Method for constructing anti-j subtype avian leukemia gene edited chicken based on adenine editor adenovirus and application

Abstract

The application provides a method for constructing an anti-J subtype avian leukemia gene editing chicken based on an adenovirus adenine editor and an application. The adenine editor is designed by taking the c.98T>C, c.100T>C, c.112T>C and c.116A>G of the chicken NHE1 gene as a target, a plasmid of the adenine editor capable of simultaneously editing all the above sites is constructed, and an adenovirus containing the above sequences is further packaged. The adenovirus is injected into HH15-18 period chicken embryos by a microinjection method, site-directed mutation of one or more of the c.98T>C, c.100T>C, c.112T>C and c.116A>G of the NHE1 gene in the chicken embryo is realized, the chicken embryo is hatched and developed to sexual maturity, and the gene editing chicken with one or more point mutations of the c.98T>C, c.100T>C, c.112T>C and c.116A>G of the NHE1 gene is obtained through mating and breeding, and it is verified that the gene editing chicken has an anti-J subtype avian leukemia trait. The application has great application value in the fields of J subtype avian leukemia prevention and purification, gene editing chicken disease resistance model research and the like.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically a method and application for constructing gene-edited chickens resistant to J subtype avian leukosis based on adenovirus editor. Background Technology

[0002] Avian leukosis is a neoplastic disease caused by avian leukosis virus (ALV). It weakens the immune function of chickens, making them more susceptible to other diseases, thus increasing mortality and reducing productivity, resulting in significant economic losses for the poultry industry. Avian leukosis virus is divided into 10 subgroups, A and J. Since its first isolation in my country in 1999, subgroup J avian leukosis virus (ALV-J) has become one of the most serious infectious diseases affecting my country's poultry industry due to its wider host range, stronger variability, and transmissibility. Currently, there are no available vaccines or effective treatments for ALV-J; prevention and control rely on pathogen detection and population eradication, a process that takes several years or even more than a decade and requires substantial human and material resources.

[0003] Research has found that the receptor for ALV-J is a type 1 Na+ / H+ exchange type 1 (NHE1) protein on the host cell membrane. By comparing ALV-J-susceptible chickens with naturally resistant Japanese quail, it was discovered that tryptophan at position 38 of NHE1 (NHE1-W38) plays a crucial role in the binding of ALV-J to the cell membrane. Deleting the NHE1-W38 site through CRISPR / Cas9 gene editing technology can confer resistance to ALV-J in the host. However, ALV-J resistance breeding based on CRISPR / Cas9 gene editing technology faces significant challenges, including: firstly, the site-directed mutagenesis of NHE1-W38 using CRISPR / Cas9 combined with homologous recombination has very low editing efficiency; secondly, conventional CRISPR / Cas9 gene editing has severe off-target effects, potentially impacting animal health and economic performance. Summary of the Invention

[0004] To overcome the problems existing in the prior art, this invention provides a method and application for constructing gene-edited chickens resistant to J subtype avian leukosis based on adenine-edited adenovirus. The method employs adenine base editing technology, fusing a small Cas9 protein with an optimized, highly active adenosine deaminase to achieve single-base conversions of adenine to guanine (A>G) or cytosine to thymine (C>T) under non-double-strand break conditions. This adenine base editing technology enables direct modification of DNA bases, offering higher precision, higher editing efficiency, and greater safety and efficiency. This invention further packages the aforementioned adenine base editor into an adenovirus, and further packages the adenovirus containing the above sequence. The adenovirus is then injected into HH15-18 stage chicken embryos via microinjection to achieve one or more site-directed mutations of c.98T>C, c.100T>C, c.112T>C, and c.116A>G in the NHE1 gene within the chicken embryo. After hatching and developing to sexual maturity, the chicken embryos are bred and selected to obtain gene-edited chickens containing one or more point mutations of c.98T>C, c.100T>C, c.112T>C, and c.116A>G in the NHE1 gene. This invention has significant application value in the prevention and eradication of J subtype avian leukosis and in the research of gene-edited chicken disease resistance models.

[0005] This invention achieves the above objective through the following technical solution: a method for constructing gene-edited chickens resistant to J subtype avian leukosis based on adenine-edited adenovirus, comprising the following steps:

[0006] (1) Integrate the adenine monobase gene editing element and guide RNA into the adenovirus vector and obtain recombinant adenovirus using the adenovirus packaging system;

[0007] (2) For chicken embryos that have developed to stage HH15-18, the equatorial plane windowing method is used to make holes with a diameter of 2.5-3 mm;

[0008] (3) The above recombinant adenovirus was introduced into the blood of chicken embryos by microinjection. After incubation until the embryos were 7 days old, the development of the chicken embryos was detected, and the genome was extracted from each organ of the chicken embryos. The gene editing efficiency was detected by enzyme digestion and sequencing.

[0009] The gene sequence of the adenine monobase gene editing element is shown in SEQ ID No. 1.

[0010] 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).

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

[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 involves using a capillary needle with a needle diameter of 30 micrometers to draw 2.5 μL of recombinant adenovirus and injecting it into a peripheral reflux vessel of the chicken embryo.

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

[0015] The gene-edited chickens possess resistance to J subtype avian leukosis.

[0016] The application of gene-edited chickens in the prevention and eradication of J subtype avian leukosis.

[0017] The application of the gene-edited chickens in disease resistance model research.

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

[0019] 1. This invention is based on an adenine base editor, which achieves gene editing through single base conversion from adenine to guanine (A>G) or from cytosine to thymine (C>T), resulting in higher editing efficiency and lower off-target effects.

[0020] 2. It can simultaneously and precisely edit four sites of the NHE1 gene: c.98T>C, c.100T>C, c.112T>C, and c.116A>G, ​​with a wider editing range and more significant disease resistance.

[0021] 3. Live gene editing can be achieved in chicken embryos, and gene-edited chickens with resistance to J subtype avian leukosis can be obtained through selective breeding, which has stronger practicality and application prospects.

[0022] 4. The adenine editor was integrated into the adenovirus vector and introduced into chicken embryo blood via microinjection, which simplified the operation process, reduced costs, and improved editing efficiency.

[0023] 5. The adenovirus provided by the adenine editor of this invention can be cryopreserved for a long time and transported over long distances under dry ice protection. When applied, it can be amplified in large quantities in 293T cells without the need for expensive equipment such as electroporators or expensive reagents such as liposomes. It has significant advantages in terms of time, space and cost. Attached Figure Description

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

[0025] Figure 2 Image of semen PCR detection.

[0026] Figure 3 The left image shows the blood test results of homozygous offspring, and the right image shows the meconium test results of the offspring.

[0027] Figure 4 Sanger sequencing peak diagram. Detailed Implementation

[0028] Example 1

[0029] This includes the construction of relevant gene-editing vectors.

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

[0031] (2) Activity verification: Four human genomic loci located in the adenine editor editing window were selected to construct expression plasmids of adenine editor guide RNA. These plasmids were co-transfected into the HEK293T cell line with the adenine editor plasmids. After extracting the genome, the editing effect was detected by first-generation sequencing.

[0032] Example 2

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

[0034] (1) DF-1 cell transfection: One day before transfection, DF-1 cells were seeded into six-well plates using complete culture medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator to achieve a confluence of 70-90% at the time of transfection. Too low or too high confluence will affect transfection efficiency. In a sterile centrifuge tube, an appropriate amount of serum-free culture medium was added, and 3, 6, and 9 μL of the adenovirus to be transfected were added in three groups, respectively. The mixture was gently stirred to dilute the adenovirus to a suitable concentration. The six-well plates were then removed from the incubator, the original cell culture medium was aspirated, and the cells were gently washed twice with PBS buffer to remove residual serum. The diluted adenovirus was then evenly added to each well of the six-well plate, and the plate was gently shaken to distribute it evenly. The six-well plates were then returned to the 37°C incubator in a 5% CO2 incubator for further incubation. Four to six hours after transfection, the culture medium was replaced with complete culture medium containing 10% fetal bovine serum to provide the necessary nutrients for cell growth.

[0035] (2) T7E1 (T7 Endonuclease I) digestion steps: Genomic DNA was extracted from the DF-1 cells after the above transfection and the untreated control sample. Then, PCR amplification was performed using specific primers to obtain DNA fragments containing the target editing site, T7E1 enzyme, DNA loading buffer, agarose, TAE buffer, and nucleic acid dyes (such as EB, GelRed, etc.).

[0036] Denaturation and annealing of PCR products: Add the sample to the PCR tube as follows:

[0037] ddH2O 8.5μL template 1μL upstream primer 2μL Downstream primer 2μL total 25μL

[0038] After denaturation and annealing reactions in a PCR instrument, add appropriate amounts of T7E1 digestion buffer and T7E1 enzyme to the PCR products. Generally, for a 20 μL reaction system, add 2 μL of 10× digestion buffer and 1 μL of T7E1 enzyme. Gently mix and briefly centrifuge to concentrate the liquid at the bottom of the tube. Incubate the reaction tube at 37°C for 30-60 minutes to allow the T7E1 enzyme to recognize and cleave mismatched regions in heteroduplex DNA. After incubation, add an appropriate amount of DNA loading buffer (e.g., 6× Loading Buffer) to terminate the digestion reaction. Carefully add the digestion product to the wells of the gel, along with 2 μL of DNA molecular weight standard (marker) as a reference. Perform electrophoresis in TAE buffer at 100V for 35 minutes. After electrophoresis, place the gel in a gel imaging system to observe and photograph the results.

[0039] Example 3

[0040] Application of adenovirus, adenopurine editor, in the production of gene-edited chickens.

[0041] (1) Equatorial windowing method for hatching eggs: After cleaning the hatching eggs with 0.1% benzalkonium chloride and drying them, incubate them for 55-60 hours at 37.8 degrees Celsius, 60% humidity, and 90 degrees Celsius per hour. The number of segments of the chicken embryo can be observed under a microscope to determine the stage of chicken embryo development, which is HH15-18. Use an electric rotary tool with a round grinding head to grind a hole with a diameter of 2.5-3 mm on the calcified layer of the eggshell on the equatorial side of the hatching egg at about 10,000 rpm. Punch a hole with a diameter of 10 mm between two layers of double-sided foam tape (6 mm thick) and stick it on the eggshell hole. Drip 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. The external environment is isolated while performing microinjection.

[0042] (2) Microinjection of recombinant adenovirus: 2.5 μl of the diluted adenovirus from Example 3 was drawn up using a capillary needle with a needle diameter of 30 μm and injected into the peripheral reflux vessels of the chicken embryo under a stereomicroscope. To ensure that the virus concentration was basically the same for each injection, the adenovirus was gently blown up with a pipette before each injection to ensure that an appropriate amount of virus was drawn up. After microinjection, the eggshell pores were sealed with sterile transparent sealing film. Sealing the pores with the sealing film can improve the hatching rate. The PBS and double-sided tape on the eggshell pores were removed, and the sealing film was fixed with sterile transparent tape in a cross shape to make a second seal. After microinjection, the chicken embryos were placed in an incubator with the air cell facing upward and the opening at a 45° angle. They were incubated at 37.8 degrees Celsius, 60% humidity, and 60 degrees Celsius rotation once per hour until hatching.

[0043] (3) Chimerism detection of gene-edited chicken embryos: Adenine editor elements and guide RNA were packaged into an adenovirus vector. After PCR detection showed that the viral genome structure was normal, the viral titer was determined in HEK293T cell line by plaque assay. The adenine editing efficiency delivered by adenovirus was detected in primary chicken embryo fibroblasts by enzyme digestion.

[0044] (4) Sequencing: The collected serum was sent for sequencing to screen live chickens that successfully converted the NHE1 gene c.98T>C, c.100T>C, c.112T>C, and c.116A>G.

[0045] (5) Semen analysis of gene-edited chickens: The chimeric roosters that were detected were raised to six months of age and their semen was collected for semen analysis. The resulting gene-edited heritable chimeric roosters were mated with wild-type hens to obtain G1 generation chicken embryos. The allantoic membrane of the chicken embryos was collected for analysis, and five heterozygous chickens were obtained.

[0046] (6) Gene-edited chicken challenge: The obtained G1 generation chickens were challenged with J subtype avian leukemia virus. After one week, two weeks, six weeks, ten weeks and fourteen weeks, the virus in the blood of the challenged chickens was collected for virus detection.

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

Claims

1. A method for constructing gene-edited chickens resistant to J subtype avian leukosis based on adenovirus editor, characterized in that, Includes the following steps: (1) Integrate the adenine editor and guide RNA into the adenovirus vector and obtain recombinant adenovirus using the adenovirus packaging system; The gene sequence of the adenine editor is shown in SEQ ID No. 1; The adenine editor can precisely edit one or more sites of the NHE1 gene, such as c.98T>C, c.100T>C, c.112T>C, and c.116A>G. (2) For chicken embryos that have developed to stage HH15-18, the equatorial plane windowing method is used to make holes with a diameter of 2.5-3 mm; (3) The above recombinant adenovirus was introduced into the blood of chicken embryos by microinjection. After incubation until the embryos were 7 days old, the development of the chicken embryos was detected, and the genome was extracted from each organ of the chicken embryos. The gene editing efficiency was detected by enzyme digestion and sequencing.

2. The method according to claim 1, characterized in that, The adenine editor is capable of single-base conversions from adenine to guanine (A>G) or from cytosine to thymine (C>T).

3. The method according to claim 1, characterized in that, The adenovirus vector has the characteristic of efficiently transfecting primary cells and can be amplified in large quantities in 293T cells.

4. The method according to claim 1, characterized in that, The microinjection method involves using a capillary needle with a needle diameter of 30 micrometers to draw 2.5 μL of recombinant adenovirus and injecting it into a peripheral reflux vessel of the chicken embryo.

5. The application of the gene-edited chickens obtained by the method of claim 1 in the prevention and eradication of J subtype avian leukosis.

6. The application of the gene-edited chickens obtained by the method of claim 1 in disease resistance model research.

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