Method for constructing anti-subtype J avian leukosis gene editing chicken based on adenine editor adenovirus and application

Abstract

The invention provides a method for constructing an anti-subtype J avian leukosis gene edited chicken based on adenine editor adenovirus and application of the anti-subtype J avian leukosis gene edited chicken. The method comprises the following steps: taking c.98Tgt of a chicken NHE1 gene; c, c, 100 Tgt; c, c.112 Tgt, c. 112 Tgt; c, c, 116Agt, c, 116Agt; the method comprises the following steps of: designing an adenine editor by taking a nucleotide sequence A and a nucleotide sequence G as targets, constructing plasmids of the adenine editor capable of simultaneously editing all the sites, further packaging an adenovirus containing the sequence, and injecting the adenovirus into an HH15-18 stage chicken embryo through a microinjection method to realize the NHE1 gene c.98Tgt in the chicken embryo; c, c, 100 Tgt; c, c.112 Tgt, c. 112 Tgt; c, c, 116Agt, c, 116Agt; the method comprises the following steps: carrying out site-directed mutagenesis on one or more of G.98Tgt and G. A chick embryo is hatched and developed to sexual maturity, and carrying out hybridization and breeding to obtain an NHE1 gene C, c, 100 Tgt; c, c.112 Tgt, c. 112 Tgt; c, c, 116Agt, c, 116Agt; g, gene edited chicken with one or more point mutations, wherein the gene edited chicken is proved to have the J subtype avian leukosis resisting character. The method has great application value in the fields of J subtype avian leukosis prevention and purification, gene editing chicken disease resistance model research and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically to a method for constructing ALV-J-resistant gene-edited chickens based on an adenovirus of an adenine editor and its application. Background Art

[0002] Avian leukosis is a neoplastic disease caused by Avian Leucosis Virus (ALV), which can lead to a decrease in the immune function of chicken flocks, making them more susceptible to other diseases, thereby increasing poultry mortality and reducing production performance, causing huge economic losses to the poultry industry. Avian leukosis virus is divided into 10 subgroups from A to J. Among them, since Avian Leucosis Virus subgroup J (ALV-J) was first isolated in China in 1999, due to its wider host range, stronger variability and transmission ability, it has become one of the most serious infectious diseases endangering the poultry industry in China. At present, there is no available vaccine and effective treatment method for ALV-J, and it can only be prevented and controlled through pathogen detection and population purification, which takes several years or even more than a decade and requires a large amount of manpower and material resources.

[0003] Studies have found that the receptor of ALV-J is the type 1 Na+ / H+ exchanger (NHE1) on the host cell membrane. By comparing chickens susceptible to ALV-J with Japanese quails with natural resistance, it was found that tryptophan at position 38 of NHE1 (NHE1-W38) plays a key role in the binding of ALV-J to the cell membrane. By using the CRISPR / Cas9 gene editing technology to edit the NHE1-W38 locus and deleting this locus, the host can obtain resistance to ALV-J. However, ALV-J disease-resistant breeding based on the CRISPR / Cas9 gene editing technology faces great difficulties, including the following aspects: First, the method of combining CRISPR / Cas9 with homologous recombination for site-directed mutagenesis of NHE1-W38 has a very low editing efficiency; in addition, conventional CRISPR / Cas9 gene editing has serious off-target phenomena, which may affect the health and economic performance of animals. Summary of the Invention

[0004] To overcome the problems existing in the above-mentioned prior art, the present invention provides a method and application for constructing anti-J subtype avian leukosis gene-edited chickens based on an adenine editor adenovirus. By using adenine base editing technology, a small Cas9 protein is fused with an optimized highly active adenosine deaminase to achieve adenine-to-guanine (A>G) or cytosine-to-thymine (C>T) single-base conversion under the condition of non-double-strand break. Through adenine base editing technology, direct modification of DNA bases can be achieved, which has higher precision, higher editing efficiency, and is safer and more efficient. The present invention further packages the above adenine base editor into an adenovirus, and further packages the adenovirus containing the above sequence. The adenovirus is injected into chicken embryos at HH15-18 stage by microinjection method to achieve site-directed mutagenesis of one or more of c.98T>C, c.100T>C, c.112T>C, c.116A>G in the NHE1 gene in the chicken embryo. The chicken embryo is hatched and developed to sexual maturity, and through breeding and selection, gene-edited chickens containing one or more point mutations of c.98T>C, c.100T>C, c.112T>C, c.116A>G in the NHE1 gene are obtained. The present invention has great application value in the fields of prevention and purification of J subtype avian leukosis, research on disease-resistant models of gene-edited chickens, etc.

[0005] The present invention realizes the above object through the following technical solutions: A method for constructing anti-J subtype avian leukosis gene-edited chickens based on an adenine editor adenovirus, comprising the following steps:

[0006] (1) Integrate the adenine single-base gene editing element and the guide RNA into the adenovirus vector, and obtain a recombinant adenovirus by using the adenovirus packaging system;

[0007] (2) Punch holes in the chicken embryo at HH15-18 stage by the equatorial plane windowing method, and the pore diameter is 2.5-3 mm;

[0008] (3) Introduce the above recombinant adenovirus into the chicken embryo blood by microinjection method, continue to incubate until the 7th embryonic day, detect the development of the chicken embryo, and sample each organ of the chicken embryo to extract the genome, and detect the gene editing efficiency by enzyme digestion and sequencing.

[0009] The gene sequence of the adenine single-base gene editing element is as shown in SEQ ID No.1.

[0010] The adenine single-base gene editing element can achieve adenine-to-guanine (A>G) or cytosine-to-thymine (C>T) single-base conversion.

[0011] The gene editing element can accurately edit one or more of the sites of c.98T>C, c.100T>C, c.112T>C, c.116A>G in the NHE1 gene.

[0012] The adenovirus vector has the characteristic of efficiently transfecting primary cells and can be amplified in large quantities in 293T cells.

[0013] The microinjection method includes using a capillary needle with a needle diameter of 30 microns to aspirate 2.5 μL of recombinant adenovirus and injecting it into the peripheral reflux blood vessels of the chicken embryo.

[0014] The gene-edited chicken obtained by the method contains one or more point mutations of c.98T>C, c.100T>C, c.112T>C, and c.116A>G in the NHE1 gene.

[0015] The gene-edited chicken has the characteristic of being resistant to subgroup J avian leukosis.

[0016] Application of the gene-edited chicken in the prevention and purification of subgroup J avian leukosis.

[0017] Application of the gene-edited chicken in the research of disease-resistant models.

[0018] Through the above technical solutions, the present invention has the following effects:

[0019] 1. Based on the adenine base editor, the present invention realizes gene editing through single-base conversion from adenine to guanine (A>G) or from cytosine to thymine (C>T), with higher editing efficiency and lower off-target effects.

[0020] 2. It can accurately edit four sites of c.98T>C, c.100T>C, c.112T>C, and c.116A>G in the NHE1 gene simultaneously, with a wider editing range and more significant disease-resistant effects.

[0021] 3. It can achieve in-vivo gene editing in chicken embryos and obtain gene-edited chickens with the characteristic of being resistant to subgroup J avian leukosis through breeding, with stronger practicability and application prospects.

[0022] 4. Integrating the adenine editor into the adenovirus vector and introducing it into the chicken embryo blood through the microinjection method simplifies the operation process, reduces costs, and improves editing efficiency.

[0023] 5. The adenine editor adenovirus provided by the present invention can be stored frozen for a long time, can be transported over long distances under dry ice protection, can be amplified in large quantities in 293T cells during application, does not require expensive equipment such as electroporators, and does not require expensive reagents such as liposomes, and has great application advantages in terms of time, space, and cost. Brief Description of the Drawings

[0024] Figure 1 Schematic diagram of gene editing sites.

[0025] Figure 2 Semen PCR detection diagram.

[0026] Figure 3 The left middle figure is the detection result diagram of homozygous blood, and the right figure is the detection result diagram of fetal meconium of the offspring.

[0027] Figure 4 Sanger sequencing peak diagram. Specific implementation mode

[0028] Example 1

[0029] Construction of relevant gene editing vectors.

[0030] (1) Construction of adenine editor plasmid vector: According to the artificially synthesized full sequence of adenine editor, a eukaryotic expression plasmid was constructed using molecular biology methods; according to the site to be edited, a guide RNA designed by the online design software E-CRISP (http: / / www.e-crisp.org / E-CRISP / index.html) was used, and the corresponding eukaryotic expression plasmid was constructed.

[0031] (2) Activity verification: Four human genomic sites located within the editing window of the adenine editor were selected, and an expression plasmid of the adenine editor guide RNA was constructed. It was co-transfected into the HEK293T cell line with the adenine editor plasmid. After extracting the genome, first-generation sequencing was used to detect the editing effect.

[0032] Example 2

[0033] NHE1 gene editing of DF-1 cells.

[0034] (1) Transfection of DF-1 cells: One day before transfection, DF-1 cells were seeded in a six-well plate, and cultured in a complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator to make the cells reach 70 - 90% confluence at the time of transfection. Too low or too high confluence will affect the transfection efficiency. In a sterile centrifuge tube, an appropriate amount of serum-free medium was added, and 3, 6, 9 μL of the adenovirus to be transfected was added in three groups and gently mixed. After diluting the adenovirus to an appropriate concentration, the six-well plate was taken out of the incubator, the original cell medium was aspirated, and the cells were gently washed twice with PBS buffer to remove the residual serum. The diluted adenovirus was evenly added to each well of the six-well plate, and the culture plate was gently shaken to make it evenly distributed. The six-well plate was then put back into the 37°C, 5% CO2 incubator for continued culture. After 4 - 6 hours of transfection, it can be replaced with a complete medium containing 10% fetal bovine serum for continued culture to provide the nutrients required for cell growth.

[0035] (2)T7E1 (T7 Endonuclease I) digestion steps: Extract genomic DNA from the DF-1 cells transfected as described above and from untreated samples used as controls. Then perform PCR amplification using specific primers to obtain DNA fragments containing the target editing sites, T7E1 enzyme, DNA loading buffer, agarose, TAE buffer, and nucleic acid dyes (such as EB, GelRed, etc.).

[0036] Denaturation and renaturation of PCR products: Add the following samples to the PCR tube:

[0037] Taq Mix 12.5 μL ddH2O 8.5 μL Template 1 μL Forward primer 2 μL Reverse primer 2 μL Total 25 μL

[0038] Add an appropriate amount of T7E1 digestion buffer and T7E1 enzyme to the PCR products obtained after performing denaturation and renaturation reactions in a PCR instrument. Generally, for a 20 μL reaction system, 2 μL of 10× digestion buffer and 1 μL of T7E1 enzyme can be added. Gently mix and briefly centrifuge to concentrate the liquid at the bottom of the tube. Place the reaction tube in an incubator at a constant temperature of 37°C for 30 - 60 minutes to allow the T7E1 enzyme to recognize and cut the mismatched regions in the heteroduplex DNA. After incubation, add an appropriate amount of DNA loading buffer (such as 6× Loading Buffer) to terminate the digestion reaction. Carefully add the digestion reaction products into the sample wells of the gel, and at the same time add 2 μL of DNA molecular weight standard (Marker) as a reference. Perform electrophoresis in TAE buffer at a voltage of 100V for 35 minutes. After electrophoresis, place the gel in a gel imaging system to observe and photograph the results for recording.

[0039] Example 3

[0040] Application of adenine editor adenovirus in the preparation of gene-edited chickens.

[0041] (1)Windowing of hatching eggs by the equatorial plane method: Clean the hatching eggs with 0.1% benzalkonium bromide and dry them. Incubate the above hatching eggs at 37.8°C, 60% humidity, and turning the eggs 90 degrees per hour for 55 - 60 hours. The developmental stage of the chicken embryo can be judged by observing the number of somites of the chicken embryo under a microscope, which is in the HH15 - 18 stage. Use an electric drill with a round grinding head to grind a hole with a diameter of 2.5 - 3 mm in the calcified layer of the eggshell on the equatorial side of the hatching egg at about 10,000 revolutions per minute. Punch a hole with a diameter of 10 mm in the middle of two layers of foam double-sided tape (with a thickness of 6 mm), stick it on the eggshell hole, drop sterile PBS buffer into the middle, and then use a scalpel to cut and remove the eggshell membrane under the eggshell hole. At this time, the chicken embryo and the peripheral blood vessels of the chicken embryo can be observed through the PBS buffer, while isolating the external environment during microinjection.

[0042] (2)Microinjection of recombinant adenovirus: Aspirate 2.5 μl of the diluted adenovirus in Example 3 with a capillary needle having a needle aperture of 30 microns, and find the peripheral reflux blood vessels of the chicken embryo under a stereomicroscope for injection. In order to ensure that the virus concentration is basically the same for each injection, before each aspiration, gently pipette with a pipette gun to ensure that an appropriate amount of virus can be aspirated. After microinjection, use a sterile transparent sealing film to seal the eggshell hole. Sinking the sealing film into the eggshell hole for sealing can improve the hatching rate. Remove the PBS and double-sided tape on the eggshell hole, and use a sterile transparent tape to fix the sealing glue in a "cross" shape for secondary sealing. Place the microinjected chicken embryo in the incubator with the air chamber facing up and the opening point at a 45° angle, and incubate until hatching under the conditions of 37.8 °C, 60% humidity, and turning the eggs 60 degrees per hour.

[0043] (3)Chimera detection of gene-edited chicken embryos: Package the adenine editor element and guide RNA into an adenovirus vector. After PCR detecting that the virus genome structure is normal, determine the virus titer by the plaque method in the HEK293T cell line, and detect the adenine editing efficiency of the adenovirus delivered by restriction enzyme digestion in primary chicken embryo fibroblasts.

[0044] (4)Sequencing: Send the collected serum for sequencing detection to screen for live chickens with successful conversions of c.98T>C, c.100T>C, c.112T>C, and c.116A>G in the NHE1 gene.

[0045] (5)Semen detection of gene-edited chickens: Raise the detected chimeric roosters to six months old and then collect semen for semen detection. Obtain gene-editable heritable chimeric roosters, mate them with wild-type hens to obtain G1-generation chicken embryos, collect the allantoic membranes of the chicken embryos for detection, and obtain 5 heterozygous chickens.

[0046] (6)Virus challenge of gene-edited chickens: Challenge the obtained G1-generation chickens. After injecting J-subtype avian leukosis virus into the wing vein, collect the virus in the blood of the challenged chickens after one week, two weeks, six weeks, ten weeks, and fourteen weeks for virus detection.

[0047] As can be seen from the above examples, the present invention has covered the adenovirus vector of the adenine editor, the obtained gene-edited chickens resistant to J-subtype avian leukosis, and their application in the prevention and purification of J-subtype avian leukosis.

Claims

1. A method for constructing anti-J-subtype avian leukosis gene-edited chickens based on an adenovirus of an adenine editor, characterized in that, It includes the following steps: (1) Integrate the adenine single-base gene editing element and the guide RNA into the adenovirus vector, and use the adenovirus packaging system to obtain recombinant adenovirus; (2) Punch holes in the chicken embryos at HH15-18 stage by the equatorial plane windowing method, with a pore diameter of 2.5-3 mm; (3) Introduce the above recombinant adenovirus into the chicken embryo blood by microinjection method. After continuing to incubate until the 7th embryonic day, detect the development of the chicken embryo, sample each organ of the chicken embryo to extract the genome, and detect the gene editing efficiency by enzyme digestion and sequencing.

2. The method according to claim 1, characterized in that, The gene sequence of the adenine single-base gene editing element is shown in SEQ ID No.

1.

3. The method according to claim 1, characterized in that, The adenine single-base gene editing element can achieve single-base conversion from adenine to guanine (A>G) or from cytosine to thymine (C>T).

4. The method according to claim 1, wherein The gene editing element can precisely edit one or more sites of c.98T>C, c.100T>C, c.112T>C, c.116A>G of the NHE1 gene.

5. The method according to claim 1, wherein The adenovirus vector has the characteristic of high-efficiency transfection of primary cells and can be amplified in large quantities in 293T cells.

6. The method according to claim 1, wherein The microinjection method includes using a capillary needle with a needle diameter of 30 microns to aspirate 2.5 μL of recombinant adenovirus and injecting it into the peripheral reflux blood vessels of the chicken embryo.

7. A gene-edited chicken obtained by the method according to any one of claims 1 to 5, characterized in that, The gene-edited chicken contains one or more point mutations of c.98T>C, c.100T>C, c.112T>C, c.116A>G of the NHE1 gene.

8. The gene-edited chicken according to claim 6, wherein The gene-edited chicken has the characteristic of being resistant to avian leukosis subtype J.

9. Use of the gene-edited chicken according to claim 6 in the prevention and purification of avian leukosis subtype J.

10. Use of the gene-edited chicken according to claim 6 in the research of disease-resistant models.

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