SgRNA for ZFY gene editing and application of sgRNA in sex regulation

Abstract

The invention discloses sgRNA for ZFY gene editing and application of the sgRNA in sex regulation and control. The invention firstly discloses sgRNA for editing the ZFY gene, and the sequence of the sgRNA is SEQ ID NO.1 and / or SEQ ID NO.4. The invention also discloses a preparation method of the sgRNA for editing the ZFY gene. The invention further discloses an application of the sgRNA in sheep sex regulation and control. A conditional lethal gene HSV-TK is knocked in before a termination codon of a ninth exon of a ZFY gene of the sgRNA targeted milk goat fibroblast through a CRISPR / Cas9 gene targeting vector expressing the sgRNA and a homologous recombination targeting vector containing the conditional lethal gene HSV-TK by utilizing a CRISPR / Cas9 technology and homologous recombination; gene-edited milk goat fibroblasts are used as somatic cell nuclear transplantation donors, and male milk goats are prepared through somatic cell cloning, so that sex regulation and control of the milk goats are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering. More specifically, it relates to an sgRNA for ZFY gene editing and its application in sex regulation. Background Art

[0002] Livestock sex control refers to a biotechnology that uses artificial means to interfere with the normal reproductive process of livestock and finally obtains offspring with the expected sex, and its application in livestock production has significantly improved sex-related traits (such as meat, milk, wool, cashmere, leather, etc.) and their economic benefits within the animal population. There are mainly two traditional livestock sex control techniques: one is sperm separation technology, that is, separating X and Y sperm before fertilization, selecting sperm of the target sex for artificial insemination or embryo transfer after in vitro fertilization, and obtaining offspring of the target sex; the other is to use embryo sex identification technology to separate female and male embryos after fertilization, select embryos of the target sex for transplantation, and obtain offspring of the target sex. Although these two methods are the most widely used at present, they both have drawbacks. The former has high technical costs, a relatively low conception rate of inseminated female livestock, and problems such as unstable screening effects and damaged acrosome structures of sperm after screening in actual production applications; the latter will cause damage to embryos to a certain extent, affecting the conception rate and the quality of offspring.

[0003] The research on animal sex determination-related genes and their regulatory mechanisms has always been a research hotspot in the fields of animal science and animal genetics, breeding, and reproduction. Among them, the genes that have received much attention are mainly the SRY gene and the ZFY / ZFX gene. ZFY / ZFX, that is, sex chromosome-related zinc finger protein transcription factor (Y / X-linked Zinc-finger containing protein), is a gene located on the short arm of the sex chromosome and related to sperm formation and occurrence that can encode zinc finger protein. Its gene family also includes ZFY located on the Y chromosome, ZFX located on the X chromosome, and ZFA located on the autosome. Among them, ZFY and ZFX are a pair of allelic genes located on the Y and X chromosomes respectively. The ZFY gene is located on the short arm of the Y chromosome (Yp11.3) and plays an important role in the growth and development of male testes and sperm formation.

[0004] In 2011, Peng Qiang et al. first used RNAi to conduct a study on RNA interference (RNAi) in mice by constructing a recombinant expression vector of small interfering RNA (siRNA) for the ZFY gene, and found that the proportion of male mice in the offspring decreased (Peng Q, Li RY, Jia B, et al. Sex control by Zfy siRNA in the mouse. Theriogenology, 2011, 76(3):507-511.). When Ma Junde used a retroviral RNAi vector to control the sex of mice, significant results were obtained, and the rate of female mice in most litters reached over 70% in the sex identification of mouse offspring (Ma Junde. Construction of RNA virus vector of mouse Zfy gene and observation on sex control effect. Shihezi: Shihezi University, 2013:6-7.). In 2017, Xi Jifeng et al. used pLL3.7-A and pLL3.7-B to interfere with the ZFY gene of Hu sheep, and the female lamb rate of the offspring reached 64% (Xi Jifeng, Zhang Yongsheng, Wang Xiangzu, et al. Study on sex control of Hu sheep by interfering with Zfy gene. Animal Husbandry & Veterinary Medicine, 2017, 49(2):47-50.). In 2019, Yuan Ligang et al. continuously treated the testes of Holstein breeding bulls with a ZFY interference gene vector, collected their semen to make sex-controlled frozen semen, and through small-scale tests and large-scale pilot applications, the female calf rates reached 76.9% and 71.6% respectively, which were 29 percentage points and 23.7 percentage points higher than those of ordinary frozen semen (Yuan Ligang, Xi Jifeng, Liu Wei, et al. Effect of Zfy interference gene on sex control of Holstein cattle. China Dairy Cattle, 2019, (3):18-21.). In 2020, Wu Yun et al. used RNAi technology, taking exosomes as the vector of ZFY gene interference fragments, and conducted sex control on Hu sheep by testicular injection. The sex ratio of the offspring in the treatment group changed significantly, and the female lamb rate reached over 66% (Wu Yun, Jia Bin, Zou Shenglei, et al. Influence of exosome-mediated sex control on Hu sheep. Experimental Research, 2020, (8):4-5.). The above studies show that the ZFY gene is expected to become a key gene in the development of sex control technology. However, due to the limited ability of RNAi technology to degrade the mRNA of the target gene, the offspring produced still cannot achieve the goal of 100% sex regulation.

[0005] The conditionally lethal gene HSV-TK is expressed as herpes simplex virus thymidine kinase, which can phosphorylate the chemical drug ganciclovir (GCV, a 9-methyl deoxythymidine) into ganciclovir monophosphate, and under the action of endogenous kinases in cells, it is diphosphorylated into ganciclovir triphosphate (a deoxythymidine triphosphate); during DNA replication, this deoxythymidine triphosphate is misincorporated into the DNA strand by DNA polymerase. Due to the lack of deoxyribose, the synthesis of the DNA strand terminates, single-strand breaks occur, the action of DNA polymerase is inhibited, and apoptosis is triggered.

[0006] Therefore, it is necessary to develop new gene editing technologies to introduce the HSV-TK gene expression sequence into cells or animal individuals, and then add ganciclovir or its analogs on the basis of its expression, so as to induce apoptosis of tissues / cells at specific tissue or developmental time points to achieve sex regulation of dairy goats. Summary of the Invention

[0007] An object of the present invention is to provide an sgRNA for ZFY gene editing, which targets the ZFY gene and achieves a relatively high targeting efficiency for ZFY gene editing.

[0008] Another object of the present invention is to provide the application of the above-mentioned sgRNA for ZFY gene editing in sex regulation.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention first provides an sgRNA for ZFY gene editing, the sgRNA targets the ZFY gene, and its sequence is SEQ ID NO.1 and / or SEQ ID NO.4.

[0011] In a specific embodiment of the present invention, the sgRNA is used to edit the 9th exon of the ZFY gene, that is, the targeting region of the sgRNA is located in the 9th exon of the ZFY gene, and the sequence of the 9th exon of the ZFY gene is as shown in SEQ ID No.5; preferably, the targeting region of the sgRNA is located before the stop codon (TAA) of the 9th exon of the ZFY gene.

[0012] The present invention also provides a biological material, and the biological material can be any one of the following A1) to A4):

[0013] A1) An expression cassette containing the sgRNA;

[0014] A2) A recombinant vector containing the sgRNA, or a recombinant vector containing the expression cassette described in A1);

[0015] A3) A recombinant microorganism containing the sgRNA, or a recombinant microorganism containing the expression cassette of A1), or a recombinant microorganism containing the recombinant vector of A2);

[0016] A4) A recombinant host cell containing the sgRNA, or a recombinant host cell containing the expression cassette of A1), or a recombinant host cell containing the recombinant vector of A2).

[0017] Among the above biological materials, the recombinant vector is a CRISPR / Cas9 gene targeting vector for the sgRNA.

[0018] In a specific embodiment of the present invention, the method for preparing the CRISPR / Cas9 gene targeting vector includes:

[0019] Design and synthesize an oligonucleotide sequence for the sgRNA that can anneal to form a fragment with sticky ends;

[0020] Anneal the oligonucleotide sequence to form double-stranded DNA;

[0021] Ligate the double-stranded DNA into a plasmid to obtain the CRISPR / Cas9 gene targeting vector for the sgRNA.

[0022] In the present invention, the plasmid is the PX330 plasmid, and the CRISPR / Cas9 gene targeting vectors for the sgRNA are Cas9-ZFY-sgRNA1 and Cas9-ZFY-sgRNA4. Among them, the sequence of Cas9-ZFY-sgRNA1 is shown in SEQ ID NO.7, and the sequence of Cas9-ZFY-sgRNA4 is shown in SEQ ID NO.10.

[0023] The present invention also provides any one of the following applications of the sgRNA or the biological material:

[0024] B1) Application in specifically recognizing and / or targeting the ZFY gene;

[0025] B2) Application in site-specific integration of foreign genes into the ZFY gene;

[0026] B3) Application in preparing a cell line with site-specific integration of foreign genes into the ZFY gene;

[0027] B4) Application in regulating the sex of sheep or preparing products for regulating the sex of sheep;

[0028] B5) Application in preparing cloned sheep;

[0029] B6) Application in molecular breeding of sheep;

[0030] B7) Applications in molecular breeding of livestock.

[0031] Furthermore, the livestock are cattle, pigs, etc.

[0032] In a specific embodiment of the present invention, the site-directed integration of the exogenous gene is the site-directed knock-in of the exogenous gene, and the exogenous gene is the conditional lethal gene HSV-TK;

[0033] The nucleotide sequence of the conditional lethal gene HSV-TK is shown as positions 804-1920 of SEQ ID NO.11 (i.e., positions 2437-3553 of SEQ ID NO.12).

[0034] Furthermore, the exogenous gene includes the guiding sequence 2A located upstream of the conditional lethal gene HSV-TK, and its nucleotide sequence is shown as positions 709-789 of SEQ ID NO.11 (positions 2342-2422 of SEQ ID NO.12).

[0035] The present invention also provides a method for constructing a cell line with the conditional lethal gene HSV-TK knocked into the ZFY gene, and the method includes:

[0036] Using the CRISPR / Cas9 technology to knock the conditional lethal gene HSV-TK into the targeting site of the ZFY gene of the recipient cell through homologous recombination.

[0037] Furthermore, the method includes the following steps:

[0038] C1) Construct a CRISPR / Cas9 gene targeting vector expressing the sgRNA;

[0039] C2) Construct a homologous recombination targeting vector containing the conditional lethal gene HSV-TK and homologous arms for homologous recombination with the upstream and downstream regions of the targeting site of the ZFY gene targeted by the sgRNA;

[0040] C3) Co-transfect the CRISPR / Cas9 gene targeting vector and the homologous recombination targeting vector into the recipient cell;

[0041] C4) Knock the conditional lethal gene HSV-TK into the targeting site of the ZFY gene of the recipient cell.

[0042] In a specific embodiment of the present invention, the nucleotide sequence of the conditional lethal gene HSV-TK is shown as positions 2437-3553 of SEQ ID NO.12.

[0043] In a specific embodiment of the present invention, the homologous recombination targeting vector further includes a guiding sequence 2A upstream of the conditional lethal gene HSV-TK, and its nucleotide sequence is as shown in positions 2342-2422 of SEQ ID NO.12.

[0044] In a specific embodiment of the present invention, the homologous arms in the homologous recombination targeting vector include a homologous left arm and a homologous right arm. The nucleotide sequence of the homologous left arm is as shown in positions 703-2329 of SEQ ID NO.12, and the nucleotide sequence of the homologous right arm is as shown in positions 3558-5070 of SEQ ID NO.12.

[0045] In a specific embodiment of the present invention, the homologous recombination targeting vector is LS-2A-TK-RS, and its sequence is as shown in SEQ ID NO.12 (positions 703-2329 of SEQ ID NO.12 are the LS sequence of the homologous left arm, positions 2342-2422 of SEQ ID NO.12 are the 2A sequence, positions 2437-3553 of SEQ ID NO.12 are the HSV-TK sequence, and positions 3558-5070 of SEQ ID NO.12 are the RS sequence of the homologous right arm).

[0046] Furthermore, the vector for co-transfecting recipient cells further includes a resistance expression plasmid for subsequent screening. In a specific embodiment of the present invention, the resistance expression plasmid is a neomycin resistance gene expression vector, PGK-Neo plasmid, and its sequence is as shown in SEQ ID NO.13.

[0047] In a specific embodiment of the present invention, the recipient cell is an ovine fibroblast.

[0048] Furthermore, the transfection method includes: transfecting into ovine fibroblasts using the Lonza primary fibroblast transfection kit.

[0049] The cell line constructed by the above method is also within the protection scope of the present invention.

[0050] The present invention also provides the application of the cell line constructed by the above method in any of the following:

[0051] E1) Application in regulating the sex of sheep or preparing products for regulating the sex of sheep;

[0052] E2) Application in preparing cloned sheep;

[0053] E3) Application in sheep molecular breeding;

[0054] E4) Application in livestock molecular breeding.

[0055] Further, the livestock are cows, pigs, etc.

[0056] The present invention further provides a method for regulating the gender of sheep. The method includes the following steps: using the cell line constructed by the above method as a somatic cell nuclear transfer donor cell, and preparing male sheep through somatic cell cloning to achieve the purpose of regulating the gender of sheep.

[0057] In the present invention, the sgRNA for editing the 9th exon of the ZFY gene in dairy goats and the homologous recombination targeting vector are applied to the CRISPR / Cas9 technology to edit the ZFY gene in dairy goats. Without affecting the expression of the ZFY gene, the conditional lethal gene HSV-TK is knocked in. Since the position where HSV-TK is knocked in is before the stop codon TAA of the last exon of the ZFY gene, not only does it not affect the normal expression of the ZFY gene, but it can accurately express the HSV-TK gene under the guidance of the 2A sequence, and the tissues / cells in which it is expressed and the copy number of the expression are also consistent with the ZFY gene. Subsequently, the male sheep can be directly treated with ganciclovir and used for breeding; or artificial insemination breeding can be carried out with the semen of the male sheep or in vitro fertilization, and then the corresponding recipient ewes or in vitro fertilized embryos are treated with ganciclovir, so as to regulate that all of their offspring are high-yielding ewes.

[0058] In the present invention, the sheep are dairy goats.

[0059] The beneficial effects of the present invention are as follows:

[0060] The present invention uses the CRISPR / Cas9 technology and homologous recombination to knock in the conditional lethal gene HSV-TK before the stop codon TAA of the 9th exon of the ZFY gene in dairy goat fibroblasts by the CRISPR / Cas9 gene targeting vector expressing the sgRNA and the homologous recombination targeting vector containing the conditional lethal gene HSV-TK, and uses the gene-edited dairy goat fibroblasts as somatic cell nuclear transfer donor cells to prepare dairy goat male breeding sheep through somatic cell cloning, so as to realize the gene editing technology strategy for regulating the gender of dairy goats. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The following further describes in detail the specific embodiments of the present invention with reference to the drawings.

[0062] Figure 1 Schematic diagram of the gene editing strategy for knocking in the 2A-TK sequence at the 9th exon of the ZFY gene in dairy goats.

[0063] Figure 2Electrophoresis results of the ZFY gene and ZFX gene amplified by PCR from dairy goats; among them, M is 1Kb DNA Ladder; Y1 is the ZFY gene sequence of dairy goats amplified by primer ZFY-DNA-1, with a fragment length of 2269bp; Y2 is the ZFY gene sequence of dairy goats amplified by primer ZFY-DNA-2, with a fragment length of 2139bp; X1 is the ZFX gene sequence of dairy goats amplified by primer ZFX-DNA-1, with a fragment length of 1975bp; X2 is the ZFX gene sequence of dairy goats amplified by primer ZFX-DNA-2, with a fragment length of 2350bp.

[0064] Figure 3 Alignment results of the ZFY gene and ZFX gene sequences in the targeting region; among them, the stop codon TAA sequence is marked by an underline.

[0065] Figure 4 Specific sgRNA sequence information (including PAM) for the targeting region of the ZFY gene in dairy goats.

[0066] Figure 5 Detection results of the targeting efficiency of sgRNA for the ZFY gene in dairy goats.

[0067] Figure 6 Bright-field image of the dairy goat monoclonal cell line.

[0068] Figure 7 Electrophoresis results of the PCR products for identifying the dairy goat monoclonal cell line; among them, M is 1Kb DNA Ladder; Wt is the wild-type dairy goat cell, and 1-87 are cell lines numbered 1-87 respectively; the amplified fragment length of the wild-type cell is 4364bp, and the amplified fragment length of the cell line with accurate knock-in of the 2A-TK sequence is 5495bp.

[0069] Figure 8 Dairy goats with accurate knock-in of the 2A-TK sequence into exon 9 (before the stop codon) of the ZFY gene.

[0070] Figure 9 Electrophoresis results of the PCR identification of 5 cloned lambs; among them, M is 1Kb DNA Ladder; Wt is the wild-type dairy goat cell, C38 is cell line No. 38; 1#-5# are the PCR identification results of the genomic DNA of the ear tissues of 5 cloned lambs (1#-5#) respectively. Detailed implementation methods

[0071] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0072] The schematic diagram of the gene editing strategy for knocking in the 2A-TK sequence at the 9th exon of the ZFY gene in dairy goats by homologous recombination using the CRISPR / Cas9 technology is as Figure 1 shown, and specifically includes the following steps:

[0073] Perform gene sequencing analysis on the 9th exon of the dairy goat ZFY gene. Based on the comparison of the dairy goat ZFY gene and the ZFX gene, determine the specific editing site of the 9th exon of the ZFY gene; design guide RNA (sgRNA) for the ZFY gene editing site and construct a CRISPR / Cas9 gene targeting vector; by transfecting the CRISPR / Cas9 gene targeting vector into dairy goat fibroblasts, use methods such as gene sequencing to compare the gene editing efficiencies of different sgRNAs; on the basis of confirming the highly efficient sgRNA and the CRISPR / Cas9 gene targeting vector, design and construct a homologous recombination targeting vector for knocking in the conditional lethal gene HSV-TK and the guiding sequence 2A, where the 2A sequence is located upstream of the conditional lethal gene HSV-TK (i.e., the 2A-TK sequence), and homologous arms for homologous recombination with the upstream region and downstream region of the targeting site of the ZFY gene are contained on both sides of the 2A-TK sequence; co-transfect the highly efficient CRISPR / Cas9 gene targeting vector, the homologous recombination targeting vector containing 2A-TK, and the resistance expression plasmid PGK-Neo into dairy goat fibroblasts, and obtain a monoclonal cell line by means of drug screening; detect whether the 2A-TK sequence is accurately knocked in front of the stop codon TAA of the 9th exon of the ZFY gene in the monoclonal cell line by PCR amplification and gene sequencing; use the monoclonal cell line with the 2A-TK sequence accurately knocked in front of the stop codon of the 9th exon of the ZFY gene as the somatic cell nuclear transfer donor, and prepare male dairy goats with the 2A-TK sequence accurately knocked in front of the stop codon of the 9th exon of the ZFY gene by somatic cell cloning.

[0074] The present invention will be described in detail below in conjunction with the embodiments.

[0075] Example 1 Method for regulating the sex of dairy goats by the gene editing strategy of knocking in the 2A-TK sequence in front of the stop codon of the 9th exon of the sgRNA-targeted dairy goat ZFY gene by using the CRISPR / Cas9 technology

[0076] I. Obtain the dairy goat ZFY and ZFX genes

[0077] According to the sequence information of the goat ZFY gene (GenBank, NW_017189885) and ZFX gene (GenBank, NW_017189516), 4 pairs of PCR primers (ZFY-DNA-1, ZFY-DNA-2, ZFX-DNA-1 and ZFX-DNA-2, primer sequences and amplified fragment lengths are shown in Table 1) were designed to amplify the 9th exon of the ZFY gene (transcript ID: XM_018044893.1) of dairy goats and the sequence fragment of the homologous ZFX gene region, respectively. Dairy goat fibroblasts were cultured, and the genomic DNA of dairy goats was extracted using the genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. The 9th exon of the ZFY gene of dairy goats and the sequence fragment of the homologous ZFX gene were amplified by PCR reaction. Among them, the PCR reaction conditions were 95°C for 5 min; 95°C for 30 s, 56°C (ZFY) / 60°C (ZFX) for 30 s, 72°C for 3 min, for 35 cycles; 72°C for 7 min, 4°C ∞. The electrophoresis results of the PCR amplification products are as Figure 2 shown. Y1 is the ZFY gene sequence of dairy goats amplified by primer ZFY-DNA-1, with a fragment length of 2269 bp; Y2 is the ZFY gene sequence of dairy goats amplified by primer ZFY-DNA-2, with a fragment length of 2139 bp; X1 is the ZFX gene sequence of dairy goats amplified by primer ZFX-DNA-1, with a fragment length of 1975 bp; X2 is the ZFX gene sequence of dairy goats amplified by primer ZFX-DNA-2, with a fragment length of 2350 bp. The PCR amplification products were subjected to Sanger sequencing. The sequencing results showed that the 9th exon sequence of the ZFY gene amplified by PCR was as shown in SEQ ID NO.5, and the ZFX gene sequence homologous to the 9th exon of the ZFY gene was as shown in SEQ ID NO.6. It was used for the next step of sgRNA design.

[0078] Table 1 Primer sequences and amplified fragment lengths

[0079]

[0080] II. Design of sgRNA sequences

[0081] The consistency of the above ZFY and ZFX gene sequencing results with the GenBank data and the specific similarity between the ZFY gene and the ZFX gene were compared. The results are as Figure 3 shown. In the region where the stop codon (the TAA sequence marked with a red underline) at the 3' end of the 9th exon of the ZFY gene is located, the specific CRISPR / Cas9 targeting region (75731 - 75951, within a region of approximately 220 bp) that is different from the ZFX gene was determined. Figure 3), using the online CRISPR design tool (http: / / crispr.mit.edu / ), following the target design principle with the PAM sequence (NGG) at the 3' end, searching for target sites within the targeting region, and designing 2 guide RNAs (sgRNAs) upstream and downstream of the stop codon TAA at the 3' end of exon 9. The results are as Figure 4 shown, named ZFY-sgRNA1, ZFY-sgRNA2, ZFY-sgRNA3, and ZFY-sgRNA4 respectively. The sgRNA sequence information without the PAM sequence is as follows: the sequence of ZFY-sgRNA1 is 5'-AGAAGTATTGCTGTTAGGGC-3' (SEQ ID NO.1), the sequence of ZFY-sgRNA2 is 5'-CTGTAGAAGTATTGCTGTTA-3' (SEQ ID NO.2), the sequence of ZFY-sgRNA3 is 5'-ACATGAAATTTCTGCTTTCA-3' (SEQ ID NO.3), and the sequence of ZFY-sgRNA4 is 5'-GATTTATACTGTGTACAGAT-3' (SEQ ID NO.4).

[0082] III. Construction of the CRISPR / Cas9 gene targeting vector

[0083] The PX330 plasmid (Addgene #42230) was extracted using the Tiangen plasmid extraction kit. Then, the PX330 plasmid was digested with the restriction endonuclease BpiI, and the linearized PX330 plasmid was recovered using a gel extraction kit. Additionally, the 5'-phosphorylated oligonucleotide (Oligo DNA) sequences of the above 4 sgRNAs were synthesized in the forms of 5'-CACCGNNNNNNNNNNNNNNNNNNNN-3' and 3'-CNNNNNNNNNNNNNNNNNNNNCAAA-5', respectively. After annealing treatment (37°C for 30 min; 95°C for 5 min; cooling to 25°C at a rate of 5°C / min), annealing products, that is, double-stranded RNAs, were formed. The formed double-stranded DNAs were diluted at a dilution ratio of 250-fold and then ligated into the linearized PX330 plasmid respectively to obtain ligation products. Finally, the ligation products were transformed into DH5α competent cells respectively, and Sanger sequencing was performed using the sequencing primer 5'-ACTATCATATGCTTACCGTAAC-3'. The sequencing results were compared with the 4 sgRNA sequences respectively, and the CRISPR / Cas9 gene targeting vectors with correct insertion of sgRNAs were selected and named Cas9-ZFY-sgRNA1 (sequencing results as shown in SEQ ID NO.7), Cas9-ZFY-sgRNA2 (sequencing results as shown in SEQ ID NO.8), Cas9-ZFY-sgRNA3 (sequencing results as shown in SEQ ID NO.9), and Cas9-ZFY-sgRNA4 (sequencing results as shown in SEQ ID NO.10).

[0084] IV. Compare the targeting efficiency of the CRISPR / Cas9 gene targeting vector targeting the ZFY gene

[0085] Goat fibroblast cells were cultured in high-glucose DMEM medium containing 10% FBS. When the cell confluence reached over 80%, the cells were digested and collected using 0.1% trypsin. Then, using the Lonza primary fibroblast transfection kit, the 4 CRISPR / Cas9 gene targeting vectors (Cas9-ZFY-sgRNA1, Cas9-ZFY-sgRNA2, Cas9-ZFY-sgRNA3, and Cas9-ZFY-Sg

[0086] RNA4) were transfected into goat fibroblast cells respectively. The transfection conditions were: 1.5×10 6Cells / group, 5 μg vector plasmid / group, and the transfection procedure was T016. The 4 groups of transfected cells were cultured in 6-well plates for 48 h, and their genomic DNA was extracted using the Tiangen Genomic DNA Extraction Kit. Using the PCR primers ZFY-DNA-3 in the stop codon region of the ZFY gene (forward: 5'-gggATTTAggCAACAgAATgAgCT-3'; reverse: 5'-CTTTCgTgCTgTAATTTAgACCTg-3'; amplified fragment length: 722 bp), the stop codon region sequence of exon 9 of the ZFY gene in the 4 groups of transfected cells was amplified by PCR using the Q5 enzyme from NEB. PCR reaction conditions: 98°C for 30 s; 98°C for 5 s, 62°C for 10 s, 72°C for 20 s, 35 cycles, 72°C for 2 min. Then, the PCR products were sent for Sanger sequencing. Using the sequencing result of the PCR product of wild-type dairy goat fibroblasts as a control, the ZFY gene targeting efficiency of Cas9-ZFY-sgRNA1, Cas9-ZFY-sgRNA2, Cas9-ZFY-sgRNA3, and Cas9-ZFY-sgRNA4 was analyzed using an online analysis software (https: / / tider.deskgen.com). The results were as Figure 5 shown. Two CRISPR / Cas9 gene targeting vectors with higher targeting efficiency (Cas9-ZFY-sgRNA1 and Cas9-ZFY-sgRNA4) were selected as the CRISPR / Cas9 gene targeting vectors to be used for gene knock-in.

[0087] V. Construction of homologous recombination targeting vector

[0088] The conditional lethal gene HSV-TK sequence in the pLOX-gfp-iresTK vector (Addgene #12243) was amplified by PCR, and NotⅠ restriction site sequences GCGGCCGC were inserted upstream and downstream of it. Among them, the primers for amplifying the conditional lethal gene HSV-TK sequence were: forward 5'-AACgCggCCgCCTgCgTTCgACCAggCTgCgCgTT-3'; reverse 5'-gAggCggCCgCggTACCCCTTCCgTgTTTCAgTTA-3'. PCR reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 64℃ for 30 s, 72℃ for 1.5 min, 35 cycles, 72℃ for 5 min. The PCR amplification product (1139 bp) was cloned into the pMD-18T vector and sent for Sanger sequencing. Then, the correctly sequenced conditional lethal gene HSV-TK sequence containing the NotⅠ restriction site was inserted into the vector containing the 2A-TK sequence prepared using the basic vector containing the 2A sequence through digestion and ligation. The sequence is shown in SEQ ID NO.11 (positions 709-789 of SEQ ID NO.11 are the 2A sequence, and positions 804-1920 of SEQ ID NO.11 are the HSV-TK sequence). Homologous arm sequences (left homologous arm LS and right homologous arm RS) targeting the caprine ZFY gene were designed and PCR amplified around the upstream and downstream regions of the targeting sites of the highly efficient ZFY-sgRNA1 and ZFY-sgRNA4 in the caprine ZFY gene. Among them, the PCR primer sequences for amplifying LS were: forward 5'-AGCGAATTCCAGGCACAGTCACAGAGTAGCAGG-3'; reverse 5'-TTGGAATTCGGGCAGGCCAACTTCTTTATGATGT-3', and the amplified fragment length was 1639 bp (including the EcoRⅠ restriction site sequences GAATTC upstream and downstream); the PCR primer sequences for amplifying RS were: forward 5'-TGCCGCGGCAGCAATACTTCTACAGTCTTTGTA-3'; reverse 5'-TTTCCGCGGCGTAGAA GAAAGCACCGTATGA -3', and the amplified fragment length was 1525 bp (including the SacⅡ restriction site sequences CCGCGG upstream and downstream). PCR reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 1.5 min, 35 cycles, 72℃ for 5 min.The PCR amplification products LS and RS were respectively cloned into the pMD-18T vector and sent for Sanger sequencing. Then LS and RS were successively inserted into the upstream and downstream of the sequence containing the 2A-TK sequence to construct the homologous recombination targeting vector LS-2A-TK-RS. The sequence is shown in SEQ ID NO.12 (positions 703-2329 of SEQ ID NO.12 are the LS sequence, positions 2342-2422 of SEQ ID NO.12 are the 2A sequence, positions 2437-3553 of SEQ ID NO.12 are the HSV-TK sequence, and positions 3558-5070 of SEQ ID NO.12 are the RS sequence).

[0089] VI. Preparation of gene knock-in goat fibroblasts

[0090] Goat fibroblasts were cultured in high-glucose DMEM medium containing 10% FBS. When the cell confluence reached over 80%, the cells were digested with 0.1% trypsin and collected. Then, using the Lonza primary fibroblast transfection kit, the highly efficient CRISPR / Cas9 gene targeting vectors (Cas9-ZFY-sgRNA1 and Cas9-ZFY-sgRNA4), the homologous recombination targeting vector LS-2A-TK-RS, and the neomycin resistance expression vector PGK-Neo plasmid (the sequence is shown in SEQ ID NO.13) were co-transfected into goat fibroblasts. The transfection conditions were: 1.2×10 6 cells, 5 μg Cas9-ZFY-sgRNA1, 5 μg Cas9-ZFY-sgRNA4, 4 μg LS-2A-TK-RS, 0.5 μg PGK-Neo, and the transfection program was T016. The transfected cells were seeded in a 10-cm culture dish at a ratio of 1:50. After 48 h of culture, 1.6 mg / mL G418 was added to the medium for drug screening. The bright-field images of goat monoclonal cells on days 6-9 of drug screening are shown in Figure 6As shown. On the 7th to 9th day of drug screening, monoclonal cell lines were picked and passaged into 24-well plates, and a total of 143 monoclonal cell lines were obtained. The monoclonal cells were expanded, and cell cryopreservation and genomic DNA extraction were carried out respectively. PCR was used to identify whether the LS-2A-TK-RS vector was accurately knocked into the 9th exon of the ZFY gene (before the stop codon TAA) in 87 dairy goat monoclonal cell lines. The sequence of the PCR identification primer (WLR) was: forward 5'-TTGCGACTTTAGGATTGTATGGTTC-3'; reverse 5'-ATTGGGTGCGGACATACATAAAGC-3'. The amplified fragment length of wild-type cells was 3662bp, and the amplified fragment length of cells with accurately knocked-in 2A-TK sequence was 4864bp. PCR reaction conditions: 98°C for 30s; 98°C for 10s, 64°C for 10s, 72°C for 3min, 35 cycles, 72°C for 7min. The electrophoresis results of the PCR products are as Figure 7 shown. Among the 87 cryopreserved dairy goat monoclonal cell lines, the 2A-TK sequence was accurately knocked into the region before the stop codon of the 9th exon of the ZFY gene in 13 cell lines (No. 5, 6, 7, 14, 19, 20, 23, 25, 38, 41, 44, 56, and 82). The PCR products were recovered using a gel recovery kit and sent for Sanger sequencing. The sequencing results were completely consistent with the PCR results, and the gene knock-in ratio was 14.94%.

[0091] VII. Preparation of male dairy goats with gene knock-in

[0092] The 38th cell line with the 2A-TK sequence accurately knocked into the region before the stop codon TAA of the 9th exon of the ZFY gene, which had the most stable cell proliferation, was selected as the donor cell. 642 cloned embryos were prepared using somatic cell nuclear transfer technology and were transplanted into the oviducts of 35 estrus-synchronized ewes in batches. Among them, 9 ewes were successfully pregnant (pregnancy rate was 25.71%), and 4 ewes reached the end of pregnancy (the proportion of ewes reaching the end of pregnancy was 44.44%, and one of them was carrying twins). A total of 5 lambs were produced. The results are as Figure 8 shown. Ear tissues of the 5 cloned lambs were collected and genomic DNA was extracted. PCR identification (primers and reaction conditions were the same as in Step VI above) and gene sequencing methods were used to identify the genotypes of the cloned lambs. The results are as Figure 9 shown. It was found that the 2A-TK sequence was accurately knocked into the region before the stop codon of the 9th exon of the ZFY gene in all 5 cloned lambs; and it was confirmed by 40K liquid-phase gene chip technology that all 5 cloned lambs were derived from the 38th cell line, that is, 5 target sex-regulated male dairy goats were successfully obtained.

[0093] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or variations can be made. It is impossible to list all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A sgRNA for ZFY gene editing, characterized in that: The sgRNA targets the ZFY gene, and its sequence is SEQ ID NO.1 and / or SEQ ID NO.

4.

2. Biomaterial, characterized in that The biological material may be any one of the following A1) to A4): A1) an expression cassette containing the sgRNA for ZFY gene editing according to claim 1; A2) a recombinant vector containing the sgRNA for ZFY gene editing according to claim 1, or a recombinant vector containing the expression cassette according to A1); A3) a recombinant microorganism containing the sgRNA for ZFY gene editing according to claim 1, or a recombinant microorganism containing the expression cassette according to A1), or a recombinant microorganism containing the recombinant vector according to A2); A4) a recombinant host cell containing the sgRNA for ZFY gene editing according to claim 1, or a recombinant host cell containing the expression cassette according to A1), or a recombinant host cell containing the recombinant vector according to A2).

3. The biomaterial according to claim 2, characterized in that The recombinant vector is the CRISPR / Cas9 gene targeting vector of the sgRNA for ZFY gene editing as described in claim 1.

4. The biomaterial according to claim 3, characterized in that The preparation method of the CRISPR / Cas9 gene targeting vector comprises: Designing and synthesizing an oligonucleotide sequence that can anneal to form a fragment with a sticky end for the sgRNA for ZFY gene editing as described in claim 1; annealing the oligonucleotide sequence to form double-stranded DNA; The double-stranded DNA is connected to a plasmid to obtain the CRISPR / Cas9 gene targeting vector of the sgRNA.

5. The sgRNA for ZFY gene editing according to claim 1, or the biomaterial according to any one of claims 2 to 4, used in any of the following: B1) Application in specific identification and / or targeting of ZFY genes; B2) Application in site-specific integration of foreign genes in ZFY genes; B3) Application in preparing a cell line with site-specific integration of foreign genes in ZFY genes; B4) Use in regulating the sex of sheep or preparing products for regulating the sex of sheep; B5) Application in the preparation of cloned sheep; B6) Application in sheep molecular breeding; B7) Application in molecular breeding of livestock.

6. A method for constructing a cell line with a conditional lethal gene HSV-TK knocked into the ZFY gene, characterized in that: The method comprises: Using CRISPR / Cas9 technology to knock in the conditional lethal gene HSV-TK into the target site of the sgRNA targeting the ZFY gene of claim 1 in the recipient cell through homologous recombination; Preferably, the method comprises the following steps: C1) constructing the CRISPR / Cas9 gene targeting vector of the sgRNA for ZFY gene editing according to claim 1; C2) constructing a homologous recombination targeting vector containing the conditional lethal gene HSV-TK and homologous arms for homologous recombination with the upstream region and downstream region of the targeting site of the sgRNA targeting the ZFY gene according to claim 1; C3) co-transfecting the CRISPR / Cas9 gene targeting vector and the homologous recombination targeting vector into the recipient cells; C4) knocking the conditional lethal gene HSV-TK into the target site of the sgRNA targeting ZFY gene in the recipient cell.

7. The method according to claim 6, characterized in that The nucleotide sequence of the conditional lethal gene HSV-TK is shown in SEQ ID NO.12, positions 2437-3553; Preferably, the homologous recombination targeting vector further includes a guide sequence 2A located upstream of the conditional lethal gene HSV-TK, and its nucleotide sequence is shown in positions 2342-2422 of SEQ ID NO.12; Preferably, the homologous arms in the homologous recombination targeting vector include a homologous left arm and a homologous right arm, the nucleotide sequence of the homologous left arm is shown in positions 703-2329 of SEQ ID NO.12, and the nucleotide sequence of the homologous right arm is shown in positions 3558-5070 of SEQ ID NO.12; Preferably, the vector for co-transfection of the recipient cell further comprises a resistance expression plasmid, and the resistance expression plasmid is a neomycin resistance gene expression vector PGK-Neo plasmid; Preferably, the recipient cells are sheep fibroblasts.

8. The cell line constructed by the method according to claim 6 or 7.

9. The cell line according to claim 8 is used in any of the following: E1) Use in regulating the sex of sheep or preparing a product for regulating the sex of sheep; E2) Application in the preparation of cloned sheep; E3) Application in sheep molecular breeding; E4) Application in molecular breeding of livestock.

10. A method for regulating the sex of sheep, characterized in that: The method comprises the following steps: using the cell line according to claim 8 as a somatic cell nuclear transplantation donor cell to prepare a male sheep by somatic cell cloning.

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