029] The above two pairs of complementary Oligo DNA strands were prepared into corresponding systems according to the annealing system shown in Table 3, a total of 5 systems. Then, they were reacted in a water bath at 98 °C for 10 min to complete annealing to form double-stranded DNA fragments with sticky ends, which were respectively denoted as sgRNA-1-g, sgRNA-2-g, sgRNA-3-g, sgRNA-4-g, and sgRNA-5-g. The annealing reaction system is shown in Table 3 below.

[0030] Table 3 Annealing reaction system of Oligo strands

[0031] The above double-stranded DNA fragments with sticky ends, sgRNA-1-g, sgRNA-2-g, sgRNA-3-g, sgRNA-4-g, and sgRNA-5-g, were respectively reacted with the pX458 vector plasmid (addgene, #48138) in the plasmid recombination ligation reaction system (Table 4) at 16 °C for 1 h. The correctly sequenced recombinant vectors were respectively denoted as pX458-CD71-sgRNA1 vector, pX458-CD71-sgRNA2 vector, pX458-CD71-sgRNA3 vector, pX458-CD71-sgRNA4 vector, and pX458-CD71-sgRNA5 vector. Each recombinant vector can transcribe sgRNAs targeting SEQ ID No.2 - SEQ ID No.6, namely sgRNA1-5. The plasmid recombination ligation reaction system is shown in Table 4 below.

[0032] Table 4 Plasmid recombination ligation reaction system

[0033] The sequences of each sgRNA are as follows: sgRNA1: AGGUCUGCCCAAAAUAAGCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc (SEQ ID No.7); sgRNA2: AGUAGCGCAAGCUUACUUGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc (SEQ ID No.8); sgRNA3: CGCAAGCUUACUUGAUGGUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc (SEQ ID No.9); sgRNA4: GUAGCCAAUCAUAAAUCCUAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc (SEQ ID No.10); sgRNA5: AGGAUUUAUGAUUGGCUACUguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc (SEQ ID No.11).

[0034] The ligation product obtained above was transformed into competent Escherichia coli DH5α cells, spread on a pre-prepared LB plate (ampicillin-resistant), and then incubated overnight in a 37°C bacterial incubator. The next morning, growing single colonies were selected for small-scale expansion by shaking, and then a part of the bacterial sample was taken for sequencing to identify the genotype of the single colonies. The sequencing results of some plasmids are shown in Figure 1 .

[0035] 3. Detection of sgRNA activity The pX458-CD71-sgRNA1 vector, pX458-CD71-sgRNA2 vector, pX458-CD71-sgRNA3 vector, pX458-CD71-sgRNA4 vector, and pX458-CD71-sgRNA5 vector prepared in the above step 2 were respectively transfected into porcine fetal fibroblasts (XU Kui, ZHOU Yan-rong, SHANG Hai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAO Shao-bo, LI Kui. Journal of Integrative Agriculture, 2023, 22(7): 2188-2199.) by nucleofection (Lonza, Nucleofector system, VPI-1002) to obtain 5 kinds of recombinant cells respectively.

[0036] For the recombinant cells obtained 48 h after transfection, total cellular DNA was extracted and PCR amplification was performed using the following primers (Table 5). The sequence of the PCR amplification product before editing was SEQ ID No. 12. The amplification reaction procedure was as shown in Table 6 below.

[0037] Table 5 PCR primers

[0038] Table 6 PCR reaction conditions

[0039] T7E1 enzyme (which can recognize and cleave incompletely paired DNA) and buffer were added to the PCR reaction product in a PCR instrument, and then the digestion reaction was carried out at 37 °C for 1 h and at 72 °C for 2 min. The reaction system was as shown in Table 7 below. The digested product was subjected to agarose gel electrophoresis, and the cleavage efficiency was calculated using ImageJ software.

[0040] Table 7 Digestion reaction system

[0041] The results were as Figure 2As shown, after digestion with T7E1 enzyme, three bands of 591 bp, 383 bp and 208 bp appeared in the PCR products of pX458-CD71-sgRNA1 transfected cells, indicating that sgRNA1 caused sequence mutations in the targeting region. The cleavage efficiency of sgRNA1 was calculated to be about 35.80% by calculating the band brightness with ImageJ software. Three bands of 591 bp, 453 bp and 138 bp appeared in the PCR products of pX458-CD71-sgRNA2 transfected cells, indicating that sgRNA2 caused sequence mutations in the targeting region. The cleavage efficiency of sgRNA2 was calculated to be about 30.59% by calculating the band brightness with ImageJ software. Three bands of 591 bp, 448 bp and 143 bp appeared in the PCR products of pX458-CD71-sgRNA3 transfected cells, indicating that sgRNA3 caused sequence mutations in the targeting region. The cleavage efficiency of sgRNA3 was calculated to be about 29.70% by calculating the band brightness with ImageJ software. Three bands of 591 bp, 345 bp and 246 bp appeared in the PCR products of pX458-CD71-sgRNA4 transfected cells, indicating that sgRNA4 caused sequence mutations in the targeting region. The cleavage efficiency of sgRNA4 was calculated to be about 44.14% by calculating the band brightness with ImageJ software. Three bands of 591 bp, 330 bp and 261 bp appeared in the PCR products of pX458-CD71-sgRNA5 transfected cells, indicating that sgRNA5 caused sequence mutations in the targeting region. The cleavage efficiency of sgRNA5 was calculated to be about 21.05% by calculating the band brightness with ImageJ software. It can be seen from the above that sgRNA1 and sgRNA4 mediated better knockout effects of Cas9 protein and were used for subsequent experiments.

[0042] Example 2. Screening of porcine ileal epithelial cell lines with CD71 gene knockout The pX458-CD71-sgRNA1 vector and pX458-CD71-sgRNA4 vector with better cleavage activity obtained in Example 1 were respectively transfected into cells. One day before transfection, porcine ileal epithelial cells (Xu Changjiang, Wang Xiaopeng, Xu Kui, Zhang Xiuling, Xiang Guangming, Zhao Haiquan, Mou Yulian, Lin Xiao, Li Kui. Construction of pAPN gene knockout IPI-2I cell line using CRISPR / Cas9 editing system. China Animal Husbandry & Veterinary Medicine, 2021, 48(7): 2282-2290) were resuscitated into a 10 cm petri dish, and cell transfection could be carried out when the cells reached 70%-80% confluence. The cell transfection method was electroporation based on a nucleofector, and the (Lonza, Nucleofector system, VPI-1002) single-well nucleofection system was used for electroporation. The specific operation process is as follows: First, cells were collected and the cell number was adjusted to 1×10 6 cells / tube. Then, they were centrifuged at 1000 rpm for 6 min to remove the culture medium as completely as possible. After 48 h of electroporation, the luminescence was observed under a fluorescence microscope (as shown in Figure 3 ). After transfection, some cells could emit green fluorescence, indicating successful cell transfection. Fluorescent positive monoclonal cells were sorted by flow cytometry. The positive cells transfected with pX458-CD71-sgRNA1 plasmid and pX458-CD71-sgRNA4 plasmid accounted for 29.8% and 25.7% of the total cells, respectively, as shown in Figure 4 . The sorted cells were continuously cultured in a cell incubator, and the culture medium was changed every 3 days. After about 14 days of culture, monoclonal cells with a confluence of about 80% would grow. The monoclonal cells were expanded in culture and subjected to PCR amplification and Sanger sequencing. The results showed that 35 monoclonal cells were obtained from the cells transfected with pX458-CD71-sgRNA1, and gene editing occurred in 6 cells, with a gene editing efficiency of 17.1%. The sequencing maps of some gene-edited cells are shown in Figure 5 . 30 monoclonal cells were obtained from the cells transfected with pX458-CD71-sgRNA4, and gene editing occurred in 8 cells, with a gene editing efficiency of 26.7%. The sequencing maps of the gene-edited cells are shown in Figure 6 . In summary, the pX458-CD71-sgRNA4 vector plasmid has a relatively high gene editing efficiency.

[0043] Example 3. Establishment of a porcine fetal fibroblast cell line with CD71 gene knockout One day before transfection, primary Large White pig fetal fibroblasts (XU Kui, ZHOU Yan-rong, SHANGHai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAOShao-bo, LI Kui. Journal of Integrative Agriculture, 2023, 22(7): 2188-2199.) were resuscitated into a 10-cm Petri dish, and cell transfection could be carried out when the cells reached 70-80% confluence. The transfection procedure was strictly carried out according to the instructions of the Basic Primary Fibroblasts Nucleofector Kit (Lonza). Specifically, 4 μg of the recombinant vector pX458-CD71-sgRNA4 obtained in Example 1 was transfected into pig fetal fibroblasts by electroporation to obtain 46 recombinant cells, and 14 cells underwent gene editing, with a gene editing efficiency of 30.4%. The sequencing diagrams of some gene-edited cells are shown as Figure 7 shown.

[0044] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A reagent for editing the porcine CD71 gene, characterized in that: The reagent contains sgRNA, and the targeting sequence of the sgRNA is SEQ ID No.5 or SEQ ID No.2 in the Sequence Listing.

2. The reagent according to claim 1, characterized in that: The sequence of the sgRNA is SEQ ID No.10 or SEQ ID No.7 in the Sequence Listing.

3. The reagent according to claim 1 or 2, characterized in that: The reagent consists of Cas9 protein and the sgRNA.

4. A recombinant vector for editing the porcine CD71 gene, which is a vector containing a coding gene of Cas9 protein and a DNA molecule of the sgRNA described in claim 1 or 2.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is a vector obtained by inserting the target sequence fragment of the sgRNA upstream of the gRNA backbone of the pX458 vector.

6. Editing method for porcine CD71 gene, comprising: Introducing the recombinant vector described in claim 4 or 5 into porcine cells to achieve editing of the porcine CD71 gene.

7. The method according to claim 6, wherein: The porcine cells are porcine fetal fibroblasts or porcine ileal epithelial cells.

8. Cells with the CD71 gene edited obtained by the method described in claim 6 or 7.

9. Use of the reagent described in any one of claims 1-3 in the editing of the porcine CD71 gene, or in the preparation of a porcine CD71 gene editing product.

10. Use of the recombinant vector described in claim 4 or 5 in the editing of the porcine CD71 gene, or in the preparation of a porcine CD71 gene editing product.

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