Method for knocking out pig E4F1 gene by using CRISPR / Cas9 system

Abstract

The invention discloses a method for knocking out a pig E4F1 gene by using a CRISPR / Cas9 system, and belongs to the technical field of gene editing. The method comprises the following steps: constructing a CRISPR / Cas9 gene knockout vector containing sgRNA by utilizing an sgRNA composition (a pair of sgRNA) of a specific targeting pig E4F1 gene, and then carrying out pig E4F1 gene knockout by utilizing the vector. According to the method disclosed by the invention, high-efficiency knockout of the pig E4F1 gene can be realized, so that the construction efficiency of E4F1 gene knockout cells and E4F1 gene knockout pigs is remarkably improved, and an effective method and basis are provided for functional research and application of the pig E4F1 gene.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and in particular relates to a method for knocking out the pig E4F1 gene by utilizing the CRISPR / Cas9 system. Background Art

[0002] E4F transcription factor 1 (E4F1) is a member of the GLI-Kruppel family of zinc finger proteins and is now widely recognized as a transcription factor. Its DNA binding activity is regulated by the action of adenovirus E1A. A 50-kDa amino-terminal product is generated from the full-length protein by proteolytic cleavage. The protein is differentially regulated by phosphorylation induced by E1A. In the absence of E1A, the full-length gene product represses transcription from the E4 promoter, while the 50-kDa form acts as a transcriptional activator in its presence. Alternative splicing results in multiple transcripts encoding different proteins. E4F1 plays a key role in regulating various cellular processes, including cell growth, proliferation, differentiation, apoptosis and necrosis, DNA damage response, and cellular metabolism. In the study of germ cells, E4F1 was found to maintain its self-renewal and homeostasis in spermatogonial stem cells by regulating mitochondrial function and metabolism, a finding that revealed the important role of E4F1 in germ cell development.

[0003] At present, there are relatively few studies on the E4F1 gene in mammals such as pigs. By using the CRISPR / Cas9 system to knock out the pig E4F1 gene and study the function of the pig E4F1 gene in vitro and in vivo, it is of great significance to study the function of the E4F1 gene in the development of pigs and its mechanism of action.

[0004] CRISPR / Cas9 is an adaptive immune defense formed by bacteria and archaea in the long-term evolution process, which can be used to fight against invading viruses and foreign DNA. CRISPR / Cas9 gene editing technology is a technology for specific DNA modification of targeted genes, which is also a relatively cutting-edge method for gene editing. CRISPR / Cas9-based gene editing technology has shown great application prospects in the application field of gene therapy for a series of diseases (such as blood diseases, tumors and other genetic diseases). The development of an efficient CRISPR / Cas9 system knockout method for the porcine E4F1 gene is of great significance for improving the efficiency of porcine E4F1 gene knockout and the efficiency of constructing E4F1 gene knockout cells or animals. Summary of the invention

[0005] In view of this, the present invention aims to provide a method for efficiently knocking out the pig E4F1 gene using the CRISPR / Cas9 system, so as to provide basic materials for studying the function and mechanism of the E4F1 gene in the development process of pigs.

[0006] In order to achieve the above object, the present invention specifically provides the following technical solutions: In a first aspect, the present invention provides an sgRNA composition specifically targeting the porcine E4F1 gene based on CRISPR / Cas9 technology, comprising a first sgRNA and a second sgRNA, wherein the first sgRNA targets a sequence from position 7750 to position 7769 of the E4F1 gene, and the second sgRNA targets a sequence from position 8167 to position 8186 of the E4F1 gene.

[0007] For the target sequence on the above-mentioned porcine E4F1 gene, the sgRNA sequences in the sgRNA composition are preferably as follows: The first sgRNA: 5'-CGCTTATCCGGCACCACCGA-3' (SEQ ID NO.3); Second sgRNA: 5'-GACGTCACCCGTGATTCACC-3' (SEQ ID NO. 4).

[0008] The experimental data of the present invention show that the sgRNA composition shown in SEQ ID NO.3 and SEQ ID NO.4 (denoted as SG3,4) can achieve excellent targeting efficiency and E4F1 gene knockout efficiency, which is conducive to the rapid and efficient construction of E4F1 gene knockout cells or animals.

[0009] A second aspect of the present invention provides a CRISPR / Cas9 gene knockout vector specifically targeting the porcine E4F1 gene. Specifically, the knockout vector contains the sgRNA specifically targeting the porcine E4F1 gene.

[0010] In some embodiments of the present invention, the CRISPR / Cas9 gene knockout vector is specifically a PX459 vector connected with the sgRNA specifically targeting the porcine E4F1 gene. Taking the PX459 vector as an example, the present invention also provides a method for constructing a CRISPR / Cas9 gene knockout vector, which may specifically include the following steps: (1) Digest the vector PX459 with BbSI to obtain a linearized vector; (2) Add BbSI restriction sites to both ends of the sgRNA, anneal the fragments of the sgRNA sequence with the BbSI restriction site to form fragments containing sticky ends of the BbSI restriction site; (3) The annealing product obtained in step (2) is connected to the linearized vector obtained in step (1) to construct a PX459 vector connected with the above-mentioned sgRNA.

[0011] Preferably, in the above construction method, the enzyme digestion reaction system of step (1) is: vector PX459 1.5 μL (1000 ng), restriction endonuclease BbSI 1 μL, Green Buffer 10× 1 μL, ddH2O to make up to 20 μL; and the enzyme digestion reaction program is: 37°C, 30 min.

[0012] Preferably, in the above construction method, the annealing reaction system of step (2) is: NEB buffer 2 μL, upstream sequence 1 μL, downstream sequence 1 μL, ddH2O to make up to 20 μL; and the annealing reaction program is: 95°C, 5 min; 37°C, 1 h.

[0013] Preferably, in the above construction method, the ligation reaction system of step (3) is: 3 μL of annealing product, 2 μL of enzyme digestion product, 1 μL of T4 DNA Ligase, 2 μL of Buf T4 Ligase, and ddH2O to make up to 20 μL; and the ligation reaction procedure is: 22°C, 1 h.

[0014] The third aspect of the present invention provides the use of the above-mentioned sgRNA composition and / or CRISPR / Cas9 gene knockout vector in efficiently knocking out the pig E4F1 gene.

[0015] Preferably, in the above application, the CRISPR / Cas9 gene knockout vectors containing the two groups of sgRNA compositions specifically targeting the porcine E4F1 gene are respectively introduced into cells together, so that the E4F1 gene is edited at two sites to cause fragment deletion, thereby obtaining positive cells. Among them, the introduction of the CRISPR / Cas9 gene knockout vector into cells can be achieved by existing technologies, including but not limited to electroporation transfection.

[0016] In the above application, positive cells can be determined by conventional methods such as PCR, etc. Preferably, they can be detected and determined by specific primers with sequences such as those shown in SEQ ID NO. 7-8.

[0017] Preferably, in the above application, the method for detecting the expression level of porcine E4F1 gene in positive cells comprises the following steps: S1, extract the total RNA of the cells to be tested and reverse transcribe it into cDNA; S2. Using the cDNA from step S2 as a template, using specific primers as shown in SEQ ID NOs. 9-10, amplify the E4F1 gene by fluorescence quantitative PCR (qPCR) to detect the expression level of the E4F1 gene.

[0018] In the above detection method, qPCR can select GAPDH as an internal reference gene; more preferably, the primer sequence used for GAPDH is shown in SEQ ID NO.11-12.

[0019] More preferably, in the above step S2, the qPCR reaction system is: 2×Universal SYBR FastqPCR Mix 10μL, ddH2O 8.2μL, 10μM upstream primer 0.4μL, 10μM downstream primer 0.4μL, cDNA template 1.0μL; and its reaction program is: 95℃ pre-denaturation for 3min; 95℃ denaturation for 5s, 60℃ annealing for 30s, 40 cycles; the instrument automatically sets the melting curve. The amplification curve and melting curve obtained by real-time monitoring of qPCR are analyzed to calculate the expression level of the E4F1 gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully constructed porcine E4F1 gene knockout cells for the first time using the CRISPR / Cas9 system, providing important materials for studying the function of the porcine E4F1 gene in vitro and in vivo. The present invention designs dual gene editing target sites for the porcine E4F1 gene, which can be specifically recognized by the Cas9 endonuclease, thereby mediating double-strand breaks, and mutating the gene through non-homologous end connection under the action of the self-repair system, and then designs an sgRNA composition, and uses tRNA-sgRNA-sgRNA scaffold as the minimum repeating unit, and uses the PTG / Cas9 strategy to construct a Cas9-E4F1 monocistronic vector, thereby achieving efficient and accurate knockout of the target gene.

[0021] According to the design principle of CRISPR / Cas9 recognition target site, a large number of sgRNAs can be designed for the porcine E4F1 gene. However, the results of transfection experiments show that the knockout efficiency of different sgRNA combinations varies significantly. The porcine E4F1 gene target site and sgRNA composition provided by the present invention can significantly improve the targeting efficiency of the CRISPR / Cas9 knockout vector, thereby significantly improving the knockout efficiency of the porcine E4F1 gene. At the same time, the present invention provides specific primers and corresponding detection methods for the quantitative analysis of the expression level of the porcine E4F1 gene, so that the expression level of the porcine E4F1 gene can be detected quickly and accurately.

[0022] The present invention systematically provides sgRNA specifically targeting the porcine E4F1 gene, a CRISPR / Cas9 gene knockout vector containing the sgRNA, a method for knocking out the porcine E4F1 gene using the vector, and a method for detecting the knockout of the porcine E4F1 gene, etc., which is highly practical and provides an effective method and basis for the functional research and application of the porcine E4F1 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a map of the PX459 vector used in Example 2 of the present invention, which has two BbSⅠ restriction sites at 245 bp and 267 bp.

[0024] Figure 2 These are the sequencing results of the sgRNA sequences with BbSI restriction sites inserted into the CRISPR / Cas9 gene knockout vectors containing four sgRNAs specifically targeting the porcine E4F1 gene in Example 2 of the present invention; wherein SG1 is a CRISPR / Cas9 gene knockout vector containing sgRNA-1, SG2 is a CRISPR / Cas9 gene knockout vector containing sgRNA-2, SG3 is a CRISPR / Cas9 gene knockout vector containing sgRNA-3, and SG4 is a CRISPR / Cas9 gene knockout vector containing sgRNA-4.

[0025] Figure 3 It is an electrophoresis diagram of DNA extracted from the E4F1 gene mutation cell line in Example 3 of the present invention; wherein, from left to right, lanes 1, 6, 8, and 13 are DL2000bp DNA Marker, lanes 2 and 9 are E4F1 gene negative controls, lanes 3-5 are knockout bands of the sgRNA-1 and sgRNA-2 combination (SG1, 2) of the E4F1 gene, and lanes 10-12 are knockout bands of the sgRNA-3 and sgRNA-4 combination (SG3, 4) of the E4F1 gene.

[0026] Figure 4 4 is the mRNA expression detection result of the E4F1 gene after 48 hours of electroporation in Example 3 of the present invention; wherein, NC is an empty vector, SG1, 2 and SG3, 4 are PX459 vectors carrying sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4, respectively, * indicates a significant difference (P<0.05), and ** indicates an extremely significant difference (P<0.01).

[0027] Figure 5 The Western blotting detection results of E4F1 protein after 48 hours of electroporation in Example 3 of the present invention; wherein, NC is an empty vector, SG1,2 and SG3,4 are PX459 vectors carrying sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4, respectively, * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The term "comprising" and any variation thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions. The terms "first" and "second" in the specification and claims of the present invention are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating a specific order or primary and secondary relationship of the indicated technical features.

[0029] In view of the lack of E4F1 gene knockout cells for studying the function of the E4F1 gene in the prior art, the present invention develops a scheme for efficiently knocking out the porcine E4F1 gene based on CRISPR / Cas9, which specifically includes the following steps: obtaining a sgRNA composition that can efficiently target the porcine E4F1 gene; constructing CRISPR / Cas9 gene knockout vectors based on the sgRNA in the composition; transfecting the CRISPR / Cas9 gene knockout vectors into cells in a combined form to knock out the porcine E4F1 gene; and detecting the expression level of the porcine E4F1 gene.

[0030] It can be understood that in the scheme of the present invention, sgRNA is one of the key factors and basis affecting the knockout efficiency of the pig E4F1 gene. Based on the analysis of the pig E4F1 gene sequence, the present invention screens the target site of the pig E4F1 gene and designs the sgRNA for the target site according to the design principle of CRISPR / Cas9 recognition of the target site, and repeatedly transfects the sgRNA of the E4F1 gene into pig kidney fibroblasts in the form of two pairs by electroporation transfection, and then screens and verifies the sgRNA composition with the best efficiency in terms of DNA, RNA and protein expression levels, and finally screens and obtains the sgRNA composition with the highest knockout efficiency.

[0031] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0032] If no specific techniques or conditions are specified in the following examples, the procedures are carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0033] Example 1 This example provides a sgRNA composition specifically targeting the porcine E4F1 gene, including a first sgRNA and a second sgRNA; a large number of experiments have shown that when the first sgRNA targets the sequence from position 7750 to position 7769 of the porcine E4F1 gene, and the second sgRNA targets the sequence from position 8167 to position 8186 of the porcine E4F1 gene, the knockout efficiency of the sgRNA composition is the highest. Specifically, the sequences of the sgRNAs in the composition (denoted as SG3,4) are as follows: sgRNA-3 (the first sgRNA): 5'-CGCTTATCCGGCACCACCGA-3' (SEQ ID NO.3); sgRNA-4 (i.e., the second sgRNA): 5'-GACGTCACCCGTGATTCACC-3' (SEQ ID NO. 4).

[0034] This example also provides another group of sgRNA combinations screened from a large number of candidate sgRNAs, which can also effectively knock out the pig E4F1 gene, but the knockout efficiency is significantly lower; its first sgRNA (referred to as sgRNA-1) targets the sequence from 841 to 860 of the pig E4F1 gene, and the second sgRNA (referred to as sgRNA-2) targets the sequence from 997 to 1016 of the pig E4F1 gene. Specifically, the coding gene sequences of sgRNA-1 and sgRNA-2 in this combination (referred to as SG1,2) are as follows: sgRNA-1: 5'-TGCGGGCCGTTGCGACATGG-3' (SEQ ID NO. 1); sgRNA-2: 5'-CTTCCTCGCTAAAGGGCGCC-3' (SEQ ID NO. 2).

[0035] Example 2 Based on the sgRNA specifically targeting the porcine E4F1 gene provided in Example 1, this example constructed a CRISPR / Cas9 gene knockout vector containing the above sgRNA, specifically comprising the following steps: (1) Pretreatment of sgRNA.

[0036] To facilitate subsequent connection with the vector, BbSⅠ restriction site sequences were added to both ends of the sgRNA, and the resulting double-stranded DNA sequence is shown in Table 1.

[0037] Table 1 Double-stranded DNA sequences corresponding to the four sgRNAs

[0038] (2) Construction of linearized vector.

[0039] The strain carrying the PX459 vector was revived and expanded, wherein the PX459 vector map is as follows Figure 1 The plasmid was extracted according to the instructions of the small-scale ultrapure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd.

[0040] The extracted product was digested with restriction endonuclease BbSI. The digestion reaction system was: vector PX459 1.5μL (1000ng), restriction endonuclease BbSI 1μL, Green Buffer 10× 1μL, ddH2O to 20μL. The digestion reaction program was: 37℃, 30min.

[0041] (3) Annealing of sgRNA sequence.

[0042] The fragments of the four pairs of sgRNA sequences carrying the BbSI restriction site shown in Table 1 were annealed separately to form fragments containing the sticky ends of the BbSI restriction site. The annealing reaction system was: NEB buffer 2μL, upstream sequence 1μL, downstream sequence 1μL, ddH2O filled to 20μL. The annealing reaction program was: 95℃, 5min; 37℃, 1h.

[0043] (4) Connection.

[0044] The sgRNA annealing product obtained in step (3) was connected to the linearized vector obtained in step (2). The ligation reaction system was: 3 μL of annealing product, 2 μL of enzyme digestion product, 1 μL of T4 DNA Ligase, 2 μL of Buf T4 Ligase, and ddH2O was added to 20 μL. The ligation reaction procedure was: 22°C, 1 h.

[0045] (5) Transformation.

[0046] The synthesized ligation product sample was diluted to 10mmol / L working solution, 20mL system was prepared, and transformed into Escherichia coli DH5α competent cells. 5mL of ligation product and 50mL of DH5α competent cells were mixed, ice bathed for 30min, heat shocked at 42℃ for 90s, and immediately placed on ice for 2min; 1mL of antibiotic-free culture medium was added, and the cells were revived in a constant temperature shaker at 37℃, 200rpm / min for 45min; centrifuged at 8000rpm / min on a desktop centrifuge for 1min to precipitate the bacteria, and then 900mL of LB culture medium was removed; the remaining 100mL sample was suspended and mixed, and then spread on an LB plate containing ampicillin (100ng / mL, Amp), and placed in a 37℃ constant temperature incubator and inverted overnight for 16h.

[0047] (6) Screen positive colonies and perform sequencing identification.

[0048] Pick the single colony obtained in step (5) for colony PCR and detect by agarose gel electrophoresis. Shake the qualified colony and send the bacterial solution to the company for sequencing using U6 primer (5'-CCGTAACTTGAAAGTATTTCG-3'). Expand the positive single clone with correct sequencing and extract the plasmid.

[0049] The PX459 vectors containing four sgRNAs that were sequenced correctly were named SG1 (PX459 vector containing sgRNA-1), SG2 (PX459 vector containing sgRNA-2), SG3 (PX459 vector containing sgRNA-3), and SG4 (PX459 vector containing sgRNA-4). The sequencing results are shown in Figure 2 shown.

[0050] Example 3 In this example, the CRISPR / Cas9 gene knockout vectors SG1, SG2, SG3, and SG4 constructed in Example 2 were used to knock out the pig E4F1 gene in pairs, and positive cells were screened; specifically, the following were included: (1) Knockout of porcine E4F1 gene.

[0051] ① Cell plating: porcine kidney fibroblasts were inoculated into six-well cell culture plates and their growth density reached about 80% before electroporation transfection.

[0052] ② Clean the spot transfer cup: rinse the electric transfer cup several times with alcohol, repeatedly scrape the inside of the spot transfer cup with the tip of the electric transfer gun until there is no cell residue, then add DPBS to clean the electric transfer cup and pour out the remaining residual liquid, finally rinse the electric transfer cup with electric transfer solution and place it in a 37℃ oven to dry.

[0053] ③ Digest cells: When the cells grow to about 80% confluence and have good morphology, use 0.25% trypsin to digest the cells (normal cell culture six-well plates are generally digested at 200mL~500mL / well of 0.25% trypsin. When the cells become round and out of contact and some cells appear to fall off and become suspended under a microscope, immediately add culture medium containing serum to terminate digestion). Repeatedly blow and beat to completely detach the adherent cells and disperse them into single cells.

[0054] ④ Collect and rinse cells: Collect the digested and dispersed cell suspension into a 1.5 mL sterile centrifuge tube, resuspend and rinse the cells with DPBS, centrifuge at 1500 rpm for 3 min, discard the supernatant, add electroporation buffer (KCl 120 mM, CaCl2 0.15 mM, Hepes 25 mM, EDTA 2 mM, anhydrous MgCl2 5 mM and K2HPO4 10 mM, pH 7.6) to re-rinse the cells, centrifuge again, centrifuge at 1500 rpm for 3 min, discard the supernatant; according to the 100 mL system (cell volume is 1 × 10 6 ) was added with an appropriate amount of electroporation buffer to 5 mg of the recombinant plasmid (prepared in Example 2), and the cells were gently pipetted to resuspend and mix evenly.

[0055] ⑤ Transfect cells by electroporation: Turn on the electroporator in advance during cell digestion, and set parameters such as voltage, electric shock time and pulse number to pre-run it. Transfer the cell electroporation mixture into a sterile and clean electroporation cup of appropriate size, and transfect the cells by electroporation at 220V / cm, 3ms, and one pulse. After electroporation, culture the cells with DMEM medium containing 12% FBS.

[0056] (2) Screening of positive cells.

[0057] The positive clone E4F1 was detected using specific primers E4F1-F1 / E4F1-R1 (for detecting SG1,2-KO) and E4F1-F2 / E4F1-R2 (for detecting SG3,4-KO), wherein the nucleotide sequences of each primer are as follows: E4F1-F1: 5'-CTCTTCTCACCTAGGGTCTA-3' (SEQ ID NO.5), E4F1-R1: 5'-CTAGGTCCAAGAGGGTGAT-3' (SEQ ID NO. 6); E4F1-F2: 5'-AAGACCTTCAAGACGGTGA-3' (SEQ ID NO.7), E4F1-R2: 5'-CCAGAACAGAGCCTGGTG-3' (SEQ ID NO. 8).

[0058] In this case, the above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and were packaged, transported, and stored in the form of dry powder; the primers were diluted with sterilized ddH2O to a 10 μmol / L solution and stored at -20°C for future use. For primers E4F1-F1 and E4F1-R1, the expected length of the original PCR product was about 579 bp, and the band after deletion was about 439 bp; for primers E4F1-F2 and E4F1-R2, the expected length of the original PCR product was about 738 bp, and the band after deletion was about 381 bp.

[0059] The method for sequencing and verifying the porcine E4F1 mutant cell line using the above primers is as follows: Cell genomic DNA was extracted according to the instructions of the mammalian genomic DNA extraction kit of Tiangen Biotechnology Co., Ltd., and its concentration was detected using a nucleic acid concentration meter and its integrity was detected by agarose gel electrophoresis. Finally, the concentration was adjusted to 500 ng / μL for use in PCR detection.

[0060] Among them, the 25μL PCR reaction system is: 2×Es Taq MasterMix (Dye) 12.5μL, upstream primer (10μmol / L) 1μL, downstream primer (10μmol / L) 1μL, DNA template (500ng / μL) 1μL, ddH2O 9.5μL. After adding the above components on ice and mixing them thoroughly, the following program is performed in the PCR instrument: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, 35 cycles; 72℃ final extension for 2min; 4℃ storage.

[0061] Take 5 μL of the PCR reaction product and run it on a 2% agarose gel at 120 V for 30 min. Observe the band size on a gel imager. Figure 3 As shown. Figure 3 As can be seen, the target band size is consistent with expectations.

[0062] (3) qPCR detection of porcine E4F1 gene expression level.

[0063] The positive cells obtained in step (2) are subjected to qPCR detection, which specifically includes the following steps: ① Cell RNA extraction and cDNA synthesis.

[0064] The total RNA of the cells to be tested was extracted by Ttizol precipitation method. The integrity of the total RNA was determined by agarose gel electrophoresis, and the concentration of the extracted RNA was determined by a concentration meter and stored at -80°C.

[0065] ②Primer design.

[0066] According to the porcine E4F1 gene mRNA sequence published by NCBI, a primer pair capable of detecting porcine E4F1 gene knockout was designed. After screening, a pair of primers E4F1 capable of achieving efficient porcine E4F1 gene detection was obtained. The specific sequence is as follows: Upstream primer, E4F1-1F: 5′-CGGAGTTCACCACCTTGGAG-3′ (SEQ ID NO. 9); Downstream primer, E4F1-1R: 5′-CCTCTTTGATGTGTCCGCCA-3′ (SEQ ID NO. 10).

[0067] At the same time, GAPDH was used as an internal reference gene, and its primer sequences are as follows: Upstream primer, F: 5′-TCGGAGTGAACGGATTTGGC-3′ (SEQ ID NO. 11); Downstream primer, R: 5'-TGACAAGCTTCCCGTTCTCC-3' (SEQ ID NO. 12).

[0068] ③qPCR amplification.

[0069] Using the cell cDNA obtained in step ① as a template, qPCR amplification was performed using the primer pair E4F1 and the internal reference GAPDH primer in step ②.

[0070] The reaction system of qPCR was: 2×Universal SYBR Fast qPCR Mix 10μL, 10μM upstream primer 0.4μL, 10μM downstream primer 0.4μL, cDNA template 1.0μL, ddH2O 8.2μL. The qPCR reaction system was prepared by the mixed addition method, that is, according to the quantity of various components required for each reaction system and the number of PCR reactions required for one reaction, the total amount of various reaction components was calculated, added to a 1.5mL centrifuge tube without RNAse, mixed thoroughly and centrifuged instantaneously, and then dispensed into 8-tube strips dedicated to fluorescent quantitative PCR, and then template cDNA was added respectively, and then centrifuged instantaneously for fluorescent quantitative PCR amplification. The entire operation process was kept away from light as much as possible.

[0071] The reaction program of qPCR was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and 40 cycles; the instrument automatically set the melting curve.

[0072] ④qPCR data analysis.

[0073] The amplification curve and melting curve of qPCR results were analyzed.

[0074] The results showed that the amplification curves of each gene were "S" shaped and reached the plateau phase, indicating effective amplification; and the melting curve peaks of each gene were single, indicating that the amplification product was specific, without primer dimers and other nonspecific amplifications, and subsequent results analysis could be performed. According to the CT values ​​of different treatment groups, the control group of NC plasmid (empty plasmid) was used as an external reference (the external reference was set to eliminate the experimental errors of different batches), and the relative quantitative method was used to calculate the relative expression of the pig E4F1 gene knockout = 2 -△△CT Independent sample T test was used to analyze and compare the expression differences of E4F1 gene in different treatments. The results are expressed as mean ± standard error. P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference.

[0075] ⑤Draw a picture.

[0076] The results were plotted using GraphPad Prism 5. Figure 4 As shown, the number of individual replicates in the figure is 2, and the number of qPCR sample addition technology replicates is 4. The results showed that compared with the control group, knocking out the E4F1 gene significantly downregulated its transcription level; among the two pairs of sgRNA-mediated porcine E4F1 gene knockouts, the combination of sgRNA-3 and sgRNA-4 mediated the lowest transcription level of the E4F1 gene in the knockout cells, indicating that the combination of sgRNA-3 and sgRNA-4 has excellent porcine E4F1 gene knockout efficiency and can achieve efficient knockout of the porcine E4F1 gene.

[0077] (5) Western blotting detection of porcine E4F1 gene expression level.

[0078] Western blotting was used to further verify the expression level of porcine E4F1 gene. The specific method is as follows: The total protein of the knockout group and the corresponding blank control group cells was extracted, and 600 ng of protein was loaded for SDS polyacrylamide gel electrophoresis. After transfer, blocking, incubation with primary and secondary antibodies, the cells were exposed using a developer, and the optical density of the bands was calculated and analyzed using the ImageJ software.

[0079] The results are as follows Figure 5 As shown, in the pig E4F1 gene knockout mediated by the two groups of sgRNA compositions, the expression level of the E4F1 gene in the knockout cells mediated by the sgRNA composition of sgRNA-3 and sgRNA-4 was the lowest, indicating that the sgRNA composition of sgRNA-3 and sgRNA-4 has excellent pig E4F1 gene knockout efficiency and can achieve efficient knockout of the pig E4F1 gene.

[0080] In summary, the sgRNA composition specifically targeting the porcine E4F1 gene provided by the present invention and the CRISPR / Cas9 gene knockout vector containing the corresponding sgRNA can achieve efficient knockout of the porcine E4F1 gene. At the same time, the specific primers provided by the present invention can achieve rapid and accurate detection of the expression level of the E4F1 gene. The present invention provides an effective method and basis for the functional study of the porcine E4F1 gene and its application.

[0081] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A sgRNA composition specifically targeting the porcine E4F1 gene, characterized in that: The sgRNA composition comprises a first sgRNA and a second sgRNA; Among them, the first sgRNA targets the sequence from position 7750 to position 7769 of the E4F1 gene, and the second sgRNA targets the sequence from position 8167 to position 8186 of the E4F1 gene.

2. The sgRNA composition according to claim 1, characterized in that The nucleotide sequence of the first sgRNA encoding gene is shown in SEQ ID NO.3, and the nucleotide sequence of the second sgRNA encoding gene is shown in SEQ ID NO.

4.

3. A CRISPR / Cas9 gene knockout vector specifically targeting the porcine E4F1 gene, characterized in that: It contains the sgRNA according to claim 1 or 2.

4. The CRISPR / Cas9 gene knockout vector according to claim 3, characterized in that Specifically, it is a PX459 vector connected with the sgRNA.

5. Use of the sgRNA composition according to claim 1 or 2 or the CRISPR / Cas9 gene knockout vector according to claim 3 or 4 in knocking out the porcine E4F1 gene.

6. A method for knocking out the pig E4F1 gene using the CRISPR / Cas9 system, characterized in that: The CRISPR / Cas9 gene knockout vector containing the sgRNA composition of claim 1 is introduced into cells to cause double-site editing of the E4F1 gene to cause fragment deletion, thereby obtaining positive cells.

7. The method according to claim 6, characterized in that The introduction method is electroporation transfection.

8. The method according to claim 6, characterized in that The cells are porcine kidney fibroblasts.

9. The method according to claim 6, characterized in that Positive cells were determined using primers whose sequences are shown in SEQ ID NO. 7-8.

10. The method according to claim 6, characterized in that In the positive cells, the method for detecting the expression level of the E4F1 gene is as follows: extracting total RNA from the cells to be tested, and reverse transcribing it into cDNA; using the cDNA as a template, and performing fluorescent quantitative PCR using primers shown in SEQ ID NO.9-10.

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