CN116769774A - UTY gene-targeted sgRNA, sheep fibroblast line integrating exogenous gene by utilizing same and application of UTY gene-targeted sgRNA - Google Patents

Abstract

The application discloses sgRNA of a target UTY gene, a sheep fibroblast line integrating exogenous genes by using the sgRNA and application thereof. Specifically discloses sgRNA targeting UTY gene, and the target sequence is SEQ ID No.1. The application also discloses a method for constructing a cell line for site-directed integration of exogenous genes, which comprises the step of site-directed integration of exogenous genes into target spots of UTY genes of receptor cells by utilizing a CRISPR/Cas9 system through HMEJ method-mediated recombination. The application screens high-efficiency sgRNA on UTY gene intron in goat fibroblasts, integrates tdTomato genes at the sites by adopting HMEJ method-mediated recombination sites, and obtains sheep fibroblasts marked with Y chromosome, which can be combined with somatic cell nuclear transfer technology to obtain transgenic sheep, thereby being used for separating XY sperms and finally being used for sex control.

Description

UTY gene-targeted sgRNA, sheep fibroblast line integrating exogenous gene by utilizing same and application of UTY gene-targeted sgRNA

Technical Field

The application relates to the technical field of genetic engineering, in particular to a sgRNA of a target UTY gene, a sheep fibroblast line integrating exogenous genes by using the sgRNA and application of the sheep fibroblast line.

Background

CRISPR/Cas is an adaptive immune system generated by prokaryotes against viral infection or phage invasion that protects bacteria from viral repeat attacks mainly by adapting, expressing, interfering with 3 basic phases. Since being discovered, researchers continuously optimize and upgrade the CRISPR/Cas system, so that the CRISPR/Cas system becomes one of important gene editing tools in the field of molecular biology, and the gene editing is realized mainly through 3 steps of specific site recognition, target gene cutting and repair. Because the II type CRISPR/Cas9 gene editing system has relatively simple composition, simple and convenient operation and high editing efficiency, the method has wide research and application in gene editing. Cas9 protein is essentially a DNA endonuclease responsible for cleaving the gene of interest to form a double strand break, also known as genetic scissors. The Cas9 protein cleaves DNA 3bp upstream of the PAM sequence, creating a blunt-ended double-strand break, followed by repair of the target gene by a non-homologous end joining or homology-directed repair pathway, resulting in sequence insertion or deletion. The CRISPR/Cas9 technology breaks through the limitation of traditional breeding, and animal varieties which cannot be bred or are difficult to breed by the traditional breeding method can be bred in a short time, so that the progress of animal genetic improvement is accelerated.

Sex control technology is of great importance in animal production. First, by controlling the sex ratio of offspring, the maximum economic benefits of sex-limited production traits (e.g., lactation) and sex-affected production traits (e.g., growth rate, meat quality, etc.) can be fully exerted. Secondly, the sex proportion of the offspring can be controlled to increase seed selection intensity, so that the breeding process is quickened. Sex control may be achieved by sperm separation. The Y chromosome-linked tetrapeptide repetitive gene (ubiquitously transcribed tetratricopeptide repeat gene Y-linked, UTY) is a single copy gene on the Y chromosome of animals, encodes histone demethylase, and the encoded protein is also a minor histocompatibility antigen. Related to spermatogenesis, it has also been used to explore genetic diversity, population structure, and origin of livestock molecules. The research and development of the efficient site-directed integration transgenic technology aiming at the gene constructs a transgenic cell line of site-directed integration exogenous DNA, can realize the separation of XY sperms, finally realize sex control, greatly reduce the molecular breeding age of livestock and poultry, and has great significance for promoting the development of modern livestock and poultry breeding technology.

Disclosure of Invention

The technical problem to be solved by the application is to provide a sgRNA of a specific targeting UTY gene with high targeting efficiency and/or provide a cell line for site-directed integration of exogenous genes and a construction method thereof. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To achieve the above object, the present application provides, first of all, sgrnas targeting the UTY gene, the target sequence of which may be SEQ ID No.1.

The target sequence of the sgRNA is positioned on an intron of the UTY gene; the sgrnas can be based on a CRISPR/Cas9 system.

The nucleotide sequence of the UTY gene may be at positions 84124-84340 of GenBank Accession No. CM022046.1 (Update Date 27-JAN-2020).

The application also provides a DNA molecule encoding the sgRNA.

The present application also provides a biomaterial which may be any one of the following:

b1 An expression cassette containing said DNA molecule;

b2 A recombinant vector comprising said DNA molecule, or a recombinant vector comprising B1) said expression cassette;

b3 A recombinant microorganism comprising said sgRNA or said DNA molecule, or a recombinant microorganism comprising B1) said expression cassette, or a recombinant microorganism comprising B2) said recombinant vector;

b4 A recombinant host cell containing said sgRNA or said DNA molecule, or a recombinant host cell containing B1) said expression cassette, or a recombinant host cell containing B2) said recombinant vector.

In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc.

In the above biological material, the host cell (also referred to as a recipient cell) may be a plant cell or an animal cell. The host cell is understood to mean not only the particular recipient cell, but also the progeny of such a cell, and such progeny may not necessarily correspond, in their entirety, to the original parent cell, but are included in the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, and the animal cells may be mammalian cells. In one or more embodiments of the application, the mammalian cells are sheep fibroblasts or goat fibroblasts.

The application also provides the use of any of the sgrnas, or the DNA molecules, or the biomaterials:

a1 Use in specific recognition and/or targeting of the UTY gene;

a2 Application of the gene in site-directed integration of exogenous genes in UTY genes;

a3 Application in preparing a cell line for site-directed integration of exogenous genes in UTY genes;

a4 Use of gene editing techniques for marking sheep or goat Y chromosomes;

a5 Use in sheep or goat XY chromosome segregation;

a6 For controlling sheep or goat gender or for the preparation of a product for controlling sheep or goat gender;

a7 Use in the preparation of transgenic sheep or goats;

a8 Use in sheep or goat molecular breeding;

a9 Application of the UTY gene in preparing a cell model or an animal model for researching functions of the UTY gene.

The method for controlling the sex of sheep or goats comprises the following steps: and constructing a cell line for integrating a reporter gene (such as tdTomato gene) in a UTY gene of sheep or goat fibroblasts at fixed points by utilizing the sgRNA, obtaining transgenic sheep or goat by utilizing the cell line combined with a somatic cell nuclear transfer technology, and screening Y sperms according to the expression condition of the reporter gene, thereby separating the XY sperms and finally realizing sex control.

The application also provides a method for constructing a cell line for site-directed integration of exogenous genes, which can comprise the following steps: exogenous genes are integrated into target points of UTY genes of receptor cells in a fixed point manner through HMEJ method mediated recombination by using a CRISPR/Cas9 system, and the nucleotide sequence of the target points can be SEQ ID No.1.

Further, the method may comprise the steps of:

e1 Constructing a vector expressing the sgRNA;

e2 Constructing a donor plasmid containing an exogenous gene and homology arms for homologous recombination with both ends of the targeting site shown in SEQ ID No. 1;

e3 Co-transfecting the recipient cells with the vector of E1) and the donor plasmid of E2).

In the above method, the exogenous gene may be a reporter gene.

Further, the reporter gene may include, but is not limited to, a green fluorescent protein gene, a red fluorescent protein gene, a Chloramphenicol Acetyl Transferase (CAT) gene, a beta-galactosidase gene, a dihydrofolate reductase gene, a luciferase gene (e.g., bacterial luciferase, firefly luciferase, renilla luciferase gene), an alkaline phosphatase gene, and the like.

In the above method, the reporter gene may be tdTomato gene (a red fluorescent protein gene).

Further, the nucleotide sequence of the tdTomato gene can be the 2529-3959 (Update Date 06-OCT-2015) of GenBank accession No. KT878736.1.

In the above method, the recipient cell may be a sheep fibroblast or a goat fibroblast.

Further, in the above method, the CRISPR/Cas9 system comprises a vector expressing an sgRNA targeting the target of the UTY gene (SEQ ID No. 1).

Further, the E1) may include the steps of:

d1 Designed to anneal to form a single-stranded oligo with cohesive end fragments: UTY-sgRNA-F (SEQ ID No. 2) and UTY-sgRNA-R (SEQ ID No. 3);

d2 Annealing the single-stranded oligo to form an annealed product (double-stranded DNA);

d3 Ligating the annealed product (double stranded DNA) into a vector to obtain a vector expressing the sgRNA.

Further, the vector in D3) may be a PX458 vector.

Further, the vector expressing the sgRNA may be PX458-UTY-sgRNA, and the PX458-UTY-sgRNA is a recombinant vector obtained by ligating the annealed product (double-stranded DNA) of D2) into the PX458 vector.

Further, the homology arms in E2) may include a left homology arm and a right homology arm, the nucleotide sequence of the left homology arm may be as shown in SEQ ID No.5 or SEQ ID No.7, and the nucleotide sequence of the right homology arm may be as shown in SEQ ID No.6 or SEQ ID No.8.

Further, the method of transfection described in E3) may comprise: the vector of E1) and the donor plasmid of E2) (molar ratio may be 1:1.5) are transfected into sheep fibroblasts or goat fibroblasts by electrotransfection.

The application also provides a cell line constructed by the method described herein and/or any of the following uses of the cell line:

c1 For controlling sheep or goat gender or for the preparation of a product for controlling sheep or goat gender;

c2 Use in the preparation of transgenic sheep or goats;

c3 Use in sheep or goat molecular breeding;

c4 Application of the UTY gene in preparing a cell model or an animal model for researching functions of the UTY gene.

The cell line described herein may be a sheep fibroblast line or a goat fibroblast line.

The animal models described herein may include sheep or goat models.

The application screens high-efficiency sgRNA on the introns of UTY genes in goat fibroblasts by CRISPR/Cas9 technology. And the tdTomato gene started by the CBh strong promoter is integrated at the site by adopting the HMEJ method-mediated recombination site, so that sheep fibroblasts marked with Y chromosome are obtained. Sex control technology is of great importance in animal production, and the cells can be used in combination with somatic cell nuclear transfer technology to obtain transgenic sheep for the separation of XY sperm for sex control.

Experiments prove that compared with the prior art, the application has the following advantages:

site-directed integration technology can enable the integration position of exogenous genes to be safe, controllable and genetically stable, but traditional homologous recombination (Homologous recombination, HR) targeting integration efficiency is low, non-homologous end joining (Non-homologous end joining, NHEJ) can generate random integration, and various forms of insertion, mutation or deletion can occur at a connecting site. The application designs and screens the sgRNA of the specific targeting UTY gene with high targeting efficiency, and utilizes the sgRNA to knock in the UTY gene successfully by combining a Homology arm-mediated end connection (HMEJ) technology through a CRISPR/Cas9 technology, thereby having higher efficiency than the existing gene knock-in strategy.

Sex control may be achieved by sperm separation. The existing technology uses different physical characteristics (volume, density, charge and mobility) of X sperm and Y sperm, and adopts different methods such as centrifugation, electrophoresis, ion exchange, cell surface antigen, etc. to realize separation of sperm, but has a common problem-poor repeatability. And these differences vary with environmental conditions. According to the application, sex control is researched from gene level, a CRISPR/Cas9 technology is combined with an HMEJ technology for the first time, a reporter gene (tdTomato gene) is successfully knocked into a UTY gene of sheep fibroblasts, a sheep fibroblast line integrating the reporter gene (such as the tdTomato gene) at fixed points is constructed, transgenic sheep can be obtained by combining the cell line with a somatic cell nuclear transplantation technology, Y sperms are screened according to the expression condition of the reporter gene, so that the XY sperms are separated, sex control is finally realized, the breeding period is reduced, and the breeding efficiency is improved.

Drawings

FIG. 1 shows the editing effect of Sanger sequencing on the sgRNA target in example 1.

FIG. 2 is a verification of pCBh-tdTomato-SV40 polyA plasmid in goat fibroblasts.

FIG. 3 shows the amplification results of UTY goat-derived homology arms.

FIG. 4 shows the sequencing results of the donor plasmid Sanger in example 2.

FIG. 5 is a fluorescent image of sheep fibroblasts co-transfected with gene targeting plasmid and donor plasmid. In FIG. 5, there are respectively a bright field image (upper left image) of transfected UTY targeting plasmid and donor plasmid, a targeting plasmid (lower left image) of transfected UTY target, a donor plasmid (upper right image) of transfected expressed CBh-tdTomato (with homology arms at both ends of UTY target site added), and co-located images of transfected targeting plasmid and donor plasmid expression (lower right image).

FIG. 6 is a flow chart of co-transfection of a gene targeting plasmid and a donor plasmid into sheep fibroblasts.

FIG. 7 is a PCR identification of sheep fibroblasts site-specifically integrating the tdTomato gene.

FIG. 8 is a Sanger sequencing validation of tdTomato positive cells.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The PX458 vector in the examples below was purchased from the adedge plasmid shared information library.

Primary goat and sheep fibroblasts in the examples below were isolated for the laboratory itself. The preparation method comprises the following steps: a small amount of otic tissue of goats or sheep within 2 weeks of birth was taken and placed in PBS. In an ultra clean bench, the ear tissue was sterilized in 75% alcohol for 1min, washed 3 times with PBS, and minced to 1mm with sterile scissors ³ Size, 200. Mu.L of foetal calf serum was added, transferred to a cell culture dish, and incubated at 37℃with 5% CO ₂ The incubator was placed upside down for 1 hour. The complete medium was carefully added, taking care not to rinse up the tissue mass. After about 1 week of culture, fibroblasts were climbed out of the tissue mass. And after the growth is completed, pancreatin is digested, and the culture is expanded, and then frozen for later use.

The nucleotide sequence of the UTY gene of sheep in the following examples was GenBank Accession No. CM022046.1 at positions 84124-84340 (Update Date 27-JAN-2020).

The nucleotide sequence of the tdTomato gene in the following example is GenBank Accession No. KT878736.1 at positions 2529-3959 (Update Date 06-OCT-2015).

Example 1 construction of CRISPR/Cas9 Gene targeting vector and efficiency detection

1. CRISPR/Cas9 gene targeting vector construction

1.1, enzyme-cleaved PX458 vector

And (3) enzyme cutting system: PX458 vector 5. Mu.g, bbsI (N.Yinlen Biotechnology (Beijing) Co., ltd.) 50units,cutsmart 10. Mu.L, ddH ₂ O was made up to 100. Mu.L. And enzyme cutting at 37 ℃ for 5 hours. After the cleavage, the cleavage product was purified by a product purification kit (guangzhou mei biotechnology limited) to obtain PX458 BbsI cleavage product.

Target sequences were synthesized as shown in Table 1 by designing target (oligo) of sgRNA against introns of the UTY gene of sheep.

TABLE 1 oligo for expression of sgRNA target sequences

1.2 oligo annealing

The designed oligo was annealed according to the following annealing system and annealing procedure, and annealed to form an annealed product (double-stranded DNA).

Annealing system: UTY-sgRNA-F (100. Mu.M) 2.5. Mu.L, UTY-sgRNA-R (100. Mu.M) 2.5. Mu.L, T4 library buffer 1. Mu.L, ddH ₂ O was made up to 10. Mu.L. Annealing procedure: the metal bath was kept at 95℃for 5min, the metal bath was closed, the lid was opened, and the metal bath was taken out after the temperature was lowered to room temperature.

1.3 connection of

The annealed product was diluted 50-fold and ligated with the PX458 BbsI cleavage product of step 1.1 according to the ligation system and ligation procedure as follows.

The connection system is as follows: PX458 BbsI cleavage 90ng, annealing product (after dilution) 1. Mu.L, T4 ligase 0.5. Mu.L, T4 ligase buffer 1. Mu.L, ddH ₂ O was made up to 10. Mu.L. The reaction was carried out at 25℃for 1h.

10 mu L of the connecting product is used for transformation, and is subjected to bacterial picking sequencing and plasmid large extraction, so that a CRISPR/Cas9 gene targeting vector (i.e. sgRNA expression vector) is constructed and obtained, and the CRISPR/Cas9 gene targeting vector is named as PX458-UTY-sgRNA.

2. Verification of target efficiency

2.1 cell electrotransformation step

Primary goat fibroblasts in good condition were transferred to a 10cm dish and cultured until the cell confluency was about 80%. Cells were harvested by pancreatin digestion into EP tubes. 100. Mu.L of electrotransfer solution (Beijing Yinggan Biotechnology Co., ltd., cat. No. 98668-20) was used for suspension, and CRISPR/Cas9 gene targeting plasmid (PX 458-UTY-sgRNA) was added and mixed uniformly. Put into Lonza Amaxa Nucleofector B cell nuclear transfection instrument, adjust to procedure A-033, electrotransfection. After completion of the electric transfer, 500. Mu.L of DMEM high-sugar medium was added to the standing horse, and the cell culture incubator was allowed to stand at 37℃for 10 minutes. Cells were plated into 6-well plates with complete medium containing 20% FBS. After 6h, the medium was changed to complete medium containing 15% FBS.

2.2 obtaining the sequence around the target site

TABLE 2 primers for sequence acquisition around target spots

Cells after 48h of electrotransformation were subjected to genomic DNA extraction using a genomic extraction kit (Guangzhou Meiyi Biotechnology Co., ltd., product No. D3018-02). 100ng of the extracted goat genomic DNA was used as a template and PCR amplification was performed using the primers shown in Table 2. The PCR amplification reaction system is as follows: 100ng of DNA template, 1. Mu.L of each primer, 25. Mu.L of PrimeSTAR (Takara Shuzo Co., ltd.) were filled with sterile deionized water to 50. Mu.L. The PCR amplification reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 20s (33 cycles); finally, the extension is carried out for 5min at 72 ℃. After the PCR is completed, the PCR product is recovered by a product recovery kit and the concentration is determined.

2.3 target fragment-to-T vector sequencing

The PCR product recovered above was ligated with pMD19-T vector (Takara Shuzo Co., ltd.), transformed, and plated for sequencing. Sequencing results showed that the target of the designed sgRNA (5'-CTAACTGCCTCGGCACGGA G-3', SEQ ID No. 1) had an editing effect, 2 mutants were detected in 10 clones, mutant 1 was a 20bp deletion, mutant 2 had a 1bp (G) insert, and the editing efficiency was 20% (FIG. 1).

EXAMPLE 2 construction of tdTomato red fluorescent protein donor plasmid

The laboratory stores pCBh-tdTomato-SV40 polyA plasmid, the construction process of which: the pROSA 26-precursor (Addgene 21710) is digested by SpeI and XbaI to obtain digested pROSA 26-precursor, and the DNA molecule shown in SEQ ID No.4 (tdTomato-SV 40polyA sequence) is connected with digested pROSA 26-precursor by seamless cloning assembly technique to obtain pROSA26-tdTomato-SV40polyA. The CBh promoter was obtained by double-cleavage of PX458 with KpnI and AgeI, and all sequences except the ROSA26 promoter were amplified using the primers in Table 3 with pRO SA26-tdTomato-SV40polyA as a template, and constructed by a seamless cloning assembly technique to obtain pCBh-tdTomato-SV40 polyA.

TABLE 3 construction of primer sequences for pCBh-tdTomato-SV40 polyA vector

The plasmid liposomes were transfected into primary goat fibroblasts. Primary goat fibroblasts were plated into 6-well plates and cultured to a confluency of about 80%. Two 500. Mu.L tubes were taken, one tube was added with 25. Mu.L of Opti-MEM medium and 1.5. Mu.L of Lipofectamine 3000 (Semer Feishmania technology Co.) and mixed well, the other tube was added with 25. Mu.L of Lopti-MEM medium, 500ng of plasmid pCBh-tdTomato-SV40 polyA and 1. Mu. L P3000, and left at room temperature for 15min after mixing well. Drop wise into goat fibroblast medium. Fluorescent expression was observed for 24 h. After 24h, cells were found to appear bright red under irradiation with excitation light by fluorescence microscopy, and the results demonstrated that the constructed pCBh-tdTomato-SV40 polyA was able to be expressed normally in goat fibroblasts (FIG. 2).

The plasmid can normally express red fluorescence in primary goat fibroblasts. The Sanger sequencing proves that UTY design targets are effective targets, so that sequences on two sides of a position (3-4 bp upstream of PAM) cut by the UTY targets are used as homology arms (102 bp of a left homology arm (HA-L) of a goat, SEQ ID No.5 of a nucleotide sequence, 106bp of a right homology arm (HA-R) of the goat, and SEQ ID No.6 of a nucleotide sequence). The left and right homology arms were amplified by PCR using the primers shown in Table 4, and then recovered by a PCR product recovery kit (Meiyi Biotechnology Co., ltd.) (FIG. 3).

Table 4, UTY left and right homology arm primers

The plasmid pCBh-tdTomato-SV40 polyA is digested, and left and right homologous arms of UTY targets are correspondingly cloned to two ends of the pCBh-tdTomato-SV40 polyA by a seamless cloning assembly technology to construct the plasmid HA-L-CBh-tdTomato-SV40 polyA-HA-R. Next, recognition sequences of the UTY target were added on the outer sides of the left and right Homology arms, and the type of donor plasmid required for HMEJ (Homology-arm mediated end ligation, homo-mediated end joining) was constructed, thereby improving the efficiency of gene knock-in, and verified by Sanger sequencing. The plasmids were sequenced and verified and Sanger sequencing results demonstrated that the donor plasmid required for the successful construction of the HMEJ cell repair type (FIG. 4). The left and right homology arms on the constructed donor plasmid are derived from goats, have extremely high homology with the corresponding sites of sheep, and the homology of the left homology arm is 100% and the homology of the right homology arm is 96% through NCBI BLAST. Wherein the nucleotide sequence of the sheep left homology arm is SEQ ID No.7, and the nucleotide sequence of the sheep right homology arm is SEQ ID No.8.

EXAMPLE 3 screening of Stable cell lines of tdTomato inserted into UTY site at fixed position

In this example, tdTomato gene is taken as an example, and the CRISPR/Cas9 gene targeting vector PX458-UTY-sgRNA constructed in example 1 and the donor plasmid HA-L-CBh-tdTomato-SV40polyA-HA-R (carrying the exogenous gene tdTomato gene, although the homology arm is derived from goat, the homology arm corresponding to sheep HAs high homology, and thus is expected to be used in sheep) constructed in the donor plasmid HA-L-CBh-tdTomato-SV40, the exogenous gene (tdTomato gene) is integrated into the targeting site (SEQ ID No. 1) of the UTY gene by the HMEJ method-mediated recombination site-specific, and a sheep fibroblast line for site-specific integration of the exogenous gene in the UTY gene is constructed. The method comprises the following specific steps:

1. gene editing plasmid and donor plasmid co-transfected sheep fibroblasts

The constructed donor plasmid HA-L-CBh-tdTomato-SV40 polyA-HA-R5000 ng and UTY target-carrying gene targeting vector PX458 (PX 458-UTY-sgRNA) 9536ng (molar ratio 1:1.5) were electrotransformed (electrotransformation procedure same as 2.1 in example 1) into primary sheep fibroblasts, after 24h tdTomato and EGFP positive primary sheep fibroblasts were flow-sorted and plated in 10cm cell culture dishes at about 500 cells per dish. After 2 weeks of culture, the cells in the cell culture dish were monoclonal digested by cloning loop into 96-well plates for culture (fig. 5 and 6).

2. Identification of sheep fibroblasts site-directed integration tdTomato

After the cell clone of the 96-well plate is full, the cells are digested, half of the original wells are left for culture, and the other half of the cells are taken into a 1.5mL centrifuge tube. 12000rpm, centrifuging for 3min, discarding the supernatant, adding 50. Mu.L of cell identification lysate (cell identification lysate preparation: tris-HCl (1M, pH=8.0) 2mL, triton X-100.45 mL, NP-40.45 mL, proteinase K0.02 g, adding deionized water to dissolve and volume to 50mL,0.22 μm filter), and fully suspending the cells, lysing according to the following procedure: 65 ℃ for 30min;95 ℃ for 15min;16 ℃ and infinity. The obtained lysate was used as a DNA template.

Primers were designed and PCR identified as shown in Table 5.

TABLE 5 site-directed integration identification primers

Amplification reaction system and amplification procedure: the total volume of the amplification reaction was 50. Mu.L, and the respective components were: 1. Mu.L of DNA template, 1. Mu.L of each primer, 10. Mu.L of PrimeSTAR (Takara Shuzo Co., ltd.) were filled with sterilized deionized water to 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 62℃for 15s, extension at 72℃for 50s (33 cycles); finally, the extension is carried out for 5min at 72 ℃. And after the PCR is finished, detecting a result by agarose gel electrophoresis.

The results showed that 14 cell monoclonals were lysed and 3 clones were identified by PCR as UTY site-directed integrated cell monoclonals (fig. 7, clones 2, 3, 13, respectively). Clone 2 and clone 3 were subsequently subjected to Sanger sequencing, and the sequencing results likewise showed site-directed integration (FIG. 8). At the same time, clones 2, 3 and 13 all fluoresce red when viewed by fluorescence microscopy. The above shows that the exogenous gene (tdTomato gene) is subjected to site-directed integration at the targeting site (SEQ ID No. 1), and a sheep fibroblast line with site-directed integration of the exogenous gene in the UTY gene was successfully constructed.

In conclusion, the result shows that the application successfully screens the sgRNA of the UTY gene, and the tdTomato gene started by the CBh promoter is integrated by utilizing the sgRNA at the UTY gene locus fixed point on the Y chromosome of sheep. And the integration position is positioned in the intron, so that the expression and the function exertion of the UTY gene are not influenced. The method successfully marks the Y chromosome, plays an important role in related researches such as XY chromosome separation, sex control, evaluation and editing efficiency and the like, and is beneficial to promoting the development of modern livestock and poultry breeding technology.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. The sgRNA targeting UTY gene is characterized in that the target sequence of the sgRNA is SEQ ID No.1.

2. A DNA molecule encoding the sgRNA of claim 1.

3. A biomaterial characterized in that the biomaterial is any one of the following:

b1 An expression cassette comprising the DNA molecule of claim 2;

b2 A recombinant vector comprising the DNA molecule of claim 2, or a recombinant vector comprising the expression cassette of B1);

b3 A recombinant microorganism comprising the sgRNA of claim 1 or the DNA molecule of claim 2, or a recombinant microorganism comprising the expression cassette of B1), or a recombinant microorganism comprising the recombinant vector of B2);

b4 A recombinant host cell comprising the sgRNA of claim 1 or the DNA molecule of claim 2, or a recombinant host cell comprising the expression cassette of B1), or a recombinant host cell comprising the recombinant vector of B2).

4. The sgRNA of claim 1, or the DNA molecule of claim 2, or any of the following uses of the biomaterial of claim 3:

a1 Use in specific recognition and/or targeting of the UTY gene;

a2 Application of the gene in site-directed integration of exogenous genes in UTY genes;

a3 Application in preparing a cell line for site-directed integration of exogenous genes in UTY genes;

a4 Use of gene editing techniques for marking sheep or goat Y chromosomes;

a5 Use in sheep or goat XY chromosome segregation;

a6 For controlling sheep or goat gender or for the preparation of a product for controlling sheep or goat gender;

a7 Use in the preparation of transgenic sheep or goats;

a8 Use in sheep or goat molecular breeding;

a9 Application of the UTY gene in preparing a cell model or an animal model for researching functions of the UTY gene.

5. A method of constructing a cell line that integrates a foreign gene at a site, the method comprising: exogenous genes are integrated into target points of UTY genes of receptor cells in a fixed point manner through HMEJ method mediated recombination by using a CRISPR/Cas9 system, and the nucleotide sequence of the target points is SEQ ID No.1.

6. The method according to claim 5, characterized in that it comprises the steps of:

e1 Constructing a vector expressing the sgRNA of claim 1;

e2 Constructing a donor plasmid containing an exogenous gene and homology arms for homologous recombination with both ends of the targeting site shown in SEQ ID No. 1;

e3 Co-transfecting the recipient cells with the vector of E1) and the donor plasmid of E2).

7. The method of claim 5 or 6, wherein the exogenous gene is a reporter gene.

8. The method of claim 7, wherein the reporter gene is tdTomato gene.

9. The method of any one of claims 5-8, wherein the recipient cell is a sheep fibroblast or a goat fibroblast.

10. A cell line constructed by the method of any one of claims 5-9, and/or any one of the following uses of the cell line:

c1 For controlling sheep or goat gender or for the preparation of a product for controlling sheep or goat gender;

c2 Use in the preparation of transgenic sheep or goats;

c3 Use in sheep or goat molecular breeding;

c4 Application of the UTY gene in preparing a cell model or an animal model for researching functions of the UTY gene.