CN111334529B - Method for preparing accurate BLG gene knockout cattle by using third generation base editor - Google Patents

Abstract

The invention discloses a method for preparing an accurate BLG gene knockout cattle by using a third generation base editor. The preparation method of the protective gene knockout dairy cow comprises the following steps: mutating 277 th basic group from C to T of the 5' end of the BLG gene in the genome of the starting dairy cow; the BLG gene is a DNA molecule shown in SEQ ID No. 1. The invention establishes a large animal point mutation technology, can carry out accurate single base mutation, and can achieve the purpose of gene knockout by skillfully designing C-T mutation to cause a stop codon to terminate protein translation in advance. Compared with the traditional gene editing technology, the point mutation technology based on BE3 does not generate genome double-strand break, thereby greatly avoiding random mutation and even genome damage and having higher safety. The invention provides important technical support for accurate gene editing and gene knockout research of cattle.

Description

Method for preparing accurate BLG gene knockout cattle by using third generation base editor

Technical Field

The invention relates to a method for preparing an accurate BLG gene knockout cattle by using a third generation base editor.

Background

Milk is an important nutritionally well-balanced health food for worldwide consumption, comprising abundant proteins, carbohydrates, fats and minerals. Although milk yield has been improved by natural breeding strategies over the years, nutrient management and quantitative genetics, milk composition has not changed significantly. With the development of biotechnology and genetic engineering, the development of animal husbandry provides more opportunities for the preparation of "designer milk" particularly suitable for human beings.

To date, various high quality transgenic cattle have been reported, such as cows expressing recombinant human lactoferrin, cows with increased milk casein content, cows with resistance to mastitis, cows with resistance to mad cow disease, and calves with no horns that improve animal welfare. However, the problem of allergic reactions in milk is not well solved at present.

Beta-lactoglobulin is a main allergen in milk, the milk allergy is most common in infant food allergy, and foreign epidemiological investigation results show that the incidence rate of infant milk protein allergy is 0.3% -3.5%. And tends to rise continuously. The absorption and utilization of the nutrient substances in the dairy products by the infants are seriously influenced. To date, researchers have tried various techniques to reduce the sensitization of BLG in cow's milk, mainly including heat treatment, high pressure, enzymatic, and glycosylation modification, among others. Although these methods reduce the sensitization of BLG to some extent, the structure and function of other proteins in milk are destroyed, the nutritional effect of milk is greatly reduced, and the sensitization of BLG cannot be completely eliminated. How to effectively reduce BLG allergenicity and simultaneously keep the original structure and function of protein as much as possible, no effective and feasible technical method exists at home and abroad at present.

The gene site-directed modification is one of the important means for editing genes, namely, the target gene can be modified and can also be used for treating human genetic diseases, so that the technology becomes a research hotspot of modern molecular biology. However, the gene targeting technology based only on homologous recombination is extremely inefficient and has limited applications. The advent of artificial endonuclease technology has changed this situation. Zinc finger endonucleases (ZFNs) were the first generation of artificial endonucleases. Zinc fingers are proteins capable of binding DNA, about half of the transcription factors of human cells contain zinc finger structures, ZFNs are endonucleases formed by fusing zinc finger proteins with the cleavage domain of the endonuclease Fok1, and it is possible to make double-stranded nicks of DNA at specific positions of various complex genomes using the same. To date, ZFNs have been successfully applied to black-tailed monkeys, rats, mice, chinese hamsters, zebrafish, drosophila, sea urchins, silkworms, arabidopsis thaliana, tobacco, corn, pigs, cattle, human iPS cells. However, ZFNs are complex to prepare, expensive, and their technical patents are controlled by a few commercial companies. Soon, the advent of a second generation artificial nuclease-like transcription activator-like effector nuclease (TALEN) largely replaced ZFNs. In 2009, scientists deciphered the base correspondence between transcription activator-like effectors (TALEs) encoded by pathogenic bacteria (Xanthamonas) of rice and DNA. In 2010, the successful application of the TALEN protein in yeast is reported for the first time, and then the TALEN protein is rapidly applied to plants, human cells, mice, zebra fish, pigs and cattle. TALEN can carry out meticulous modification to complicated genome like ZFN, and its structure is comparatively simple simultaneously, and the specificity is higher, has consequently received scientific research worker's favor. In 2012, TALENs were rated as one of ten scientific breakthroughs by the journal of science. The principles of the two artificial nucleases are the same regardless of ZFN or TALEN, and the two artificial nucleases are formed by fusing DNA binding protein and endonuclease Fok1, so that a large amount of vector construction work is required, and the multi-gene knockout operation is difficult to perform.

In the beginning of 2013, a brand-new artificial endonuclease (CRISPR)/CRISPR-associated) Cas9 appears, which is mainly formed by modifying an acquired immune system based on bacteria, and has the characteristics of simple manufacture, low cost and high effect. Two subject groups of Feng Zhang and George m. Church of MIT in 2013 successfully edited human cells first using this technique and could perform multigene knockout, and then this technique was rapidly applied to mice, zebrafish, nematodes, rats, rabbits, dogs, pigs, cattle and plants. However, the technology only determines the target specificity and has 20bp sequence and off-target phenomenon. Although researchers subsequently upgraded nickase versions of dCas9 and high fidelity Cas9 in this technology to greatly reduce off-target effects, these technologies all utilize DSBs made on host genomic DNA, which are then progressively repaired by NHEJ or HDR, with certain drawbacks. The efficiency of NHEJ is far higher than HDR, however, the repair randomness of NHEJ is strong, random fragment deletion or insertion of a target gene is often caused, and accurate point mutation preparation is difficult to perform; HDR repair, while highly accurate, is extremely inefficient and restricted to mitotic cells, limiting the application of HDR repair. Therefore, the precise point mutation technique remains a big problem. Because most of mutations existing in nature are point mutations, establishing an efficient point mutation technology for repair and quality improvement is significant. The problem group of David R. Liu at Harvard university in the United states is published in Nature, and a single-Base Editor (BE) based on CRISPR/Cas9 is innovatively developed to effectively solve the problems. This group connects rat cytosine nucleoside deaminase rAPOBEC1 converting cytosine (C) to uracil (U) to the N-terminus of catalytically inactive Cas9 (catharticy end Cas9, dCas9) via linker sequence XTEN, constructing first generation single base editor (BE1) (rAPOBEC 1-XTEN-dCas 9), followed by further structural optimization such as adding UGI and restoring a cleavage domain of Cas9 (rAPOBEC 1-XTEN-dCas 9-UGI), successfully preparing third generation single base editor base editors (BE3) that can efficiently mutate C to T at the human cellular level. This technology was subsequently successfully applied in mice, zebrafish and plants.

Disclosure of Invention

The invention aims to provide a method for preparing a precise BLG gene knockout cattle by using a third generation base editor.

In a first aspect, the method for preparing a knockout bovine having a protected gene of the present invention comprises the steps of: mutating 277 th base of BLG gene from 5' end to T; the BLG genes on two homologous chromosomes of the gene knockout cattle have the same point mutation; the BLG gene is a DNA molecule shown in SEQ ID No. 1.

The difference between the gene knockout cattle and the starting cattle is only that single-point mutation is introduced into the BLG gene of the starting cow (namely the 277 th nucleotide of the DNA molecule shown in SEQ ID No.1 from 5' end is changed from C mutation to T mutation, and the two chromosomes are subjected to the same mutation).

Further, the method comprises the steps (1) and (2):

(1) preparing a gene knockout cell: introducing a third generation single base editor BE3 and gRNA for identifying BLG gene into bovine fibroblasts, and preparing gene knockout cells through gene editing; all 277 bases of the BLG genes on two homologous chromosomes of the gene knockout cell from the 5' end are mutated from C to T; the target sequence of the gRNA is shown as 273 to 295 th positions from the 5' end of SEQ ID No. 1;

(2) and (2) cloning the single cell of the gene knockout cell obtained in the step (1) by a somatic cell nuclear transfer technology to obtain the gene knockout cattle.

In the step (2), the gene knockout cell single cell is used as a donor cell to be injected into the bovine oocyte with the cell nucleus removed, a reconstructed embryo is developed and formed, then the reconstructed embryo is transferred into the cow uterus, and the gene knockout cow is obtained through delivery.

Further, the present invention also provides a method for knocking out a BLG gene in a cell, comprising the steps of: introducing a third generation single base editor BE3 and gRNA for identifying BLG gene into a target cell, and preparing a gene knockout cell through gene editing; the 277 th base of the BLG gene on two homologous chromosomes of the gene knockout cell from the 5' end is mutated from C to T; the target sequence of the gRNA is shown as 273 to 295 th positions from the 5' end of SEQ ID No. 1; the BLG gene is a DNA molecule shown in SEQ ID No. 1.

The target cell may be specifically a bovine fibroblast.

Any one of the gRNAs is shown in SEQ ID No. 4. Any one of the above gRNAs can be obtained by in vitro transcription of a template, which is shown in SEQ ID No. 3.

Any one of the above third generation single base editors BE3 is mRNA transcribed in vitro from a vector expressing the third generation single base editor BE3 or a protein translated from the mRNA.

The vector for expressing the third generation single base editor BE3 can BE pCMV-BE3 vector.

The gRNA and the mRNA are introduced into bovine fibroblasts in an electric shock transformation mode, and the mass ratio of the gRNA to the mRNA is 1: 1.

After the gRNA and mRNA are introduced into bovine fibroblasts, a single cell clone (gene knockout positive cell) is prepared by an infinite dilution method.

In a second aspect, the invention provides the use of a method according to any one of the first aspects for breeding cows producing hypoallergenic milk.

The method for breeding the dairy cattle producing hypoallergenic milk comprises the following steps: the gene knockout dairy cow prepared by the method in the first aspect is the dairy cow producing hypoallergenic milk.

In a third aspect, the present invention provides a protection kit comprising any one of the grnas described above, or a gene encoding the gRNA, or an expression cassette containing the encoding gene; the kit is used for preparing gene knockout cattle.

The kit also comprises a vector for expressing the third generation single base editor BE3 or mRNA obtained by in vitro transcription of the vector or protein obtained by translation of the mRNA.

In a fourth aspect, the invention protects the application of the kit in the third invention in breeding cows producing hypoallergenic milk.

In each of the above aspects, the cow may be a holstein cow.

The possibility that an exogenous BE3 plasmid is integrated into a receptor genome exists when a BE3 plasmid is used for preparing a gene editing animal, so that the transfection is carried out by using mRNA of BE3, the problem is avoided, the biological safety problem caused by screening of a marker gene is avoided, and then the BLG double allele C-T point mutation positive cell clone without exogenous DNA integration is finally obtained by using a gene editing method without a screening marker, and the early termination of AA is caused by the mutation of C-T, so that the purpose of BLG gene knockout is achieved. The positive cells are subjected to somatic cloning to prepare the BLG gene knockout cattle with accurate editing. When the cow normally lactates, the experiment proves that the milk of the cow does not contain BLG protein.

The invention establishes a large animal point mutation technology which can carry out accurate single base mutation and achieve the purpose of gene knockout by skillfully designing C-T mutation to cause a stop codon to terminate protein translation in advance. Compared with the traditional gene editing technology, the point mutation technology based on BE3 does not generate genome double-strand break, thereby greatly avoiding random mutation and even genome damage and having higher safety. The invention provides important technical support for accurate gene editing and gene knockout research of cattle.

Drawings

FIG. 1 is a technical scheme of the present invention.

FIG. 2 is a schematic diagram showing the target region sequence of BE3 single base modification technique and the achievement of BLG gene knockout by the single base modification technique of BLG gene.

FIG. 3 shows the results of the sequencing alignment in example 2.

FIG. 4 shows the results of SDS-PAGE in example 3.

FIG. 5 shows the Western blot detection results in example 3.

FIG. 6 is the statistical results of the anal temperature changes of the mice in example 4.

FIG. 7 shows the results of ELISA assay in example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

pCMV-BE3 vector: addgen, Cat number: # 73021.

In the pCMV-BE3 vector, the CMV promoter drives the expression of the third generation base editor BE3 (rAPOBEC 1-XTEN-Cas9 n-UGI-NLS).

Example 1 preparation of BLG Diallelic mutant cell lines Using third Generation base editor

This example utilizes the third generation base editor (BE3) to prepare BLG biallelic mutant cell lines. Considering the later industrialization and avoiding the biosafety problem, the invention does not use a screening marker gene and simultaneously uses the mRNA of BE3, so that a BLG biallelic gene mutant cell line without exogenous DNA integration can BE prepared, and the technical route is shown in figure 1. The target region sequence of BE3 single base modification technology and the schematic diagram of BLG gene knockout achieved by the single base modification technology of BLG gene are shown in FIG. 2.

According to the BLG gene sequence (SEQ ID No. 1), the region of the target sequence with base editing is positioned on the exon 1 (SEQ ID No. 2) of the BLG gene sequence, and the binding sequence of the target region is cacccagaccatgaagggccTGG (SEQ ID No.1 at position 273 and 295 from the 5' end,97-119 th from the 5' end of SEQ ID No. 2), wherein tgg is a PAM sequence, and the underlined site is the expected mutation site.

First, preparation of in vitro transcribed BE3 mRNA

1. In vitro transcribed mRNA systems were configured at room temperature (table 1). After the prepared in vitro transcription mRNA system is completely mixed, the mixture is incubated for 1 h at 37 ℃, 1 mu L of TURBO DNase is added into the reaction system, and the incubation is carried out for 15min at 37 ℃.

TABLE 1

Components	Volume of
		Linearized pCMV-BE3 vector treated with Ahd1	1μL（0.1-1μg）
2×NTP/CAP	10μL
		10×Buffer	2μL
RNA synthetase	2μL
		ddH2O	Make up to 20 mu L

All of the above reagents were obtained from MEGAscript T7 Transcription Kit, Ambion, cat # SEQ ID NO: AM 1333.

2. Taking the reaction product obtained in the step 1, configuring an in vitro transcription mRNA and polyA system (table 2), and incubating for 1 h at 37 ℃ after complete mixing.

TABLE 2

Components	Volume of
		Reaction product of step 1	20μL
5×E-PAP Buffer	20μL
		MnCl2(25mM)	10μL
ATP(10mM)	10μL
		E-PAP	4μL
ddH2O	36μL
		Total of	100μL

All from Poly (A) labeling Kit, Ambion, cat # K: AM 1350.

3. In vitro transcription of mRNA purification by column chromatography

(1) And (3) adding 350 mu L of binding buffer into the reaction product obtained in the step (2), blowing, sucking and uniformly mixing, transferring the sample into a column, and centrifuging at 10000g for 1min at room temperature.

(2) Discarding the filtrate, re-packing the column, rinsing the column with 500 μ L of eluent, centrifuging at 10000g at room temperature for 1min, and repeatedly rinsing once; the filtrate was discarded and centrifuged on an empty column for 15 s.

(3) The column was placed in a new centrifuge tube, 50. mu.L of the eluent was added to the center of the column, the lid was closed and incubated at 60 ℃ for 10min, and 10000g was centrifuged at room temperature for 1 min.

Both the reagents and the column were from clear ­ Transcription clear-Up Kit, Ambion, cat #: AM 1908.

BE3 mRNA was obtained according to the procedure described above.

Second, preparation of in vitro transcribed gRNA

1. A gRNA in vitro transcription template (SEQ ID No. 3) was synthesized.

In SEQ ID No.3, the T7 promoter is located at positions 1 to 20 from the 5' end, the transcription initiation site is located at position 19, the target sequence is located at positions 21 to 40, and the sgRNA is located at positions 41 to 120.

2. Taking the gRNA in-vitro transcription template obtained in the step 1, configuring an in-vitro transcription system (table 3), and incubating for 2 h at 37 ℃.

TABLE 3

Components	Volume of
		Form panel	8 μL(2μg)
2×ATP/CTP/UTP/GTP	2/2/2/2μL
		10×Reaction Buffer	2 μL
Enzyme Mix
		2 μL
Total of			20 μL

3. After completion of step 2, 1. mu.L of TURBO DNase was added to the reaction system and incubated at 37 ℃ for 15 min.

4. After the step 3 is completed, 2 times of volume of absolute ethyl alcohol is added to precipitate the transcription product, the transcription product is kept stand at the temperature of minus 20 ℃ for 30min, the transcription product is centrifuged at 13000rpm for 30min, the supernatant is discarded, and the precipitate is taken.

5. And (3) fully washing the precipitate obtained in the step (4) by using 75% (v/v) ethanol water solution, removing supernatant, fully drying the precipitate by using an ultra-clean bench, and fully dissolving the gRNA (the gRNA sequence is shown as SEQ ID No. 4) by using nucleic-free water.

All of the above reagents were obtained from MEGASHORTCRIPT-T7 Transcription Kit, Ambion, cat #: AM 1354.

Third, obtaining bovine fibroblasts

Collecting ear skin tissue of Holstein cow, removing hair from lower edge back side of ear, cleaning with 70% (v/v) ethanol water solution, and picking with blade with area of 1cm²The skin was transported to the laboratory as soon as possible in DMEM/F12 medium at 0 deg.C, washed several times with PBS buffer and 70% (v/v) alcohol in water and chopped to 1mm³The left and right small blocks were washed with DMEM medium for 2 times and then planted in batches in 1mL DMEM medium flasks (25 cm in specification) containing 10% (v/v) FBS²) After the tissue blocks adhere firmly, the DMEM medium containing 10% (v/v) FBS is supplemented to 6mL, the temperature is 37 ℃, and the CO content is 5 percent₂The culture box is cultured for 6-7 days, the culture solution is changed 1 time every 2 days, after the cells grow and are confluent, the cells are digested and passaged for 2-3 times by 0.25% trypsin, and the cells are frozen in DMEM culture medium containing 20% (v/v) FBS and 10% (v/v) DMSO in batches. Thus, the Holstein cow fibroblast cell line is established through in vitro culture operations such as primary culture, subculture, freezing and the like.

IV, BE3 mRNA transfection

(1) 2 days before transfection, the bovine fibroblast cell line obtained in step three was trypsinized into single cells, and 1 × 10 was added⁶The bovine fibroblasts were transferred to a culture flask (100 mL), 4mL of DMEM medium containing 10% (v/v) fetal bovine serum was added thereto, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing to logarithmic growth phase.

(2) After the step (1) is completed, taking bovine fibroblasts in logarithmic growth phase, digesting the bovine fibroblasts with trypsin, centrifuging the bovine fibroblasts for 5min at 1000g, and collecting precipitates.

(3) After completion of step (2), the pellet was taken, washed 1 time with PBS buffer, and then resuspended with 100 μ L of shock solution to obtain a cell suspension.

(4) Taking cell suspension (containing 1 × 10)⁶Cell), 4. mu.g of BE3 mRNA obtained in the first step and 4. mu.g of gRNA obtained in the second step were thoroughly mixed and transferred into an electric shock cup for electric shock (electric field strength 1.2KV/cm, pulse time 1 ms).

(5) After the step (4) is completed, the cells after electric shock are transferred into a culture bottle (the specification is 100 mL), 4mL of DMEM culture solution containing 10% (v/v) fetal calf serum is added, the temperature is 37 ℃, and 5% CO is added₂Culturing for 1-2 days to obtain transfected cells.

Five, infinite dilution method for preparing single cell clone and genotype identification

(1) And (3) culturing the transfected cells obtained in the fourth step until the fusion rate reaches 80-90%, digesting the cells by using 0.1% pancreatin at 37 ℃, stopping digestion by using a DMEM medium containing 10% (v/v) FBS, and centrifuging to collect the cells.

(2) The cells were resuspended in a DMEM medium containing 20% (v/v) FBS, a portion of the cells were counted, and the cells were diluted to 100 cells/mL to obtain a cell suspension. To 20 dishes to which 9mL of 20% (v/v) FBS-containing DMEM medium had been added, 1mL of each cell suspension was added at 37 ℃ with 5% CO₂And culturing under saturated humidity condition.

(3) When the cell clone in the culture dish grows to be more than 2mm in diameter, removing the culture medium, washing with DPBS, covering the clone cluster with a clone ring, dripping about 100 mu L of 0.1% pancreatin at 37 ℃ into the clone cluster, digesting for about 3min, dripping DMEM culture medium containing 20% (v/v) FBS to stop digestion, slightly blowing and then transferring the cell clone into a 48-well plate for culture expansion.

(4) When the cell fusion rate in the 48-well plate reaches 90%, half of the cells are taken for cell clone genotype identification after digestion, and the rest half are continuously cultured in the well plate.

(5) After the cells for identifying the genotype are centrifuged for 5min at 1000g, the supernatant is discarded, and 10-20 mul of cell lysate is added according to the amount of the cell precipitate.

(6) mu.L of cell lysate was pipetted as a template for PCR identification, and PCR amplification was performed using a primer pair consisting of primer P1 and primer P2 (designed and synthesized based on the bovine BLG gene).

Primer P1: 5'-AGGCCTCCTATTGTCCTCGT-3', respectively;

primer P2: 5'-GCAAAGGACACAGGGAGAAG-3' are provided.

The reaction system was 20. mu.L, consisting of 1.0. mu.L of DNA template, 0.4. mu.L of primer P1 (10. mu.M), 0.4. mu.L of primer P2 (10. mu.M), 0.4. mu.L of dNTP, 0.3. mu.L of LA DNA polymerase, 2.0. mu.L of 10 XPCR Buffer, and 15.5. mu.L of ddH 2O.

Reaction procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 52 ℃, 1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 5min and 4 deg.C.

And purifying and sequencing the PCR amplification product. If a mutation is generated at the target site, a bimodal phenomenon appears in the sequencing result, and the cell line is determined to be a mutant. And connecting the PCR product of the mutant to a pMD-19t plasmid vector, transforming the competence of escherichia coli, selecting a plurality of single colonies for sequencing, and comparing the sequencing result with the wild type BLG gene to obtain detailed mutation information.

The results showed that of the 32 single cell clones, one biallelic C-T mutation was obtained and AA prematurely terminated. The sequencing is carried out, and the BLG gene in the genome of the wild-type fibroblast is the wild-type BLG gene (shown in SEQ ID No. 1). The BLG gene in the genome of mutant fibroblast cells differs from the wild-type BLG gene only in that a single point mutation (i.e., the 277 th nucleotide from the 5' end of the DNA molecule shown in SEQ ID No.1 is mutated from C to T, and the same mutation occurs in both chromosomes) is introduced into the wild-type BLG gene.

Example 2 preparation of BLG double allele knock-out cattle

1. Taking the mutant fibroblast cells in logarithmic growth phase, and digesting with 0.25% trypsin for 5min to obtain single cells.

2. Collecting ovaries of adult Holstein cows from slaughter houses, cleaning in PBS (phosphate buffer solution) at 37 ℃ for three times, extracting follicles with the diameter of 2-8mm by using a needle with the diameter of 0.7mm, recovering cumulus-oocyte-complex (COCs) with uniform shape and compact structure, washing twice by using mature liquid (M199+10% FBS +0.01U/mL bFSH +0.01U/mL bLH +1 mu g/mL estradiol), putting the cumulus-oocyte-complex into a four-well plate containing the mature liquid at 38.5 ℃ and 5% CO for 50-60 holes, and washing by using a washing machine₂After the mature culture in the incubator for 18-20h, placing the mature oocyte in a tube of 0.1% hyaluronidase, oscillating for 2-3min, and lightly blowing and beating with a glass tube to completely separate the cumulus cell from the oocyte, and selecting the oocyte with complete shape, uniform cytoplasm and discharged first polar body as a cytoplasmic receptor.

Transferring the oocyte with the first polar body into an operation liquid containing M199+10% FBS +7.5 mug/mL cytochalasin B, cutting a small opening on the upper part of the polar body by using a glass needle under a 200-time microscope, sucking off chromosomes in the first polar body and the oocyte under the first polar body by using a glass tube with the inner diameter of 20 mug, washing the first polar body and the oocyte under the first polar body for three times by using a solution of M199+20% FBS, and placing the first polar body in an incubator for later use.

3. Transferring the single cell obtained in the step 1 into an enucleated oocyte transparent band, firstly placing the enucleated oocyte transparent band into Zimmerman liquid (100 mL Zimmerman liquid is composed of sucrose 0.9854g, magnesium acetate tetrahydrate 10.7mg, calcium acetate monohydrate 1.8mg, dipotassium hydrogen phosphate 7.4mg, reduced glutathione 3.1mg, bovine serum albumin 1.0mg and water) for balancing for 3-5min, then placing the enucleated oocyte transparent band into a fusion tank to rotate the oocyte to enable donor cells to be in contact with the oocyte prepared in the step 2 to be vertical to an electric field, simultaneously, rapidly transferring the oocyte into M199 liquid (product of Gibco company) containing 10% (v/v) FBS in a direct current pulse field with the field intensity of 2.5kV/cm under the conditions that the pulse time is 10 mu s, the pulse frequency is 2 times and the pulse interval is 1s, and culturing for 30min at 37 ℃ and 5% CO2 to obtain the embryo.

4. Taking the reconstructed embryo obtained in the step 3, adding CR1aa culture solution (100 mL of CR1aa culture solution is composed of 0.67g of sodium chloride, 0.023g of potassium chloride, 0.22g of sodium bicarbonate, 2mg of sodium pyruvate, 100 mul of phenol red and water) of calcium ionophore A23178 with the concentration of 5 mul, and treating for 5 min; discarding the liquid phase, adding CR1aa culture solution containing 5 μ g/mL cytochalasin B and 10 μ g/mL cycloheximide, and treating for 5h (for activating recombinant embryo); discarding the liquid phase, adding 5% (v/v) FBS-containing CR1aa culture solution, 37 deg.C, 5% CO₂Culturing for 48h, observing the cleavage rate, and observing the blastocyst development rate in 7-8 days.

5. The morphologically excellent transgenic cloned blastocysts cultured for 7 days in step 4 were transferred into the uterine horns of the contemporary recipient cows (3-head recipient cow was co-transplanted), and BLG double allele knock-out cows (i.e., No. #170923 cows) were obtained.

The genomic DNA of the ear tissue of a cattle to be detected (cattle No. 170923 or a wild Holstein cow) is extracted by adopting a blood and tissue genomic kit (QIAGEN company), and is used as a template, and a primer pair consisting of a primer P1 and a primer P2 (shown in Table 1) is adopted for PCR amplification, so that a PCR amplification product is obtained. And purifying and sequencing the PCR amplification product.

Sequencing results show that the genotype of a wild Holstein cow (i.e. a non-transgenic cow) based on the BLG gene is wild type (BLG +/+), and the genotype of a cattle #170923 based on the BLG gene is biallelic mutant type (BLG-/-). The biallelic mutant is characterized in that 277 nucleotides of wild-type two homologous chromosomes (the wild-type BLG gene is shown as a sequence 1 in a sequence table) are mutated from C to T from 5' end, and the transcription and translation of mRNA of the BLG gene are terminated early due to the base mutation of C-T, so that the function of BLG protein is lost.

The sequencing alignment results are shown in FIG. 3.

Cattle #170923 survived normally at present, see FIG. 3.

Example 3 analysis of BLG protein expression in milk from BLG double allele knock-out cattle

First, SDS-PAGE detects whether BLG protein exists in milk of BLG double allele knock-out cattle

1. Extracting total protein from milk produced from 1 st to 7 th days of lactation period of cattle to be detected (BLG double allele knock-out cattle or wild Holstein cow).

2. And (3) taking the total protein of the cattle to be detected obtained in the step (1) and a BLG protein standard (a product of Sigma company) respectively, and performing SDS-PAGE.

The results are shown in FIG. 4 (M is protein Marker and BLG is BLG protein standard). The results show that the total protein of the BLG double allele knock-out cattle has no expression of BLG protein detected, and the milk produced by the BLG double allele knock-out cattle has no BLG protein, so that the BLG gene is knocked out at a DNA level, and the BLG is also effectively removed at a protein level.

Secondly, detecting whether BLG protein exists in milk of cattle with BLG double allele knockout by Western blot

1. Extracting total protein from milk produced on 1 st, 3 rd, 5 th or 7 th day of lactation period of cattle (BLG double allele knock-out cattle or wild Holstein cow) to be detected.

2. And (3) respectively taking the total protein of the cattle to be detected obtained in the step (1) and the BLG protein standard substance, and carrying out Western blot. The BLG protein was detected using a Bovine beta-lactoglobulin Antibody (product of BETHYL corporation) as a primary Antibody and a goat Antibody (product of China fir Jinqiao corporation) as a secondary Antibody.

The results of the experiment are shown in FIG. 5 (in FIG. 5, BLG is a standard BLG protein). The results show that the total protein of the BLG double allele knock-out cattle can not detect the expression of the BLG protein, and the milk produced by the BLG double allele knock-out cattle can not have the BLG protein.

The results show that the milk produced by the BLG gene disruption of the cattle with the BLG double allele knockout does not contain BLG protein.

Example 4 sensitization assay of BLG double allele knock-out cow milk

To further verify whether the milk produced by BLG knockout cattle had low allergenicity, the milk sample from #170923 was made into dry powder for later sensitization experimental studies, while milk powder from wild type milk was used as a control.

Animal model: clean-grade female Balb/c mice, Beijing Wittingle laboratory animal technology Ltd, certification number SCXK (Jing) 2012-0001.

The clean-grade common feed is prepared by mixing the levels of main nutritional ingredients and processing into pellet feed; then pass through⁶⁰Co radiation sterilization is carried out, so that the feed reaches a clean level. The level of the clean-grade common feed reaches the experimental animal nutrition standard listed in GB 14924-2010.

Feeding conditions are as follows: the experiment is carried out in SPF animal houses of food and nutrition engineering college of China agricultural university, and the qualification number is as follows: SYXK (Kyoto) 2010-0036. The environment temperature is 22-25 ℃, the relative humidity is 40% -60%, the artificial illumination/dark cycle is carried out for 12 h, and the ventilation frequency is 15 times/h. Animals in the same group were fed 4 animals per cage, with free access to food and water.

Taking 96 clean-grade female Balb/c mice with the weight of 16-20g, after the mice are fed with clean-grade common feed adaptively for 5 days, randomly dividing the mice into 8 groups of a positive control group, a negative control group, a non-transgenic milk powder group 1, a non-transgenic milk powder group 2, a non-transgenic milk powder group 3, a beta-LG double allele knockout milk powder group 1, a beta-LG double allele knockout milk powder group 2 and a beta-LG double allele knockout milk powder group 3 according to the weight, and carrying out the following treatment (the average weight of the mice needs to be weighed before each gastric lavage for adjusting the concentration of gastric lavage fluid):

positive control group (allergen Ovalbumin (OVA) solution): each mouse was gavaged orally with 0.2 mL OVA solution on day 0, 7, 14, 21 and 28 of the experiment, respectively; on day 42 of the experiment, each mouse was gavaged with 2 mL OVA solution orally; the concentration of OVA solution is 5 mg/mL, the solvent is PBS buffer solution with pH7.2 and 0.01 mM;

negative control group (non-allergen PBS buffer): on experiment days 0, 7, 14, 21 and 28, respectively, each mouse was gavaged with 0.2 mL PBS buffer per oral route; on day 42 of the experiment, each mouse was gavaged with 2 mL PBS buffer per mouth;

non-transgenic milk powder group 1: on the 0 th day, the 7 th day, the 14 th day, the 21 st day and the 28 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test object, and the dosage is 0.5 mg total protein/g BW); on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test substance, and the dosage is 5 mg total protein/g BW);

non-transgenic milk powder group 2: on the 0 th day, the 7 th day, the 14 th day, the 21 st day and the 28 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test object, and the dosage is 1mg total protein/g BW); on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test substance, and the dosage is 10mg total protein/g BW);

non-transgenic milk powder group 3: on the 0 th day, the 7 th day, the 14 th day, the 21 st day and the 28 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test object, and the dosage is 2mg total protein/g BW); on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of wild type non-transgenic milk powder solution (total protein content in wild type non-transgenic milk powder is used as a test substance, and the dosage is 20 mg total protein/g BW);

BLG biallelic knockout milk powder group 1: on the 0 th day, the 7 th day, the 14 th day, the 21 st day and the 28 th day of the experiment, each mouse is orally gavaged with 0.2 mL of BLG double allele knockout milk powder solution (the total protein content in the BLG double allele knockout milk powder is used as a test object, and the dosage is 0.5 mg of total protein/g BW); on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of BLG double allele knockout milk powder solution (total protein content in BLG double allele knockout milk powder is used as a test substance, and the dose is 5 mg total protein/g BW);

BLG double allele knock-out milk powder group 2: orally intragastrically perfusing 0.2 mL of BLG double allele knockout milk powder solution (total protein content in BLG double allele knockout milk powder is used as a test object, and the dosage is 1mg total protein/g BW) for each mouse on days 0, 7, 14, 21 and 28 respectively; on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of BLG double allele knockout milk powder solution (total protein content in BLG double allele knockout milk powder is used as a test substance, and the dose is 10mg total protein/g BW);

BLG biallelic knockout milk powder group 3: orally intragastrically perfusing 0.2 mL of BLG double allele knockout milk powder solution (total protein content in BLG double allele knockout milk powder is taken as a test object, and the dosage is 2mg total protein/g BW) for each mouse on days 0, 7, 14, 21 and 28 respectively; on the 42 th day of the experiment, each mouse was orally gavaged with 0.2 mL of BLG double allele knock-out milk powder solution (total protein content in BLG double allele knock-out milk powder was used as the test substance, and the dose was 20 mg total protein/g BW).

Allergen Ovalbumin (OVA), purchased from sigma.

During the experiment, the growth conditions of the mice (including the appearance, the feed intake amount, the mental state, and the like of the mice) were observed every week.

On day 42 of the experiment, the body temperature change of the mice before and after the stimulation was measured using an electronic thermometer with a probe coated with appropriate vaseline. The results are shown in FIG. 6. The results showed that mice in each protein-treated group, except negative control group non-allergenic PBS, had a significant decrease in anal temperature after stimulation (. about.p < 0.05). However, the temperature drop of the wild non-transgenic milk powder is higher, and the temperature drop of the BLG double allele knock-out milk powder after treatment is lower than that of the positive control milk powder and the common milk powder group, and the result proves that the sensitization of the BLG double allele knock-out milk powder is reduced compared with that of the wild non-transgenic milk powder.

Subsequently, the mice were tested by ELISA for the important immunoglobulin IgE at day 42 (purchased from Thermo, PA 1-84764).

The results are shown in FIG. 7. The result shows that the immunoglobulin level of the positive control group is obviously higher than that of the negative control group (P < 0.05), and the successful establishment of the sensitization animal model is proved. Meanwhile, 3 doses of the BLG double allele knock-out milk powder group all caused a significant increase in IgE level, but the IgE level was low compared with that of the wild type non-transgenic milk powder and the positive control group, and these results indicate that the allergenicity of the BLG double allele knock-out milk powder is lower than that of the wild type non-transgenic milk powder.

Sequence listing

<110> university of agriculture in China

<120> method for preparing accurate BLG gene knockout cattle by using third generation base editor

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>4894

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

tcctgcttgg cctcgaatgg aagaaggcct cctattgtcc tcgtagagga agcaacccca 60

gggcccaagg ataggccagg ggggattcgg ggaaccgcgt ggctgggggc ccggcccggg 120

ctggctggct ggccctcctc ctgtataagg ccccgagccc actgtctcag ccctccactc 180

cctgcagagc tcagaagcgt gaccccagct gcagccatga agtgcctcct gcttgccctg 240

gccctcactt gtggcgccca ggccctcatt gtcacccaga ccatgaaggg cctggatatc 300

cagaaggttc gagggtgccc gggtgggtgg tgagttgcag ggcaggcagg ggagctgggc 360

ctcagagacc aagggaggct gtgacgtctg ggattcccat cagtcagcta gagccgcctg 420

acaaatcgcc cgccagggca gcttcaacca ggcgtttagt gtcttgcatt ctggaggctg 480

gaagcctgca atccgggcat cggcccagct ggcttctcct gcggccactc tccggggagc 540

agacagccat cttctccctg tgtcctttgc gtgccctggt ttcctcttcc tgtgaggtca 600

ccaggcctgc tggatccacg cccgcccaca cagcctcacg taacctttgt catctcttta 660

aaggccgtgt ctccagtcct gtgttgaggt tctgggggtt aatgggacac agttcagccc 720

ctaaaagagt cactctgccc ctcaaatttt ccccacctcc agctatgtct ccccaagatc 780

caaatgttgc cacgtgtgcg ggggctcatc tgggtccctc tttgggctca gagtgagtct 840

ggggagagca ttcctcaggg tgccgagttg gggggagcat ctcagggctg cccaggccag 900

ggtgggacag agagcccact gtggggctgg gggccccttc ccgcccctgg agtgcagctc 960

aaggtccctc cccaggtggc ggggacttgg tactccttgg ccatggcggc cagcgacatc 1020

tccctgctgg acgcccagag tgcccccctg agagtgtatg tggaggagct gaagcccacc 1080

cctgagggcg acctggagat cctgctgcag aaatggtggg cgtccccccc aaaaaaagca 1140

tggaaccccc actccccagg gatatggacc cccccggggt ggggtgcagg agggaccagg 1200

gccccagggc tggggaacgg ggcttggagt ttcctggtac ccctggaggt ccacccaagg 1260

ctgcttatcc agggctttct ctttcttttt ttcccccaac ttttattaat ttgatgcttc 1320

agaacatcat caaacaaatg aacacaaaac atcattttcg ttaacttgga aggggagata 1380

aaatccactg aagtggaaat gcataggaaa gatacataca gtaaggcagg tattctgaat 1440

tcgctgttag tttgaggatt acaaatgcac ttgagcaaca gagagacgtt ttcattattt 1500

ctggtctgaa cagctcagta tctaaaatga acaagatgtc atggagacaa agccggcggg 1560

ggagaggccc gtgtgaaggc cgctgggcgg ctgcagacct gggtcctcgg ggcccaggca 1620

gttcccacta ccagccctgt ccaccctcag acgggggtca gagtgcagga gagagctggg 1680

tgggtgtggg ggcagagatg gggacctgaa ccccaggact gccttttggg gtgcctgtgg 1740

tcaaggctct ccccaacctt ttctccctgg ctccatctga cttctcctgg cccatccacc 1800

cggtcacctg tggccccaga ggtgacagtg agtgcagcca aggccggttg gccagccggc 1860

cccctatgcc cacgccaccc gcctccagcc cctcctgggg ccgccttctg cccctggccc 1920

tcagttcatc ctgatgaaaa tggtccatgc ccgtggctca gaaagcagct gtctttcagg 1980

gagaacggtg agtgtgctca gaagaagatc attgcagaaa aaaccaagat ccctgcggtg 2040

ttcaagatcg atggtgagtg ctgggtcccc aggggacgcc caccaccccc cagggactgt 2100

gggcaggtgc agggggctgg cgtcaggccc cgagatgcta aggggctggt ggtgatgaag 2160

acactgccgt gccacctgct tccctggcct ccctgccacc tgcccggggc cttggggccg 2220

gtggccgtgg gcaggtcccg gctgggcagg tctgacaccc cagggtgaca cccgagctct 2280

ctttgctgag ggtggggtgg tgctcggggc cctcaggctg agctcaggag gtccctgtgc 2340

ccacccaggg gtaaccgaga gccgctgccc gctccagggg tccaggtgcc ccacgatccc 2400

agcccacccc acggctcctt catctcctga agacgaactc tgtccgccct cgctcattca 2460

cttgtttgtc ctaaatccaa gatgagaaag cttcgaggtg gggttggggt tccatcaggg 2520

cctgcccttc cgccgggcag cctgggccac atctgccctt ggcctcttca ggactcactc 2580

tgactggagg ccctgcactg actgatgcca gggtgcccag cccagggtct cctgtgccat 2640

ccggctgcac ggggtttgga tgctggtcct gcccccaagc tgcccagaca ctgcagggca 2700

gctggggcca cccgcaggcc tcggtcaggg agagccccag ctgcccccgc tcagcgctgc 2760

cccccaacaa ttccccagtc ctcaggacgc atccctcttc ccttgctggg cagtgttcag 2820

ccccacccga gatcggggga agccctattt cttgaccact ccggtccctg ggggagggcg 2880

gcctcagact gagtggtgag tgttcccaag tccaggaggt ggtggagggt ccctggcgga 2940

tccagagttg ggcttccaga gtgagggctt cctgggcccc atgtgcctgg cagtggcagc 3000

agggaagggg ccacaccatt ttggggctgg gggatgccag agggcgctcc ccaccccgtc 3060

ctcaccaagt ggtgaccccg ggggagcccc gctggttgtg gggggcgctg ggggctgacc 3120

agaaaccccc ctcctgctgg aactcacttt cctcctgtct tgatctctac cagccttgaa 3180

cgagaacaaa gtccttgtgc tggacaccga ctacaaaaag tacctgctct tctgcatgga 3240

gaacagtgct gagcccgagc aaagcctggt ctgccagtgc ctgggtgggt gccaaccctg 3300

gctgcccagg gagaccagct gtgtggtcct cgctgcaacg gggccggggg ggacggtggg 3360

agcagggagc ttgattccca ggaggaggag ggatgggggg tccccgagtc ccgccaggag 3420

agggtggtca tataccggga gccggtgtcc tgggggtctg tgggtgactg gggacggggg 3480

ccagacacac aggctgggag acggggggct gcagcgctct ggtgtgacca tcacgatgga 3540

gccggcggtc actatgaatc taacagcctt tgttaccggg gagtttcaat tatttcatca 3600

aataagaact caggcacaaa gctgtctttc aactgtcacg tcctgaaaac aaatggcagg 3660

tgacattttc catgccatag cagtgccact gggcattttc agggcccatg tgccaggagg 3720

gcgtgggcat cggcgagtgg aggctcctgg ccgtgtcagc tggcccaggg ggaggagggg 3780

acccagacag ccagaggtgg ggagcaggct ttccccctgt gacgctgcag acccaccgca 3840

ctgccctggg aggaagggga gggaactggg ccaaggggga agggcaggtg tgctggaggc 3900

caaggcagac ctgcacacca ccctggagag caggggttga ccccgtcccg gccccacagt 3960

caggaccccg gaggtggacg acgaggccct ggagaaattc gacaaagccc tcaaggccct 4020

gcccatgcac atccggctgt ccttcaaccc aacccagctg gagggtgagc acccaggccc 4080

caccctgctc ctggggcagg aagccacccg gcccaggacc acctcctccc atggtgaccc 4140

ccagctcccc aggcctcccg ggaggatgga gacggggtgc agggccccga ggtggccccc 4200

tccccacccc ctccccagct ccctctgtcc tggggtgtcc agtcccatcc tgacgctccc 4260

ccgccacggc tctccctccc ccacagagca gtgccacatc taggtgagcc cctgccggcg 4320

cctctggggt aagctgcctg ccctgcccca cgtcctgggc acacacatgg ggtaggggtt 4380

cttggttggg cccgggagcc cccattaggc cctggggtcc ccccgtagga atggctggaa 4440

gctggggtcc ttcctggaga ctacagagcc ggctggccac atgctcgctc ttgtggggtg 4500

acctgtgtcc tggcctcact cacacgctga tctcctccac ctccttcctg gcagacctaa 4560

gggccaaggt ggaggctcag gaagtggaca cctaaggggg aggctagggg ggtccttctc 4620

ccaaggaggg gccgtcctga atccccagcc acggacaggc tggcaagggt ctggcaggta 4680

ccccaggaat cacaggggag ccccatgtcc atttcagagc ccgggagcct tggcccctct 4740

ggggacagac gatgtcatcc ccgcctgccc catcagggga ccaggaggaa ccgggaccac 4800

attcacccct cctgggaccc aggcccctcc aggcccctcc tggggcctcc tgcttggggc 4860

cgctcctcct tcagcaataa aggcataaac ctgt 4894

<210>2

<211>132

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

actccctgca gagctcagaa gcgtgacccc agctgcagcc atgaagtgcc tcctgcttgc 60

cctggccctc acttgtggcg cccaggccct cattgtcacc cagaccatga agggcctgga 120

tatccagaag gt 132

<210>3

<211>120

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ttaatacgac tcactatagg cacccagacc atgaagggcc gttttagagc tagaaatagc 60

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120

<210>4

<211>102

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gcacccagac caugaagggc cguuuuagag cuagaaauag caaguuuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

Claims (8)

1. The preparation method of the knockout cattle comprises the following steps (1) and (2):

(1) preparing a gene knockout cell: introducing a third generation single base editor BE3 and gRNA for identifying BLG gene into bovine fibroblasts, and preparing gene knockout cells through gene editing; all 277 bases of the BLG genes on two homologous chromosomes of the gene knockout cell from the 5' end are mutated from C to T;

the BLG gene is a DNA molecule shown as SEQ ID No. 1;

the target sequence of the gRNA is shown as 273 to 295 th positions from the 5' end of SEQ ID No. 1;

the gRNA is shown in SEQ ID No. 4;

(2) and (2) cloning the single cell of the gene knockout cell obtained in the step (1) by a somatic cell nuclear transfer technology to obtain the gene knockout cattle.

2. The method of claim 1, wherein: the third generation single base editor BE3 is mRNA obtained by in vitro transcription of a vector for expressing the third generation single base editor BE 3.

3. A method for knocking out a BLG gene in a cell, comprising the steps of: introducing a third generation single base editor BE3 and gRNA for identifying BLG gene into a target cell, and preparing a gene knockout cell through gene editing; the 277 th base of the BLG gene on two homologous chromosomes of the gene knockout cell from the 5' end is mutated from C to T; the target sequence of the gRNA is shown as the 273 to 295 th positions from the 5' end of SEQ ID No. 1; the BLG gene is a DNA molecule shown as SEQ ID No. 1; the gRNA is shown in SEQ ID No. 4.

4. Use of the method according to any one of claims 1 to 3 for breeding cows producing hypoallergenic milk.

5. The method for breeding the dairy cattle producing hypoallergenic milk comprises the following steps: the method of any one of claims 1 to 3, wherein the knockout dairy cow is a dairy cow producing hypoallergenic milk.

6. A kit for preparing a knockout cow comprises gRNAs shown as SEQ ID No.4 or an expression cassette containing gRNAs shown as SEQ ID No. 4.

7. The kit of claim 6, wherein: the kit also comprises a vector for expressing the third generation single base editor BE3 or mRNA obtained by in vitro transcription of the vector.

8. Use of the kit according to claim 6 or 7 for breeding cows producing hypoallergenic milk.

Images