Method for realizing precise editing of herbivorous livestock oocyte single allele and application

Abstract

The invention belongs to the technical field of cell engineering and gene engineering, and discloses a method for realizing precise editing of a single allele of an oocyte of herbivorous livestock and application, and the method for realizing precise editing of the single allele of the oocyte comprises the following steps: preparing sgRNA for target oocyte gene editing; carrying out cytoplasm microscopic co-injection on the oocytes; and carrying out in-vitro fertilization and detection on the gene editing oocytes. According to the method, the herbivorous livestock oocyte monoalleles can be accurately deleted. According to the method for specifically knocking out the oocyte gene, sgRNA and Cas9mRNA capable of specifically modifying the oocyte gene in a targeted manner are co-transfected to an oocyte, so that the oocyte gene is knocked out; the co-transfection mode specifically can be microscopic co-injection; the oocytes can be specifically sheep oocytes, and the co-injection time of the oocytes can be 20 hours of in-vitro maturation.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering and genetic engineering, and in particular relates to a method and application for realizing precise editing of single alleles in sheep oocytes. Background Art

[0002] The CRISPR / Cas9 system is an adaptive immune defense system formed by bacteria and archaea during the long-term evolution process, which can be used to fight against invading viruses and foreign DNA. The CRISPR / Cas9 gene editing technology is a technology for specific DNA modification of targeted genes. This gene editing technology system is mainly composed of Cas9 nuclease and sgRNA. Cas9 nuclease has an active domain that cuts double-stranded DNA, causing double-stranded DNA breaks (DSB); sgRNA is a chimeric RNA formed by the combination of tracrRNA with a scaffolding function and specific crRNA. sgRNA targets and recruits the Cas9 / sgRNA complex to the target gene through RNA-DNA base complementary pairing, and Cas9 then cuts the double-stranded DNA at a fixed point by recognizing the PAM motif (5'-NGG-3') on the target gene, thereby editing the target gene.

[0003] Since 2014, the Institute of Bioengineering of Xinjiang Academy of Animal Husbandry Sciences has used fertilized egg microinjection technology and Cas9 / sgRNA gene editing technology to target edit sheep fertilized eggs, and obtained hundreds of gene-edited sheep. It is one of the few laboratories in China that can carry out large-scale in vitro production of sheep embryos, embryo transplantation and sheep genome editing breeding. The edited target genes are mostly important functional genes related to reproduction, meat production, coat color and hair length, such as fibroblast growth factor 5 (FGF5), which is closely related to the wool production and hair length of sheep and camels. However, it has been found in production practice that the deletion and modification of some important functional genes may affect the development and pregnancy rate of the embryo, and even cause abnormal development of the embryo, thereby inducing miscarriage. Therefore, it is of great significance to only perform single allele deletion on oocytes for the study of embryonic development and gene function. Summary of the invention

[0004] In response to the problems existing in the prior art, the present invention provides a method and application for achieving precise editing of a single allele in an oocyte.

[0005] The present invention is achieved by providing a method for achieving precise editing of monoallelic genes in oocytes of herbivorous livestock, the method comprising the following steps:

[0006] Step 1: Preparation of sgRNA targeting oocyte gene editing;

[0007] Step 2: in vitro maturation of oocytes and cytoplasmic microinjection;

[0008] Step three: in vitro fertilization and testing of gene-edited oocytes.

[0009] Further, in step 1, the preparation of the sgRNA includes:

[0010] (1) BbsⅠ single enzyme digestion plasmid 42230Cas9 plasmid concentration 510ug / ml OD = 1.83; 10×FastDigestBuffer2, Cas9 plasmid 0.8, FastDigest enzyme 1, ddH2O 16.2, a total of 20Ul; 37℃ enzyme digestion for 2h, enzyme digestion product electrophoresis, gel cutting, QIAGEN kit gel recovery, and 30ul Nuclease-free water column elution;

[0011] (2) Primer annealing: CF71, CR71, 10× buffer A 2, ddH2O 16, 20ul in total; 95°C for 5min, cooled to room temperature at 0.1°C / s;

[0012] (3) Connect the linearized plasmid and the annealed product, Solution I 5ul, plasmid 2, annealed primer 1, ddH2O 2, a total of 10ul; connect at 16 degrees for 1h;

[0013] (4) Transform DH5α, coat Amp+ plates, 100 μl of DH5α E. coli competent cells, slowly thaw on ice, take 5 μl of the ligation product and add it to 50 μl of DH5α E. coli competent cells, gently flick with your fingers to mix, and place on ice for 30 min;

[0014] (5) Pick a single colony on the plate and perform colony PCR verification, 10× buffer 2.5ul, dNTP2, TF7 or CF7 0.5ul, TR7 0.5ul, template 1ul, TaqE 0.125ul, ddH2O 18.375ul, a total of 25ul; PCR program: 94℃ 4min; 94℃ 30s, 55℃ 30s, 72℃ 30s, 72℃ 10min; 2.0% agarose gel electrophoresis, product size 100bp;

[0015] (6) Amplify and label the verified plasmid or PCR product, and send it for sequencing; perform PCR amplification using the plasmid; PrimeSTAR Max Premix (2×) 25ul, TF 70.5ul, TR 70.5ul, Template 0.5ul, ddH2O 23.5ul, totaling 50ul; thermo amplification using TAQ enzyme; purify with QIAGEN PCR product purification kit and use as a transcription template;

[0016] (7) Using MEGA shortscript TM Kit for in vitro transcription; T710×Reactionbuffer2ul, T7 ATPsolution 2ul+C+G+U 2ul each, mix to 8ul, Template DNA 8ul, T7 Enzyme mix2ul, a total of 20ul; Incubate the reaction at 37℃ for at least 2h, preferably more than 3h;

[0017] (8) Use MEGAclear after transcription TM Kit purification; first preheat 200ul Elution solution at 96 degrees, and finally perform electrophoresis detection and cryopreservation.

[0018] Another object of the present invention is to provide a method for specifically knocking out an oocyte gene, the method comprising:

[0019] The sgRNA and Cas9 mRNA capable of specifically targeting and modifying oocyte genes are co-transfected into oocytes, thereby knocking out oocyte genes.

[0020] Furthermore, the co-transfection method is co-injection.

[0021] Furthermore, the oocyte may be a sheep oocyte, and the oocyte is injected for 20 hours of in vitro maturation.

[0022] Another object of the present invention is to provide an application of the method for specifically knocking out oocyte genes in precise editing of oocyte genes.

[0023] Another object of the present invention is to provide an application method of the specific knockout of oocyte genes, the application method of the specific knockout of oocyte genes comprising:

[0024] (1) The pX330 plasmid was digested with BbsI to obtain a digestion vector, and the target gene sgRNA obtained by annealing was connected to the digestion vector to obtain a recombinant plasmid;

[0025] (2) The target gene sgRNA in step (1) may also be an mRNA targeting FGF5 gene editing;

[0026] (3) using the sgRNA recombinant plasmid obtained in step (2) to obtain FGF5-sgRNA for microinjection by in vitro transcription and purification;

[0027] (4) The FGF5-sgRNA and Cas9 protein obtained in step (3) are co-injected into oocytes to achieve sgRNA targeted editing of oocyte genes.

[0028] Furthermore, in step (4), the concentration of the FGF5-sgRNA is 50 ng / μl, and the concentration of the Cas9 protein is 100 ng / μl.

[0029] Furthermore, the oocyte microinjected with the Cas9 protein / sgRNA may be an oocyte that has been matured and cultured in vitro for 20 hours.

[0030] Furthermore, the Cas9 protein / sgRNA can also be co-injected into oocytes with ssODN to achieve HDR precise editing of oocyte target genes.

[0031] In combination with all the above technical solutions, the advantages and positive effects of the present invention are as follows: the method for realizing precise editing of oocyte monoallelic genes provided by the present invention can realize precise deletion of oocyte monoallelic genes. The present invention protects a method for specifically knocking out oocyte genes, which is to co-transfect cells with the sgRNA and Cas9 mRNA that can specifically target and modify oocyte genes, thereby knocking out oocyte genes. The co-transfection method can be specifically co-injection. The oocyte can be specifically a sheep oocyte and a camel oocyte, and the time for co-injection of the oocyte can be 20 hours of in vitro maturation. The present invention also provides the application of the above technology in precise editing of oocyte genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of gene editing targeting the first exon region of the oocyte FGF5 gene provided by an embodiment of the present invention.

[0033] Figure 2 It is a schematic diagram of the PCR amplification product of FGF5-sgRNA provided in an embodiment of the present invention.

[0034] Figure 3 It is a schematic diagram of randomly selecting 3 recombinant plasmid FGF5-sgRNA PCR products for in vitro transcription to obtain mRNA provided by an embodiment of the present invention.

[0035] FIG. 4 is a schematic diagram of gene editing of in vitro matured oocytes and embryo development provided by an embodiment of the present invention.

[0036] FIG. 4( a ) is a schematic diagram of an oocyte matured in vitro for 20 hours provided in an embodiment of the present invention.

[0037] FIG4( b ) is a schematic diagram of co-injection of sgRNA and Cas9 mRNA into oocytes matured in vitro for 20 hours, as provided in an embodiment of the present invention.

[0038] Figure 4(c) is a schematic diagram of a gene-edited embryo at the cleavage stage provided in an embodiment of the present invention.

[0039] Figure 4(d) is a schematic diagram of a gene-edited embryo that has developed to the blastocyst stage provided in an embodiment of the present invention.

[0040] Figure 5 It is a schematic diagram of the single embryo slot PCR amplification results provided by an embodiment of the present invention;

[0041] In the figure: M is a marker, the horizontal bar indicates a negative control, and the numbers 1-15 are embryo numbers.

[0042] Figure 6 This is a sequencing peak diagram of the oocyte gene editing PCR product provided in an embodiment of the present invention.

[0043] Figure 7 It is a schematic diagram of the FGF5 gene sgRNA sequence and ssODN provided in the embodiments of the present invention.

[0044] Figure 8 This is the sequencing peak diagram of the monoallelic GG deletion in the gene-edited sheep oocyte provided in an embodiment of the present invention.

[0045] Figure 9(a) is a peak diagram of monoclonal sequencing of gene-edited sheep oocyte PCR products provided in an embodiment of the present invention.

[0046] Figure 9(b) is a peak diagram of monoclonal sequencing of PCR products of gene-edited camel oocytes provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0048] The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.

[0049] The method for achieving precise editing of a single allele in an oocyte provided by an embodiment of the present invention comprises the following steps:

[0050] S101, preparation of sgRNA for targeted oocyte gene editing;

[0051] S102, oocyte cytoplasmic microco-injection was performed;

[0052] S103, perform in vitro fertilization and testing of gene-edited oocytes.

[0053] The technical scheme provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, see "Molecular Cloning (Third Edition)" for details. The experimental materials used in the following embodiments, unless otherwise specified, were purchased from conventional biochemical reagent companies.

[0054] The oocyte monoallelic precise editing method provided by the present invention realizes the editing of specific monoallelic genes in oocytes through precise gene editing technology. The core of the method is to design specific sgRNA targeting oocytes, and combine microinjection technology to achieve accurate delivery of editing tools, so that the gene editing process is more accurate. First, for the monoallelic characteristics of the target gene, a specific sgRNA is designed and synthesized to ensure that the editing process can accurately act on the target gene sequence to avoid nonspecific interference to other genes.

[0055] Next, sgRNA and gene editing tools (such as CRISPR / Cas9 system) are injected into the cytoplasm of oocytes using cytoplasmic microinjection technology. As a high-precision tool, microinjection enables sgRNA to be quickly and stably introduced into cells, ensuring that the editing system can directly act on the target gene region. At the same time, microinjection can effectively reduce the degradation and loss of editing tools, thereby improving the efficiency and success rate of gene editing.

[0056] After the injection is completed, the edited oocytes are fertilized in vitro, and genetic testing is performed when the embryo develops to the appropriate stage. Genetic testing can confirm whether the editing of the monoallelic gene is successful and verify the accuracy of the editing. This step is crucial to ensure the editing effect. Through testing, non-target effects can be excluded and the expected mutation of the required monoallelic gene can be confirmed, ensuring the specificity and efficiency of the editing.

[0057] The overall process of this method is simple and efficient. Conventional biotechnology methods are used from sgRNA design, microinjection to in vitro fertilization and detection, which is easy to promote and apply. This method overcomes the difficulties of traditional gene editing methods in single allele editing, achieves higher editing accuracy, and provides new technical support for gene research, genetic disease treatment and other fields.

[0058] Example 1: Preparation of FGF5-sgRNA

[0059] (1) According to the design principle of CRISPR / Cas9 technology, sgRNA was designed for the sheep FGF5 gene using online software (http: / / www.genome-engineering.org / crispr / ). Based on the potential off-target sites and comprehensive scores of each site, the sequence with low predicted off-target effect was selected as the sgRNA template sequence. Figure 1 As shown, the sgRNA sequence targeting the FGF5 gene includes a 20 nt sequence paired with the first exon of the FGF5 target gene (SEQ ID NO: 1: 5'-gatcaccctaggatataccc-3').

[0060] Figure 1 Schematic diagram of gene editing targeting the first exon region of the oocyte FGF5 gene, where the underlined portion represents the 20 nt sequence recognized by the FGF5-sgRNA pairing, and the 3 red nt are the PAM sequences required for Cas9 to recognize and bind to the target site.

[0061] (2) Use restriction endonucleases according to the instructions for use to digest the px330 plasmid (Addgene 42330). Perform electrophoresis on the digestion products and use the QIAGEN kit to recover the correct digestion products according to the instructions.

[0062] (3) Add 1 μl of each forward and reverse primer (i.e., SEQ ID NO: 2) (10 μM) as shown in Table 1, incubate at 95 degrees for 5 min, and cool to room temperature at 0.1 degrees / s to obtain primer annealing products. The underlined portion is the sgRNA sequence paired with the target gene, and caccg is the restriction site connected to the BbsI restriction plasmid.

[0063] Table 1 Primers used to prepare FGF5-sgRNA

[0064]

[0065] (4) The enzyme digestion product (10 μl) obtained in step (2) and the primer annealing product (10 μl) obtained in step (3) were connected according to conventional procedures to obtain a recombinant plasmid.

[0066] (5) Using the recombinant plasmid obtained in step (4) as a template (randomly selecting 7 recombinant plasmid clones), conventional PCR amplification was performed using the primers described in Table 2 (SEQ ID NO: 3) (where the underlined portion of TF-sgRNA is the sgRNA sequence paired with the target gene, and the T7 promoter sequence required for in vitro transcription of mRNA is before the sgRNA sequence). The size of the obtained PCR product is 120 bp, and the electrophoresis results are shown in 2% agarose conventional gel electrophoresis. Figure 2 .

[0067] Table 2 Primer list

[0068]

[0069] (6) Take 1 μg of the PCR product obtained in step (5) above (PCR products of three recombinant plasmids were randomly selected) as a template and perform in vitro transcription according to the instructions of the in vitro transcription kit MEGA shortscriptTM kit (ambion AM1354) to obtain a dedicated sgRNA targeting the FGF5 gene. All steps involving in vitro mRNA manipulation must strictly avoid mRNA enzyme contamination.

[0070] (7) The sgRNA obtained in step (6) was purified according to the RNA purification kit (ambionAM1908) and the purified product was subjected to 2% gel electrophoresis. The electrophoresis results are shown in Figure 3 , the qualified sgRNA was stored at -80℃.

[0071] Example 2: Maturation, cytoplasmic microinjection and fertilization of sheep oocytes

[0072] (1) Sheep ovaries were collected from a slaughterhouse, sterilized and washed 3 to 4 times with saline, oocytes were extracted, washed 3 to 4 times with maturation solution, and oocytes wrapped with more than 3 layers of granulosa cells were selected and randomly divided into 3 groups. Oocytes were transferred into maturation solution for in vitro maturation for 16 h, 20 h and 24 h, respectively (the volume of maturation solution was 700 μl / well, 70 oocytes were placed in each well, and the following oocyte operations, fertilization and embryo culture were all of the same volume), and then placed in a 38.6°C incubator containing 5% CO2 (the following incubator culture was all carried out under the same conditions).

[0073] The composition of the maturation medium: TCM199 culture medium + 10% FBS by volume + 0.05 IU / ml FSH + 0.05 IU / ml LH + 1 μg / ml estradiol + 24.2 μg / ml sodium pyruvate + 0.1 mM / L cysteine ​​+ 10 ng / ml EGF + 100 IU / ml penicillin + 100 IU / ml streptomycin.

[0074] (2) The oocytes (parts) matured in vitro for 20 h in step (1) were Figure 4a ), gently blow with 0.1% hyaluronidase to remove granulosa cells, wash three times with oocyte handling solution, and then put into the balanced oocyte handling solution.

[0075] Micromanipulation fluid composition: M199 + 10% FBS + 100IU / ml double antibody

[0076] (3) The Cas9mRNA / FGF5-sgRNA mixture described in Examples 1 and 2 was injected into the cytoplasm of sheep oocytes using a NIKON micromanipulator (see Figure 4b ), each oocyte was injected with about 10pL of Cas9mRNA / FGF5-sgRNA mixture, and this step strictly required RNase-free operation. The co-injected oocytes were placed in oocyte maturation solution for further maturation culture (i.e., the total in vitro maturation time of the three groups of co-injected oocytes was still 24h).

[0077] (4) In vitro fertilization of gene-edited oocytes: Take frozen semen, thaw it in a water bath, transfer it to the upstream of the balanced fertilization solution for 20-25 minutes, then aspirate the upper semen, centrifuge it at 1500 rpm for 4-5 minutes, discard the supernatant, and obtain sperm sediment (for sperm counting). Gene-edited oocytes that have been mature for 24 hours are placed in the fertilization solution that has been balanced for more than 3 hours for group fertilization. The sperm concentration is 1-5×106 / ml, and in vitro fertilization is carried out at 38.6℃ for 24 hours.

[0078] The composition of the fertilization solution: SOF solution + 20% estrus sheep serum + 6IU / ml heparin sodium + 100IU / ml gentamicin. SOF solution: contains 6.29mg / ml NaCl, 0.534mg / ml KCl, 0.162mg / ml KH2PO4, 0.6μl / ml sodium lactate, 0.089mg / ml MgSO4, 2.1mg / ml NaHCO3, 0.0357mg / ml sodium pyruvate and 0.299mg / ml CaCL2·2H2O.

[0079] (5) 24 h after fertilization, the fertilized eggs of each group were washed 2-3 times with embryo culture medium, transferred to a four-well culture plate filled with embryo culture medium, marked, and cultured in vitro in a carbon dioxide incubator at 38.6°C, 5% CO2, 5% O2, 90% N2, and saturated humidity. The cleavage rate of each group was calculated 48 h after fertilization ( Figure 4c ), after 168h of culture, the blastocyst rate of each group was counted ( Figure 4dThe statistical results of cleavage rate and blastocyst rate of each group are shown in Table 3. The cleavage rate and blastocyst rate of embryos in the co-injection group with oocytes matured for 20 hours were significantly higher than those in the control group with oocytes matured for 16 hours and 24 hours (p<0.01).

[0080] The composition of the embryo culture medium: SOF medium + 1% NEAA (non-essential amino acid) + 2% BAA (essential amino acid) + 100 IU / ml double antibody + 5 mg / ml BSA (bovine serum albumin).

[0081] Table 3 Effects of co-injection of oocytes at different maturation times on embryo cleavage rate and blastocyst rate

[0082]

[0083] Note: Lowercase letters in the same column indicate no significant difference (p>0.05); different letters in the same column indicate significant difference (p<0.05)

[0084] Example 3: Detection of target gene editing efficiency in embryos

[0085] (1) After 168 h of embryo culture, the gene-edited embryos were collected, each embryo was washed twice with PBS buffer, and then placed in 5 μL of lysis buffer and lysed at 37°C for 3 h.

[0086] Lysis buffer composition: the solvent is Tris-HCl (50 mM, pH 8.0), containing 0.5% (V / V) Triton X-100 and 1 mg / mL Proteinase K.

[0087] The cleavage product of step (1) was used as a template to perform slot PCR on the FGF5 gene editing region (primers were redesigned within the first PCR amplification product (SEQ ID NO: 4) for secondary amplification (SEQ ID NO: 5). The information of the first amplification primers and the annealing temperature are shown in Table 4. The results of the single embryo slot PCR are shown in Figure 5 .

[0088] Table 4 Primers for FGF5 gene editing embryo detection

[0089]

[0090] (3) The PCR amplification product of step (2) was sent to Shanghai Sangon Biotechnology Co., Ltd. for PCR product sequencing and identification. The sequencing results are shown in Figure 6 The gene editing positive rates of oocytes injected at different maturation times are shown in Table 5.

[0091] Table 5 Gene editing efficiency of oocytes at different injection times.

[0092]

[0093] Example 5: Application of ssODN in precise gene editing of oocytes

[0094] In another embodiment of the present invention, the FGF5-ssODN sequence is simultaneously added to the Cas9 / FGF5-sgRNA for co-injection into the oocyte cytoplasm, thereby improving the efficiency of precise editing HDR. Figure 6 As shown, the added FGF5-ssODN is 120 bp in size (58 nt on the left homology arm and 62 nt on the right homology arm). The underlined part is the FGF5-sgRNA sequence paired with the target gene. The red mark is the PAM sequence of 3 Gs. FGF5-ssODN introduces a deletion of 2 Gs (GG-deletion) in the PAM sequence, thereby introducing a frameshift mutation in the FGF5 gene. HDR precise editing PCR sequencing diagram is shown in Figure 7 , monoclonal sequencing map see Figure 8 and Figure 9.

[0095] Example 6

[0096] The method for preparing sgRNA provided in the embodiment of the present invention comprises the following steps:

[0097] 1. BbsⅠ single restriction plasmid 42230Cas9 plasmid concentration 510ug / ml OD = 1.83

[0098] 10×FastDigest Buffer2, Cas9 plasmid 0.8, FastDigest enzyme 1, ddH2O 16.2, a total of 20Ul; enzyme digestion at 37 degrees for 2h, electrophoresis of the digestion product, gel cutting, QIAGEN kit gel recovery, and elution through the column with 30ul Nuclease-free water.

[0099] 2. Primer annealing: CF7 1, CR7 1, 10×bufferA 2, ddH2O 16, 20ul in total; 95 degrees for 5 minutes, and cool to room temperature at 0.1 degrees / s.

[0100] 3. Connect the linearized plasmid and the annealed product, Solution I 5ul, plasmid 2, annealed primer 1, ddH2O 2, a total of 10ul; connect at 16 degrees for 1h.

[0101] 4. Transform DH5α, apply Amp+ plates, 100ul of DH5α E. coli competent cells, slowly thaw on ice, take 5μl of the ligation product and add it to 50μl of DH5α E. coli competent cells, gently flick with your fingers to mix, and place on ice for 30 minutes.

[0102] Keep in 42℃ warm water for 45s, quickly take out and put on ice for 1-2min, add 450μl LB culture medium without Amp at room temperature, and shake culture at 37℃ for 1-2h. Centrifuge, discard the supernatant, take 100ul heat shock transformation product to plate, and culture at 37℃ overnight. A total of one plate was coated, half of which was CF7 and the other half was CF8. After overnight culture, the number of clones grown was relatively small, and 15 clones were picked from CF7 and CF8 respectively.

[0103] 5. Pick a single colony on the plate and perform colony PCR verification, 10× buffer 2.5ul, dNTP2, TF7 (or CF7) 0.5ul, TR7 0.5ul, template 1ul, Taq E 0.125ul, ddH2O 18.375ul, a total of 25ul; PCR program: 94 degrees for 4min;

[0104] 94 degrees for 30 seconds, 55 degrees for 30 seconds, 72 degrees for 30 seconds, 72 degrees for 10 minutes; 2.0% agarose gel electrophoresis, the product size is about 100 bp;

[0105] 6. Amplify and number the verified correct plasmid (or PCR product) and send it for sequencing;

[0106] 7. Use plasmid for PCR amplification; PrimeSTAR Max Premix (2×) 25ul, TF7 0.5ul, TR7 0.5ul, Template 0.5ul, ddH2O 23.5ul, totaling 50ul; thermo's TAQ enzyme amplification;

[0107] After purification with QIAGEN PCR product purification kit, use it as a transcription template (it is best to send it for sequencing again).

[0108] 8. Use MEGA shortscript TM kit (Cat. No.: AM1354) for in vitro transcription;

[0109] 9. Use MEGAclear after transcription TM kit (Cat. No.: Life Technologies, cat. no. AM1908) for purification;

[0110] 10. Electrophoresis detection and cryopreservation are the same as Cas9.

[0111] In the description of the present invention, unless otherwise specified, "plurality" means two or more than two; the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0112] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for achieving precise editing of a single allele in an oocyte, characterized in that: The method for achieving precise editing of single alleles in oocytes, mainly oocytes of large animals such as herbivorous livestock, comprises the following steps: Step 1: Preparation of sgRNA targeting oocyte gene editing; Step 2, oocyte cytoplasmic microinjection; Step three: in vitro fertilization and testing of gene-edited oocytes.

2. The method for achieving precise editing of a single allele in an oocyte according to claim 1, characterized in that: In step 1, the preparation of the sgRNA includes: (1) BbsⅠ single enzyme digestion plasmid 42230Cas9 plasmid concentration 510ug / ml OD = 1.83; 10×FastDigestBuffer2, Cas9 plasmid 0.8, FastDigest enzyme 1, ddH2O 16.2, a total of 20Ul; 37℃ enzyme digestion for 2h, enzyme digestion product electrophoresis, gel cutting, QIAGEN kit gel recovery, and 30ul Nuclease-free water column elution; (2) Primer annealing: CF71, CR71, 10× buffer A 2, ddH2O 16, 20ul in total; 95°C for 5min, cooled to room temperature at 0.1°C / s; (3) Connect the linearized plasmid and the annealed product, Solution I 5ul, plasmid 2, annealed primer 1, ddH2O2, a total of 10ul; connect at 16 degrees for 1h; (4) Transform DH5α, coat Amp+ plates, 100 μl of DH5α E. coli competent cells, slowly thaw on ice, take 5 μl of the ligation product and add it to 50 μl of DH5α E. coli competent cells, gently flick with your fingers to mix, and place on ice for 30 min; (5) Pick a single colony on the plate and perform colony PCR verification, 10× buffer 2.5ul, dNTP2, TF7 or CF7 0.5ul, TR7 0.5ul, template 1ul, TaqE 0.125ul, ddH2O 18.375ul, a total of 25ul; PCR program: 94℃ 4min; 94℃ 30s, 55℃ 30s, 72℃ 30s, 72℃ 10min; 2.0% agarose gel electrophoresis, product size 100bp; (6) Amplify and label the verified plasmid or PCR product, and send it for sequencing; perform PCR amplification using the plasmid; PrimeSTAR Max Premix (2×) 25ul, TF 70.5ul, TR 70.5ul, Template 0.5ul, ddH2O 23.5ul, totaling 50ul; thermo amplification using TAQ enzyme; purify with QIAGEN PCR product purification kit and use as a transcription template; (7) Using MEGA shortscript TM Kit for in vitro transcription; T710×Reactionbuffer2ul, T7 ATPsolution 2ul+C+G+U 2ul each, mix to 8ul, Template DNA 8ul, T7 Enzyme mix2ul, a total of 20ul; Incubate the reaction at 37℃ for at least 2h, preferably more than 3h; (8) Use MEGAclear after transcription TM Kit purification; first preheat 200ul Elution solution at 96 degrees, and finally perform electrophoresis detection and cryopreservation.

3. A method for specifically knocking out an oocyte gene, characterized in that: The method for specifically knocking out oocyte genes comprises: The sgRNA and Cas9 mRNA capable of specifically targeting and modifying oocyte genes are co-transfected into sheep cells, thereby knocking out oocyte genes.

4. The method for specifically knocking out an oocyte gene as claimed in claim 3, characterized in that: The co-transfection method is co-injection.

5. The method for specifically knocking out an oocyte gene as claimed in claim 3, characterized in that: The oocytes may be sheep oocytes or camel oocytes, and the oocytes may be injected for 20 hours of in vitro maturation.

6. Use of the method for specifically knocking out oocyte genes as described in any one of claims 3 to 5 in precise editing of oocyte genes.

7. A method for applying the method for specifically knocking out oocyte genes as claimed in claim 6 in accurately editing oocyte genes, characterized in that: The application method of specifically knocking out oocyte genes comprises: (1) The pX330 plasmid was digested with BbsI to obtain a digestion vector, and the target gene sgRNA obtained by annealing was connected to the digestion vector to obtain a recombinant plasmid; (2) The target gene sgRNA in step (1) may also be an mRNA targeting FGF5 gene editing; (3) using the sgRNA recombinant plasmid obtained in step (2) to obtain FGF5-sgRNA for microinjection by in vitro transcription and purification; (4) The FGF5-sgRNA and Cas9 protein obtained in step (3) are co-injected into oocytes to achieve sgRNA targeted editing of oocyte genes.

8. The method for applying the method for specifically knocking out oocyte genes as claimed in claim 7 in accurately editing oocyte genes, characterized in that: In step (4), the concentration of the FGF5-sgRNA is 50 ng / μl, and the concentration of the Cas9 protein is 100 ng / μl.

9. The method for applying the method for specifically knocking out oocyte genes as claimed in claim 8 in accurately editing oocyte genes, characterized in that: The oocyte microinjected with the Cas9 protein / sgRNA may be an oocyte that has been matured and cultured in vitro for 20 hours.

10. The application method for specifically knocking out oocyte genes according to claim 8, characterized in that: The Cas9 protein / sgRNA can also be co-injected into oocytes with ssODN to achieve HDR precise editing of oocyte target genes.

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