Method for obtaining genetically modified progeny of bird animals

Abstract

The present invention relates to a method for obtaining a genetically modified progeny of an avian animal by packaging a gene edited or genetically modified vector in a recombinant adenovirus and injecting into the dorsal aorta of an embryo when PGCs migrate to a developing gonad; compared with the existing disclosed method for transforming the PGCs in vitro, the operation method disclosed by the invention not only can stably and effectively infect the PGCs in vivo, but also has the advantages that more importantly, the probability of generating gene editing offspring is higher, and particularly, the breeding efficiency of cultivating the transgenic chicken offspring with KRT75L4 gene and MSTN gene knockout is higher; and on the other hand, the reporter gene or crispr-cas protein gene does not need to be integrated into the genome of the bird and the non-native protein does not need to be expressed, so that the operability is higher from the commercial level.

Description

Technical Field

[0001] The invention relates to a method for obtaining genetically modified bird offspring, and belongs to the technical field of bird breeding and genetic engineering. Background Art

[0002] Transgenic birds, particularly transgenic chickens, are increasingly used in biotechnology and serve as excellent model organisms for biological research. Currently, transgenic chickens can be used as bioreactors for producing pharmaceutical proteins. Furthermore, those skilled in the art are also interested in developing new breeds with disease resistance (e.g., resistance to avian influenza) and / or superior quality (e.g., chicken and egg quality) through gene editing and modification.

[0003] Currently, regarding the methods of obtaining transgenic animals, the somatic cell nuclear transfer (SCNT) method has been successfully applied to mammals because it is relatively easy to obtain single oocytes from mammals. However, this method is not applicable to birds.

[0004] After ovulation, the oocyte, heavily yolk-attached, is transported to the oviduct and fertilized within the infundibulum. The resulting fertilized egg rapidly proliferates, reaching approximately 55,000 cells within 24 hours of spawning. This makes it difficult to obtain a single fertilized egg or oocyte for in vitro genetic modification. Surgically obtaining a single oocyte attached to the yolk and then transplanting it back into the yolk after genetic modification is extremely difficult and uneconomical.

[0005] In the existing technology, the transgenic operation method of chickens mainly includes three steps. The first step is to establish a stable primordial germ cell (PGCs) culture system through isolation and in vitro culture. The second step is to modify PGCs in vitro and screen genetically modified PGCs. The third step is to transplant the genetically modified PGCs into recipient embryos (such as embryonic blood vessels, subembryonic cavity) or the testicles of adult roosters to obtain offspring with edited germ cells.

[0006] However, the above method still has many problems in actual practice. For example, establishing a stable PGCs culture system requires high technology and cost. Currently, only a few species such as chickens and quails have mature in vitro PGCs culture technology. In addition, transplanted genetically modified PGCs can also compete with endogenous PGCs, ultimately leading to a decrease in the efficiency of effectively obtaining offspring with edited germ cells.

[0007] Currently, researchers in this field are also trying to develop methods for in vivo transformation of PGCs, for example, using transposon plasmids to stably transform primordial germ cells in vivo (which can pass transgenes to the next generation) to produce transgenic offspring that express reporter genes or CRISPR-Cas protein genes carried in the transposon. However, integrating reporter genes or CRISPR-Cas protein genes into the genome of poultry and expressing non-native proteins is unacceptable for commercial poultry production and complicates safety regulations for the consumption of poultry and their eggs.

[0008] Therefore, the art hopes to develop a new method for genetically modifying avian animals and obtaining genetically modified offspring. Summary of the Invention

[0009] In order to solve the above technical problems, the present invention provides a method for obtaining genetically modified avian offspring, wherein:

[0010] The method comprises the following steps:

[0011] Step 1) incubating the fertilized eggs of the bird until the embryo forms the dorsal aorta;

[0012] Step 2) Injecting the packaged recombinant adenovirus into the dorsal aorta of the embryo by microinjection when PGCs migrate to the developing gonads;

[0013] The recombinant adenovirus is packaged with a gene editing substance targeting a target gene, or is packaged with an exogenous gene vector;

[0014] The titer of the recombinant adenovirus was 1×10 12 ~1×10 13 VP / ml;

[0015] Step 3) Continue incubation and collect semen from sexually mature male individuals for testing; screen male F0 generation individuals whose semen is detected to have gene editing or gene modification of the target gene, and collect their semen for artificial insemination of a wild-type female population;

[0016] Step 4) Screening and obtaining heterozygous female F1 generation individuals, mating them with the male F0 generation individuals, and screening and obtaining homozygous genetically modified offspring.

[0017] In a preferred embodiment of the present invention, the gene editing material uses a gene editing material selected from the group consisting of ZFN, TALEN, CRISPR and megaTAL methods; preferably, the gene editing material is a site-directed knockout gene editing material, or a site-directed knock-in gene editing material.

[0018] In a more preferred embodiment of the present invention, in step 2), the recombinant adenovirus is a type 5 recombinant adenovirus, which is packaged with a CRISPR gene editing material for site-directed knockout; preferably, the CRISPR gene editing material comprises sgRNA and Cas9 protein targeting the target gene.

[0019] In a preferred embodiment of the present invention, the bird is a Galliformes, Anseriformes, Otidiformes, Columbiformes or Struthioniformes animal; preferably, the bird is a Galliformes animal; more preferably, the Galliformes animal is selected from the Numididae or Phasianidae animals; more preferably, the Phasianidae animals include Gallus or Meleagris animals.

[0020] More preferably, the avian animal belongs to the order Galliformes, family Phasianidae, genus Gallus; and in step 2), microinjection is performed when the embryo is hatched to stage 14 to 17 of the Hamburger-Hamilton staging system.

[0021] In a preferred embodiment of the present invention, in step 1), newly laid fertilized eggs are incubated at 37.8°C, 60% humidity, and rotated 90 degrees every 2 hours with the blunt end facing upward; in step 2), when the embryos are incubated to stages 14 to 17 of the Hamburger-Hamilton staging system, a window is opened at the blunt end to expose the dorsal aorta of the embryo.

[0022] In a preferred embodiment of the present invention, the bird belongs to the order Galliformes, family Phasianidae, genus Gallus;

[0023] The target gene is KRT75L4 gene, and the sequence of the sgRNA is as shown in SEQ ID NO.1 to 8; preferably the sequence shown in SEQ ID NO.4 to 7; more preferably the sequence shown in SEQ ID NO.5; or,

[0024] The target gene is the MSTN gene, and the sequence of the sgRNA is shown in SEQ ID NOs. 9 to 15; preferably, the sequence shown in SEQ ID NOs. 13-14.

[0025] More preferably, in step 2), the titer of the recombinant adenovirus is 1×10 12 ~2×10 12 VP / ml, 1-2 μL of recombinant adenovirus was injected into the left dorsal aorta of the embryo.

[0026] In a more preferred embodiment of the present invention, the type 5 recombinant adenovirus is packaged with a CRISPR adenovirus vector; preferably, the CRISPR adenovirus vector uses a pAV[CRISPR]-hCas9:P2A:EGFP-U6 vector inserted with sgRNA targeting the target gene.

[0027] Another aspect of the present invention provides a genetically modified avian offspring, wherein the avian offspring is obtained using the above method.

[0028] The present invention provides a method for obtaining genetically modified avian offspring. Compared with the currently disclosed methods for converting PGCs in vitro, the operating method of the present invention can not only stably and effectively infect PGCs in vivo, but more importantly, it has a higher probability of producing gene-edited offspring, especially the breeding efficiency of transgenic chicken offspring with KRT75L4 gene and MSTN gene knockout. On the other hand, there is no need to integrate reporter genes or CRISPR-Cas protein genes into the bird's genome and express non-native proteins, which makes it more operational from a commercial perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : The electrophoresis results of the bands after the cell genome was cut with the knockout vector and digested with T7E1 in Example 2;

[0030] Figure 2 : TA cloning and Sanger sequencing results of the PCR products corresponding to sgRNAs 5 and 6 in Example 2;

[0031] Figure 3 and Figure 4 This is an analysis chart of the gene editing status of various chicken embryo tissues collected from Example 1 and Example 2 groups.

[0032] Figure 5 Before microinjection in Example 1 and Example 2, a circular window with a diameter of no more than 1 cm was cut in the egg with the blunt end facing upwards;

[0033] Figure 6 The dorsal aorta of the embryo was exposed under a microscope before direct microscopy in Example 1 and Example 2 (red arrow). DETAILED DESCRIPTION

[0034] The following definitions are provided to help explain the "Implementation" section below.

[0035] As used herein, "about," "approximately," and "substantially" should be understood to refer to a number within a numerical range, for example, within the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, and most preferably -0.1% to +0.1% of the referenced number.

[0036] In addition, all numerical ranges herein are understood to include all integers or fractions (including endpoints) within the range. In addition, these numerical ranges are understood to provide support for claims involving any number or subset of numbers within the range. For example, the disclosure of 1 to 10 is understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0037] As used herein and in the appended claims, unless the context clearly dictates otherwise, the singular form of a word includes the plural form. Thus, references to "a," "an," and "the" generally include the plural form of the corresponding term. For example, reference to "a component" or "a method" includes reference to a plurality of such components or methods. The term "and / or" employed in the context of "X and / or Y" should be interpreted as meaning "X" or "Y" or "X and Y." Similarly, "at least one of X or Y" should be interpreted as meaning "X" or "Y" or "both X and Y."

[0038] Similarly, the words "comprises / comprising / containing" are to be interpreted inclusively rather than exclusively. Likewise, the terms "comprises / comprising / containing" and "or" should be considered inclusive unless the context clearly prohibits such an interpretation. However, the embodiments provided by the present disclosure may not include any elements not expressly disclosed herein. Therefore, the disclosure of one embodiment defined by the terms "comprises / comprising / containing" is also the disclosure of multiple embodiments consisting essentially of and consisting of the disclosed components.

[0039] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0040] The term "examples / embodiments" used herein is for illustration only and should not be considered exclusive or comprehensive. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein, unless otherwise explicitly indicated.

[0041] Explanation of "gene editing", "gene modification" and "genetic modification":

[0042] ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR, and megaTAL are all effective tools for gene editing. The term "gene editing agent" in this application refers to agents used in gene editing methods such as ZFNs, TALENs, CRISPR, and megaTAL.

[0043] "Genetic modification" includes genome changes / hereditary changes resulting from gene editing or gene modification. Gene editing includes "gene knock-in" and "gene knock-out."

[0044] In some embodiments, the CRISPR-Cas9 system is introduced into PGC as a transient episomal plasmid or as a recombinant sgRNA-Cas9 protein complex (RNP). Using the adenovirus delivery method of the present application, in addition to genome editing itself, no foreign DNA traces of the CRISPR system are left. Producing DNA breaks can trigger DNA repair mechanisms, thereby causing INDELs at the break sites. These INDELs can inactivate the target gene. This inactivation can be achieved by non-homologous end joining repair mechanisms. On the other hand, the homologous recombination repair mechanism promotes the targeted integration (site-directed knock-in) of exogenous DNA sequences, which can replace the genomic DNA sequences flanking the breakpoints. The CRISPR system expression plasmid and the site-directed knock-in mass co-transfection PGC containing the exogenous DNA sequence to be introduced.

[0045] In some embodiments, the gene editing method for producing transgenic offspring includes a gene knockout method. In some embodiments, the above-mentioned genome editing technology (e.g., ZFN, TALEN, CRISPR and megaTAL or specific gene knockout of other methods known in the art) is applicable to knocking out a gene (e.g., silencing its expression), including but not limited to removing, replacing, inactivating, mutating, etc. to knock out or otherwise silence or inactivate a gene.

[0046] In the embodiment of the present application, a viral vector (adenoviral vector) is used and injected into the embryo by microinjection.

[0047] In some embodiments, methods for producing transgenic offspring include genetic modification that alters the genome. In some embodiments, the altered genome comprises DNA sequences or genes from different species to produce transgenic offspring. Genetic modification methods include, but are not limited to, methods described herein, including the use of endonucleases and genome modifiers, such as CRISPR, TALEN, etc., with or without a combination of homologous recombination repair mechanisms.

[0048] Implementation Plan

[0049] Example 1

[0050] The KRT75L4 gene encodes a keratin protein, a member of the intermediate filament protein family. Keratins are important structural proteins found throughout the epidermal cells of mammals, birds, and reptiles. They play a key role in the formation and maintenance of the cytoskeleton and are closely associated with biological processes such as cell differentiation, migration, and signal transduction.

[0051] The protein encoded by the KRT75L4 gene is a keratin subtype primarily expressed in the feathers and skin of chickens. This gene plays an important role in the growth and development of chickens, particularly in feather formation and skin protection, and may be associated with the chicken's disease resistance and growth rate.

[0052] The KRT75L4 gene may be an important candidate gene in chicken genetics and breeding research. Research on this gene can help us understand the growth, development, and disease resistance mechanisms of chickens, thereby providing a theoretical basis for genetic improvement and breeding of chickens.

[0053] 1. Design sgRNA targeting KRT75L4 gene and construct pX459-sgRNA expression vector

[0054] sgRNA design: The KRT75L4 gene sequence of the White Leghorn chicken (a laying hen breed) was obtained from the Ensembl website (https: / / asia.ensembl.org / index.html). Four sgRNAs were designed targeting exons 1 and 2 of the KRT75L4 gene using the CHOPCHOP V3 tool website (http: / / chopchop.cbu.uib.no / ). The specific sequences are shown in Table 1 below.

[0055] Table 1

[0056]

[0057] Construction of pX459-sgRNA plasmid DNA:

[0058] The sgRNA sequences listed in Table 1 were added to the sticky ends and complementary strands of Bbs I (for ligation to the CRISPR / Cas9 protein expression vector) to synthesize single-stranded DNA oligos. The Oligo-F and Oligo-R reaction components were then subjected to a PCR annealing procedure to ligate the synthesized single-stranded DNA into double-stranded DNA with sticky ends.

[0059] The pX459 plasmid was digested with Bbs I, and the linearized plasmid after digestion was rapidly ligated with the double-stranded DNA (oligonucleotide dimer) with sticky ends obtained above (reaction at 25° C. for 5 min) to obtain plasmid DNA.

[0060] Transform E. coli and amplify the pX459-sgRNA expression vector:

[0061] Remove the Fast-T1 E. coli competent cells from the -80°C ultra-low temperature storage box and place them on ice to thaw. Add 2uL of the DNA to be transformed into the competent cells, gently flick the tube wall to mix (avoid using a pipette), and let it stand on ice for 30 minutes; then place it in a 42°C water bath for 45 seconds and then quickly place it on ice for 2 minutes; then add 900uL of LB culture medium without antibiotics, mix well, and place it in a shaker at 37°C and 200rpm to recover for 1 hour; centrifuge at 5000rpm for 3 minutes, discard 900uL of supernatant, resuspend the bacteria with the remaining culture medium, take 50ul and evenly spread it on the LB solid plate containing the corresponding antibiotics, and place it in a 37-degree Celsius shaker for overnight culture;

[0062] A single colony was picked and placed in a 1.5 mL centrifuge tube to which 1 mL of ampicillin-resistant LB liquid medium had been added in advance. The tube was then cultured in a shaker at 37°C and 200 rpm for 8–12 hours. Sanger sequencing was then performed using the HU6F universal primer to determine whether the sgRNA scaffold region had been inserted into the desired sgRNA.

[0063] For each sgRNA, a correctly inserted colony was selected for expansion culture. The plasmid was extracted according to the instructions of the endotoxin-free plasmid miniprep kit to obtain the successfully constructed Cas9 / sgRNA expression vector.

[0064] 2. Screening of sgRNA targeting KRT75L4 gene

[0065] Cell plating: DF-1 cells frozen in liquid nitrogen were revived and cultured until the cells were in good condition. The cells were then plated in six-well plates for subsequent use.

[0066] Cell transfection: When the confluence of DF-1 cells in a six-well plate reaches about 70%, dilute the Lipofectamine 3000 reagent and the successfully constructed Cas9 / sgRNA expression vector in advance according to the instructions of the Lipofectamine 3000 liposome transfection reagent. Then, thoroughly mix the diluted reagent and DNA and incubate at room temperature for 10-15 minutes. Then, add the liposome-DNA complex to the cells and continue to culture in the incubator. Change the culture medium 6 hours after transfection;

[0067] Cell drug screening: When the confluence of the transfected cells reaches 95%, use a culture medium without dual-antibody containing 2 μg / mL puromycin for drug screening. Replace the culture medium containing puromycin every two days. After the cells in the blank control group die, replace them with complete culture medium without puromycin and continue culturing.

[0068] Cell DNA collection: After screening, collect cells when the cell confluence reaches 100%. Discard the waste liquid from each well and wash twice with 1mL DPBS. Then, add 500μl of trypsin to each well. After digestion is complete in a 37°C incubator, add 1mL of complete culture medium to terminate digestion. Then, blow off the cells and collect the liquid.

[0069] Extract sample DNA according to the Tissue DNA kit instructions;

[0070] Prepare a PCR amplification system using primers containing different barcode sequences. The PCR amplification program is shown in Table 2 below, and the amplification primers are shown in Table 3 below:

[0071] Table 2

[0072]

[0073] Table 3

[0074]

[0075] Purify and recover DNA from the PCR reaction system according to the Cycle Pure Kit instructions. The resulting DNA was tested for concentration and purity using a Nanodrop 2000. The product was then sent to the company for high-throughput sequencing of the amplicon.

[0076] After quality testing and data analysis of the amplicon library, the editing efficiency of each sgRNA was obtained, as shown in Table 4 below:

[0077] Table 4

[0078]

[0079]

[0080] 3. Recombinant adenovirus

[0081] Select E2-sgRNA2 with the highest in vitro gene editing efficiency in Table 4 above.

[0082] E2-sgRNA2 was packaged into adenovirus type 5 vector.

[0083] The pAV[CRISPR]-hCas9:P2A:EGFP-U6>gRNA (hereinafter referred to as AdV-CRISPR-EGFP, viral titer: 1.79×10 12 VP / ml) and control adenovirus pAV[Exp]-CMV>EGFP (hereinafter referred to as AdV-EGFP, virus titer: 1.79×10 12 VP / ml);

[0084] 4. Incubation and Microinjection

[0085] Use fertilized eggs from white Leghorn chickens.

[0086] In a specific embodiment of the present invention, newly laid fertilized eggs are incubated in an egg incubator at 37.8° C. and 60% humidity, with the eggs rotated 90 degrees every 2 hours and the blunt end facing upward, to HH14-17 stages (about 55-60 hours).

[0087] Use 75% alcohol to disinfect the whole egg, and place the egg with the blunt end facing up. First, use scissors to pierce the eggshell slightly in the center of the blunt end, and then use elbow scissors to slowly cut a circular window with a diameter of no more than 1 cm along the rupture (see Figure 5 ), then use bent-tip forceps to tear open the shell membrane covering the embryo and expose the dorsal aorta of the embryo (see Figure 6 );

[0088] The recombinant adenovirus AdV-CRISPR-EGFP (titer of 1.79×10 12 VP / ml) was aspirated into a hydraulic microinjector and 1-2 μl of the recombinant adenovirus was injected into the dorsal aorta (left dorsal aorta) of the chick embryo under a stereomicroscope. The blank control group was injected with the same amount of recombinant adenovirus AdV-EGFP.

[0089] Then, a drop of double-antibody (penicillin-streptomycin mixed solution) was dripped into the circular window, and then the window was quickly sealed with pressure-sensitive tape (see Figure 5 ), cut a 1cm x 1cm piece of black tape and place it over the pressure-sensitive tape to cover the circular window. Seal the black tape with a pressure-sensitive plastic bag and place the eggs in the incubator to continue incubation. Observe the eggs daily.

[0090] Example 2

[0091] The MSTN (myostatin) gene is an important gene that plays a key role in muscle growth and development in chickens.

[0092] MSTN, also known as Growth / Differentiation Factor 8 (GDF-8), is a member of the transforming growth factor beta (TGF-β) superfamily. It not only negatively regulates muscle growth but also regulates fat deposition, bone growth and development, and the strength of tendons and ligaments.

[0093] Studies have shown that the polymorphism of the MSTN gene is significantly correlated with the production performance of chickens.

[0094] Overexpression of the MSTN gene will hinder muscle proliferation, differentiation, growth and development, while its absence or reduced expression will lead to muscle hypertrophy and the formation of double muscle phenomenon.

[0095] Knockout of the MSTN gene can affect muscle growth in chickens, which has important implications for poultry breeding and meat production.

[0096] 1. Design sgRNA targeting the MSTN gene and construct the pX459-sgRNA expression vector

[0097] The MSTN gene sequence of Hy-Line White chicken (a laying hen breed) was obtained from the Ensembl website (https: / / asia.ensembl.org / index.html). Single-sgRNAs targeting exons of the MSTN gene were designed using the CHOPCHOPV3 tool website (http: / / chopchop.cbu.uib.no / ). Seven sgRNA sequences were selected based on the software's sgRNA scores, as shown in Table 5 below.

[0098] Table 5

[0099]

[0100] Construction of pX459-sgRNA plasmid DNA:

[0101] The sgRNA sequences listed in Table 1 were added to the sticky ends and complementary strands of Bbs I (for ligation to the CRISPR / Cas9 protein expression vector) to synthesize single-stranded DNA oligos. The Oligo-F and Oligo-R reaction components were then subjected to a PCR annealing procedure to ligate the synthesized single-stranded DNA into double-stranded DNA with sticky ends.

[0102] The pX459 plasmid was digested with Bbs I, and the linearized plasmid after digestion was rapidly ligated with the double-stranded DNA (oligonucleotide dimer) with sticky ends obtained above (reaction at 25° C. for 5 min) to obtain plasmid DNA.

[0103] Transform E. coli and amplify the pX459-sgRNA expression vector:

[0104] Remove the Fast-T1 E. coli competent cells from the -80°C ultra-low temperature storage box and place them on ice to thaw. Add 2uL of the DNA to be transformed into the competent cells, gently flick the tube wall to mix (avoid using a pipette), and let it stand on ice for 30 minutes; then place it in a 42°C water bath for 45 seconds and then quickly place it on ice for 2 minutes; then add 900uL of LB culture medium without antibiotics, mix well, and place it in a shaker at 37°C and 200rpm to recover for 1 hour; centrifuge at 5000rpm for 3 minutes, discard 900uL of supernatant, resuspend the bacteria with the remaining culture medium, take 50ul and evenly spread it on the LB solid plate containing the corresponding antibiotics, and place it in a 37-degree Celsius shaker for overnight culture;

[0105] A single colony was picked and placed in a 1.5 mL centrifuge tube to which 1 mL of ampicillin-resistant LB liquid medium had been added in advance. The tube was then cultured in a shaker at 37°C and 200 rpm for 8–12 hours. Sanger sequencing was then performed using the HU6F universal primer to determine whether the sgRNA scaffold region had been inserted into the desired sgRNA.

[0106] For each sgRNA, select a correctly inserted colony for expansion culture, extract the plasmid according to the instructions of the endotoxin-free plasmid extraction kit, and obtain the successfully constructed Cas9 / sgRNA expression vector

[0107] 2. Screening of sgRNA targeting the MSTN gene

[0108] Cell plating: DF-1 cells frozen in liquid nitrogen were revived and cultured until the cells were in good condition. The cells were then plated in six-well plates for subsequent use.

[0109] Cell transfection: When the confluence of DF-1 cells in a six-well plate reaches about 70%, dilute the Lipofectamine 3000 reagent and the successfully constructed Cas9 / sgRNA expression vector in advance according to the instructions of the Lipofectamine 3000 liposome transfection reagent. Then, thoroughly mix the diluted reagent and DNA and incubate at room temperature for 10-15 minutes. Then, add the liposome-DNA complex to the cells and continue to culture in the incubator. Change the culture medium 6 hours after transfection;

[0110] Cell drug screening: When the confluence of the transfected cells reaches 95%, use a culture medium without dual-antibody containing 2 μg / mL puromycin for drug screening. Replace the culture medium containing puromycin every two days. After the cells in the blank control group die, replace them with complete culture medium without puromycin and continue culturing.

[0111] Cell DNA Collection: After screening, cells were collected when the confluence reached 100%. Discard the waste liquid from each well and wash twice with 1 mL of DPBS. Add 500 μl of trypsin to each well and digest in a 37°C incubator. After digestion is complete, add 1 mL of complete culture medium to terminate the digestion. Disperse the cells and collect the liquid. Extract sample DNA according to the Tissue DNA kit instructions and perform PCR amplification. Amplification primers are shown in Table 6 below:

[0112] Table 6

[0113]

[0114] DNA was purified and recovered from the PCR reaction system according to the instructions of the Cycle Pure Kit, and the concentration and purity of the obtained DNA were tested using Nanodrop 2000;

[0115] T7E1 digestion: Double-stranded DNA should be re-paired before using T7E1 digestion verification. The annealing reaction procedure is shown in Table 7 below:

[0116] Table 7

[0117]

[0118] Add 1 μL of T7E1 endonuclease to the annealed product, mix, and incubate the system in a PCR instrument at 37°C for 30 minutes to perform the enzyme digestion reaction. Add Gel Loading Dye Purple to the system to terminate the reaction. Run the resulting reaction product on a 2% agarose gel electrophoresis and use a gel imaging system to photograph and record. If the cell genome has been cut with the knockout vector, the experimental group will show enzyme digestion bands after T7E1 digestion, such as Figure 1 shown.

[0119] The T7E1 enzyme digestion bands showed that sgRNA 5 and 6 had higher efficiency. The corresponding PCR products were cloned by TA and sequenced by Sanger to analyze the gene editing efficiency. 8-9 single colonies were randomly selected for each sgRNA for sequencing. The results are shown in the figure below. Figure 2 As shown, drug screening confirmed that the editing efficiencies of sgRNA 5 and sgRNA 6 were 100% and 87.5%, respectively.

[0120] 3. Recombinant adenovirus

[0121] sgRNAs 5 and 6 were packaged into adenovirus type 5 vector to obtain pAV[CRISPR]-hCas9:P2A:EGFP-U6>gRNA (hereinafter referred to as AdV-CRISPR-EGFP, virus titer: 1.15×10 12 VP / ml) and control adenovirus pAV[Exp]-CMV>EGFP (hereinafter referred to as AdV-EGFP, virus titer: 1.15×10 12 VP / ml);

[0122] 4. Incubation and Microinjection

[0123] Take fertilized eggs from Hy-Line White chickens.

[0124] Newly laid fertilized eggs were incubated in an egg incubator at 37.8°C and 60% humidity, with the eggs rotated 90 degrees every 2 hours and the blunt end facing upwards, until HH14-17 (approximately 55-60 hours).

[0125] Use 75% alcohol to disinfect the whole egg, and place the egg with the blunt end facing up. First, use scissors to pierce the eggshell slightly in the center of the blunt end, and then use elbow scissors to slowly cut a circular window with a diameter of no more than 1 cm along the rupture (see Figure 5 ), then use bent-tip forceps to tear open the shell membrane covering the embryo and expose the embryo's dorsal aorta (see Figure 6 );

[0126] The recombinant adenovirus AdV-CRISPR-EGFP (titer of 1.15×10 12 VP / ml) was aspirated into a hydraulic microinjector and 1-2 μl of the recombinant adenovirus was injected into the left dorsal aorta of the chicken embryo under a stereomicroscope. The blank control group was injected with the same amount of recombinant adenovirus AdV-EGFP.

[0127] Then, a drop of double-antibody (penicillin-streptomycin mixed solution) was dripped into the circular window, and then the window was quickly sealed with pressure-sensitive tape (see Figure 5 ), cut a 1cm x 1cm piece of black tape and place it over the pressure-sensitive tape to cover the circular window. Seal the black tape with a pressure-sensitive plastic bag and place the eggs in the incubator to continue incubation. Observe the eggs daily.

[0128] Performance data

[0129] In Example 1 and Example 2, 300 eggs were processed in each group, of which 47% and 58% of the chicken embryos died after hatching, respectively.

[0130] Of the remaining surviving eggs, three were randomly selected, and tissues including liver, spleen, heart, kidney, lung, intestine, stomach, intestinal tract, breast muscle, brain, skin, and gonads were collected from chicken embryos incubated to HH41–45 (15–20 days);

[0131] 16 chicks were randomly selected and their blood samples were collected after they were hatched into chicks.

[0132] In Example 1 group and Example 2 group, each group had 20 and 18 male individuals, respectively. Semen samples were collected from the male individuals after they reached sexual maturity.

[0133] The collected tissue samples were subjected to Tissue DNA Kit, and the collected blood and semen samples were subjected to NRBC Blood DNA Kit for genomic DNA extraction.

[0134] The gene editing efficiency of each sample was determined using amplicon sequencing and other techniques containing PCR amplified target fragments. The results of Example 1 and Example 2 are shown in Tables 8 and 9 below.

[0135] Table 8

[0136]

[0137] Table 9

[0138]

[0139]

[0140] For Example 1 and Example 2, the gene editing conditions of the collected chicken embryo tissues were analyzed in detail. Figure 3 and Figure 4 .

[0141] from Figure 3 It can be seen that the 8bp deletion accounts for the highest proportion.

[0142] from Figure 4 As can be seen in the figure, a 50 bp deletion was detected.

[0143] From Tables 8 and 9 above and Figure 3 and 4 The results show that, using the methods of Examples 1 and 2 of the present application, whether knocking out the chicken KRT75L4 gene or the chicken MSTN gene, in addition to finding successfully edited gametes in the semen of sexually mature roosters, it was unexpectedly found that other tissues of the embryo, especially the gonads, also achieved a higher gene editing efficiency.

[0144] This shows that, by using the method of the present invention, the sgRNA targeting the target gene knockout with high gene editing efficiency obtained by in vitro experiment screening is used to construct a Cas9 / sgRNA expression vector, which is packaged into an adenovirus and injected into a specific location (inside the dorsal aorta of the embryo) when PGCs migrate to the developing gonads. It was unexpectedly found that the adenovirus can not only infect migrating PGCs in vivo, but also infect other tissues of the chicken embryo, especially the gonadal tissue, achieving a higher gene editing efficiency. This means that the probability of producing gene-edited offspring is higher. From a commercial perspective, it can greatly reduce the time to obtain transgenic chicken offspring and improve breeding efficiency.

[0145] Compared with the currently disclosed methods for transforming PGCs in vitro, the operating method of the present invention can not only stably and effectively infect PGCs in vivo, but more importantly, it has a higher probability of producing gene-edited offspring, especially the efficiency of cultivating transgenic chicken offspring with KRT75L4 gene and MSTN gene knockout. On the other hand, there is no need to integrate reporter genes or CRISPR-Cas protein genes into the genome of poultry and express non-native proteins, which is safer and more operational from a commercial perspective.

[0146] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0147] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for obtaining genetically modified avian offspring, characterized in that: The method comprises the following steps: Step 1) incubating the fertilized eggs of the bird until the embryo forms the dorsal aorta; Step 2) Injecting the packaged recombinant adenovirus into the dorsal aorta of the embryo by microinjection when PGCs migrate to the developing gonads; The recombinant adenovirus is packaged with a gene editing substance targeting a target gene, or is packaged with an exogenous gene vector; The titer of the recombinant adenovirus was 1×10 12 ~1×10 13 VP / ml; Step 3) Continue incubation and collect semen from sexually mature male individuals for testing; screen male F0 generation individuals whose semen is detected to have gene editing or gene modification of the target gene, and collect their semen for artificial insemination of a wild-type female population; Step 4) Screening and obtaining heterozygous female F1 generation individuals, mating them with the male F0 generation individuals, and screening and obtaining homozygous genetically modified offspring.

2. The method according to claim 1, wherein: The gene editing material uses a gene editing material selected from the group consisting of ZFN, TALEN, CRISPR and megaTAL methods; preferably, the gene editing material is a gene editing material for site-directed knockout, or a gene editing material for site-directed knockin.

3. The method according to claim 2, wherein: In step 2), the recombinant adenovirus is a type 5 recombinant adenovirus, which is packaged with a CRISPR gene editing material for site-directed knockout; preferably, the CRISPR gene editing material contains sgRNA and Cas9 protein targeting the target gene.

4. The method according to any one of claims 1 to 3, wherein: The bird is a Galliformes, Anseriformes, Otidiformes, Columbiformes or Struthioniformes animal; preferably, the bird is a Galliformes animal; more preferably, the Galliformes animal is selected from the Numididae or Phasianidae animals; more preferably, the Phasianidae animals include Gallus or Meleagris animals.

5. The method according to claim 4, wherein: The bird belongs to the order Galliformes, family Phasianidae, genus Gallus; in step 2), microinjection is performed when the embryo is hatched to stage 14 to 17 according to the Hamburger-Hamilton staging system.

6. The method according to claim 5, wherein: In step 1), newly laid fertilized eggs are incubated at 37.8° C., 60% humidity, and rotated 90 degrees every 2 hours with the blunt end facing upward; in step 2), when the embryos are incubated to stages 14 to 17 according to the Hamburger-Hamilton staging system, a window is opened at the blunt end to expose the dorsal aorta of the embryo.

7. The method according to claim 3, wherein: The bird belongs to the order Galliformes, family Phasianidae, genus Gallus; The target gene is KRT75L4 gene, and the sequence of the sgRNA is as shown in SEQ ID NO.1 to 8; preferably the sequence shown in SEQ ID NO.4 to 7; more preferably the sequence shown in SEQ ID NO.5; or, The target gene is the MSTN gene, and the sequence of the sgRNA is shown in SEQ ID NOs. 9 to 15; preferably, the sequence shown in SEQ ID NOs. 13-14.

8. The method according to claim 3, wherein: In step 2), the titer of the recombinant adenovirus is 1×10 12 ~2×10 12 VP / ml, 1-2 μL of recombinant adenovirus was injected into the left dorsal aorta of the embryo.

9. The method according to claim 3, wherein: The type 5 recombinant adenovirus is packaged with a CRISPR adenovirus vector; preferably, the CRISPR adenovirus vector uses a pAV[CRISPR]-hCas9:P2A:EGFP-U6 vector inserted with sgRNA targeting the target gene.

10. A genetically modified avian offspring, characterized in that: The avian offspring is obtained by the method according to any one of claims 1 to 9.

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