SgRNA specifically targeting safe site rosa26 of capra hircus and application thereof

Abstract

The application provides sgRNA which specifically targets the Rosa26 safe site of a cashmere goat and application of using CRISPR / Cas9 to complete site-directed knockout and site-directed integration of an EGFP gene. The application firstly uses a bioinformatics method to predict the complete sequence of the Rosa26 site, then designs two sgRNA aiming at the site, constructs a targeting vector based on the CRISPR / Cas9 system, verifies the guiding efficiency of the sgRNA, simultaneously constructs a homologous integration EGFP gene vector, co-transfects the sgRNA and the homologous integration vector into cashmere goat fetal fibroblasts, and obtains a cell strain of the Rosa26 site site-directed integration of the EGFP gene. The efficiency of the sgRNA of the application in specifically guiding Cas9 to cut the Rosa26 site reaches about 40%, effectively reduces the off-target phenomenon existing in the CRISPR / Cas9 system, and further reduces the mutation of non-target gene sequences caused by non-specific cutting. The Cas9 / gRNA expression vector can realize the specific knockout or knock-in of the Rosa26 site at the cell, embryo or even individual level, so as to study the expression of specific genes and provide technical support for the cultivation of new goat breeds.

Description

Technical Field

[0001] This invention relates to "sgRNA specifically targeting the safe site Rosa26 in cashmere goats and its application," primarily involving the fields of bioinformatics and genetic engineering. It includes the prediction of the Rosa26 site, sgRNA design, construction of CRISPR / Cas9 vectors, cell transfection, efficiency of TA cloning and sequencing detection of sgRNA, construction of homologous integration vectors, and screening of positive cells.

[0002] In the late 1990s, Philipe Soriano and his colleagues accidentally discovered that a mouse strain with a randomly integrated LacZ gene efficiently expressed β-galactosidase in various tissues without affecting its own growth. The researchers then located this insertion site on mouse chromosome 6, naming it the Rosa26 site. In the following years, through a series of efforts, researchers successively identified the Rosa26 site in humans, cattle, pigs, and sheep. Unlike the H11 site, the Rosa26 site contains a promoter, allowing for systemic RNA expression but not protein production. Furthermore, the insertion of a foreign gene at this site does not affect the animal's healthy growth, making it a potential "safe site."

[0003] Gene editing technology is a technique for sequence-specific modification of DNA within cells, primarily involving the insertion, deletion, and replacement of DNA fragments. Precise genome editing based on programmable nucleases has opened up the possibility of achieving desired genome editing results both in vitro and in vivo, ensuring its safe and rapid application in cross-biological genome engineering. Gene editing technologies mainly include zinc finger nucleases (ZFNs), TALENs, and CRISPR / Cas9. Research has found that gene editing technology has great potential in improving animal production performance, disease resistance, and in treating human diseases and constructing disease models. ZFNs and TALENs require a series of cloning and subcloning steps for assembly, while CRISPR / Cas9 can be easily assembled in a single cloning step. This difference makes the assembly of ZFNs and TALENs more time-consuming; therefore, CRISPR / Cas9 technology, as a means of gene editing, makes the CRISPR / Cas system a powerful genome editing tool. Its structure includes the Cas9 gene, three functional genes, a leader sequence, and a spacer repeat sequence.

[0004] If the Rosa26 site in the cashmere goat genome can be identified as a "safe site" for inserting foreign genes in gene editing, and if sgRNA sequences that can specifically target the Rosa26 site and efficiently guide Cas9 nuclease to target and edit this site can be screened, it will provide strong technical support for the preparation of gene-edited cashmere goats that integrate foreign genes at the Rosa26 site, promote the research and application of gene editing in cashmere goats, and promote the healthy development of the cashmere goat industry. Summary of the Invention

[0005] The purpose of this invention is to provide a specific sgRNA targeting the safe site Rosa26 in cashmere goats with high targeting efficiency and low off-target rate, and its application.

[0006] The present invention first determined that the safe site Rosa26 is located on chromosome 22 of goats and retrieved its nucleotide sequence from NCBI, as shown in SEQ ID NO.1.

[0007] This invention provides an sgRNA that specifically targets the Rosa26 site in cashmere goats, the RNA sequence of which is shown in SEQ ID NO. 2 or SEQ ID NO. 3. Specifically, the DNA sequence of the sgRNA transcribed is shown in SEQ ID NO. 4 (transcribed SEQ ID NO. 2) or SEQ ID NO. 5 (transcribed SEQ ID NO. 3).

[0008] This invention provides a CRISPR / Cas9 targeting vector containing the fluorescent selection marker EGFP and the drug selection marker Puro, which can enrich more positive cells in a short time.

[0009] Extensive preliminary experiments revealed that electrotransfer at 250V-2.5ms resulted in the highest transfection efficiency. Therefore, this transfection condition was used in the experiments.

[0010] In this invention, to avoid false positive results that may occur when using T7E1 and Surveyor enzyme digestion, a direct TA cloning and sequencing method was used to detect the targeting efficiency.

[0011] This invention provides the application of the sgRNA or its CRISPR / Cas9 targeting vector and homologous integration vector in the specific recognition and targeted modification of the safe site Rosa26 in cashmere goats, wherein the targeted modification includes gene knockout, site-directed integration of exogenous genes or site-directed overexpression of endogenous genes.

[0012] This invention provides the application of the sgRNA or its CRISPR / Cas9 targeting vector and homologous integration vector in the preparation of transgenic animals or the improvement of livestock production performance and disease resistance.

[0013] This invention provides the application of the sgRNA or its CRISPR / Cas9 targeting vector and homologous integration vector in the breeding of new gene-edited cashmere goats.

[0014] This invention application identifies an sgRNA sequence that specifically targets the Rosa26 site and efficiently guides the Cas9 nuclease to cleave it at that site, achieving a targeting efficiency of over 40%. This effectively reduces off-target effects caused by low sgRNA specificity, improving gene editing safety while minimizing gene mutations at non-target sites due to non-specific cleavage. This provides a new and safe option for the insertion of exogenous genes. While CRISPR / Cas9 gene editing technology can efficiently produce gene-edited cashmere goats, the insertion of exogenous genes can directly affect the structure or function of endogenous genes. Finding a site in the genome that can efficiently express exogenous genes without affecting the normal expression of endogenous genes would be the perfect way to overcome the risks of gene editing; such a site is called a "safe site." The sgRNA specifically targeting the cashmere goat safe site Rosa26 provided in this application, combined with the CRISPR / Cas9 system, can be used for both site-specific gene knockout and site-specific overexpression of exogenous or endogenous genes. This provides a feasible method and approach for studying the function of important genes in livestock, promoting research and development in the livestock industry. Attached Figure Description

[0015] Figure 1 Locations of the Rosa26 gene in various species in the Ensembl database.

[0016] Figure 2 shows the alignment results of the goat genome with the mouse Rosa26 promoter and exon 1. A and B represent the alignment results of Ensembl and NCBI, respectively.

[0017] Figure 3 The predicted complete sequence of the first exon of the goat Rosa26.

[0018] Figure 4 Comparison of the Rosa26 gene sequence of sheep with that of goats.

[0019] Figure 5 Comparison of the homology of Rosa26 first exon among different species.

[0020] Figure 6 The relative expression levels of the Rosa26 gene in various tissues and organs.

[0021] Figure 7 Sequencing results of the Cas9 / gRNA co-expression vector.

[0022] Figure 8 Schematic diagram of Cas9 / gRNA co-expression vector.

[0023] Figure 9 Image showing the effect of cell electroporation.

[0024] Figure 10 Semi-quantitative analysis results of fluorescence intensity (ImageJ).

[0025] Figure 11 Image showing the enrichment effect of positive cells.

[0026] Figure 12 Figure showing the Rosa26-sgRNA1 / 2 mutation efficiency results.

[0027] Figure 13 shows the sequencing alignment results of PCR products from 5 potential off-target sites with wild-type PCR products. A, B, C, D, and E represent the alignment results of Chr2, Chr16, Chr26, Chr28, and Chr15 sites, respectively.

[0028] Figure 14 Electrophoresis image of the homologous arm of the Rosa26 site homologous integration vector.

[0029] Figure 15 Schematic diagram of homologous integration carrier.

[0030] Figure 16 Diagram of target practice.

[0031] Figure 17 Electrophoresis diagram for identifying positive monoclonal cells.

[0032] Figure 18 Image of positive monoclonal cells.

[0033] Figure 19 The composition and working principle of the CRISPR / Cas9 system. Detailed Implementation

[0034] The screening process of this invention will be described in detail below with reference to experiments. The following experiments are for illustrative purposes only and are not intended to limit the scope of this invention. Those skilled in the art can make various modifications and substitutions to this invention without departing from its spirit and purpose.

[0035] Unless otherwise specified, all experimental methods used in this experiment are standard methods. All materials and reagents used are commercially available unless otherwise specified.

[0036] Example 1: Identification of the Rosa26 locus in cashmere goats

[0037] Name: Rosa26 locus; Location: Chr 22 (17030817 to 17033107). Based on the partial gene sequences of Rosa26 in mice and sheep, we found that the Rosa26 gene is located between the SETD5 and THUMPD3 genes, as shown below. Figure 1 As shown. Using the mouse Rosa26 promoter (1kb) and exon 1 sequence (NC_000072.6) as templates, a search of the Ensembl goat database revealed a homology of 109 bases (92.66%) on goat chromosome 22, of which 99 bases are part of exon 1 of the goat Rosa26 gene, as shown. Figure 2A As shown. After expanding the alignment range using NCBI, it was found that the mouse Rosa26 promoter and exon 1 sequences share 90% homology with 590 bases from goat, including 130 bases from goat Rosa26 exon 1. Figure 2B As shown. Based on the above comparative analysis, we predicted the complete sequence of the first exon of the goat Rosa26 gene, as follows. Figure 3 As shown. Using the known sheep Rosa26 gene sequence as a template, a search of the Ensembl goat database revealed 98.78% homology with 2295 bases (Chr22: 17030817 to 17033107) on goat chromosome 22. Figure 4 As shown in the figure. Combined with the previously determined exon 1 sequence, the complete sequence of goat Rosa26 was finally determined and retrieved from Genebank. Its nucleotide sequence is shown in SEQ ID NO.1. Alignment of the first exon of the goat Rosa26 gene with known first exons of the mouse and sheep Rosa26 genes showed that the homology between the first exons of the mouse and sheep Rosa26 genes and the first exon of the goat Rosa26 gene was 60.11% and 76.74%, respectively. This indicates that the Rosa26 gene is highly conserved across species, further proving the accuracy of our predicted goat Rosa26 sequence. Figure 5 As shown.

[0038] Example 2: Expression levels of the Rosa26 gene in various tissues and organs of adult cashmere goats

[0039] Studies have found that the Rosa26 gene can express non-coding RNA in various tissues and organs in other species. Therefore, we hypothesized that it could also be widely expressed in cashmere goats. To confirm this hypothesis, we used real-time PCR to detect it. First, total RNA was extracted from the heart, liver, spleen, lungs, kidneys, muscles, brain, stomach, and skin of wild-type adult cashmere goats. The RNA quality and concentration met the requirements. After synthesizing cDNA, real-time PCR experiments were performed. The results showed that the Rosa26 gene was expressed in all the tissues and organs we examined. After setting the expression level in the heart to 1, we found that the expression levels in the spleen and lungs were significantly higher than in the heart (P<0.01), with the highest expression level in the spleen. The expression levels in muscles and brain were significantly lower than in the heart (P<0.01), with the lowest expression level in muscles. The expression level in skin was significantly lower than in the heart (P<0.05). Figure 6 As shown, non-coding RNA can still be expressed systemically at the Rosa26 site in cashmere goats.

[0040] Example 2 Construction of sgRNA expression vector

[0041] Based on the design principles of sgRNA, two sgRNA target sites and their corresponding PAM sequences (NGG) were screened at the Rosa26 site by combining three design websites [HTTP: / / CRISPOR.TEFOR.NET / CRISPOR.PY; CRISPRDIRECT(DBCLS.JP); HTTPS: / / CCTOP.COS.UNIHE-IDELBERG.DE:8043 / ].

[0042] SgRNA target site 1 (named SH Rosa26-sgRNA1): CCATAATCGAGGAGAG-ACTCGAC; the corresponding nucleotide sequence in the sgRNA sequence that identifies this target site is shown in sequence SEQ ID NO.2 in the sequence listing; the DNA sequence encoding the above sequence is shown in sequence SEQ ID NO.4.

[0043] SgRNA target site 2 (named SH Rosa26-sgRNA2): CCCCGCGATCTCGTCAT-CGCCTC; the corresponding nucleotide sequence in the sgRNA sequence that identifies this target site is shown in sequence SEQ ID NO.3 in the sequence listing; the DNA sequence encoding the above sequence is shown in sequence SEQ ID NO.5.

[0044] The gRNA primer oligo was designed according to the following format, synthesized by BGI Genomics, and purified by PAGE.

[0045] Primer formats: Target-Sense: 5'-AAACACCG-gRNAsense, Target-Anti: 5'-CTCTAAAAC-gRNAanti, specific sequences are shown in Table 1.

[0046] Table 1. gRNA primers Oligo

[0047]

[0048] Oligo dimer formation: The above primers were diluted and then subjected to annealing hybridization. System: Target-Sense 1 μL, Target-Anti 1 μL, Solution I 5 μL, H₂O 3 μL, total volume 10 μL. Program: 95℃ for 3 min, 85℃ for 1 min, 75℃ for 1 min, 65℃ for 1 min, 55℃ for 1 min, 45℃ for 1 min, 35℃ for 1 min, 25℃ for 1 min, 4℃ for 5 min, cooling from 95℃ to 25℃ at a rate of 0.1℃ / s.

[0049] The Oligo dimer obtained in the previous step was ligated into the Cas9 / gRNA co-expression vectors (named Cas9 / gRNA-Rosa26-1 and Cas9 / gRNA-Rosa26-2). The system composition was: 1 μL Cas9 / gRNA Vector, 2 μL Oligo dimer obtained in the previous step, 7 μL H2O, and a total volume of 10 μL. The program was 25℃ for 10 min.

[0050] Transformation: The Cas9 / gRNA co-expression vector constructed in the previous step was transformed into competent cells (Trans1-T1Phage Resistant Chemically Competent Cell) using the heat shock method: the Cas9 / gRNA co-expression vector was mixed with 50 μL of competent cells and incubated on ice for 30 min; heat-shocked at 42℃ for 30 s and then incubated on ice for 2 min; then 500 μL of sterile, antibiotic-free LB medium was added and activated at 37℃ and 200 rpm for 60 min; then plated on LB solid medium containing ampicillin and cultured at 37℃ for 6-8 hours.

[0051] Single clone selection and sequencing: After single colonies have grown, select 5 Cas9 / gRNA-Rosa26-1 and 5 Cas9 / gRNA-Rosa26-2 colonies, propagate them by shaking at 37℃, preserve them, and then send them for sequencing. Sequencing primers: TGAGCGTCGATTTTTGTGATGCTCGTCAG. Sequencing results are as follows: Figure 7As shown. Plasmids from correctly sequenced Cas9 / gRNA-Rosa26-1 and Cas9 / gRNA-Rosa26-2 strains were extracted using the PureYield Plasmid Midiprep System for later use. A schematic diagram of the Cas9 / gRNA co-expression vector is shown below. Figure 8 As shown.

[0052] Plasmid extraction: (1) Inoculate 10 μL of E. coli containing the Cas9 / gRNA co-expression vector into 50 ml of LB medium containing ampicillin and shake for 18 h; (2) Centrifuge at 4500 rpm for 15 min and collect the precipitate; (3) Resuspend in 2 ml of Cell Resuspension solutin; (4) Add 2 ml of Cell lysis solution and gently invert 5-7 times, then incubate at room temperature for 3 min; (5) Add 3.3 ml of Neutralization (6) Gently invert the solution 3-5 times, let it stand at room temperature for 2-3 minutes, centrifuge at 5000 rpm for 15 minutes, discard the precipitate, and keep the liquid; (7) Place the blue adsorption column into a new centrifuge tube, then pour the liquid obtained in step (5) into the blue adsorption column, incubate for 2 minutes to allow residual cell debris to float to the top, then centrifuge at 1500 rpm for 5 minutes, and keep the liquid; (8) Place the white adsorption column into a centrifuge tube, then pour the liquid obtained in step (6) into the white adsorption column, centrifuge at 4000 rpm for 3 minutes, and discard the filtrate; (9) Add 5 ml of Endotixion Reneral Wash Solution to the white centrifuge tube, centrifuge at 1500 rpm for 3 minutes, and discard the filtrate; (10) Add 20 ml of Colum Wash, centrifuge at 1500 rpm for 15 min, discard the filtrate, and then centrifuge at 1500 rpm for 10 min; (10) Take out the white adsorption column, place it at room temperature for 10 min to ensure no ethanol residue, then add 600 μL of enzyme-free water to the white adsorption column, incubate at room temperature for 2 min, and centrifuge at 4500 rpm for 2 min; (11) After measuring the DNA concentration, store it at -20℃.

[0053] Example 3 Cell Transfection

[0054] (1) Cell preparation

[0055] Resuscitate goat fetal fibroblasts (GFF) with cell culture medium containing 10% FBS, passage them once before transfection to bring them into the logarithmic growth phase, and collect the cell pellet for plasmid transfection when the cells grow to 70%-80% of the volume of a 100 mm dish.

[0056] (2) Comparison of the effects of different cell transfection conditions

[0057] This invention uses electroporation to transfect cells. The instruments used were a NEPA GENE and a NEPA21 TYPE II. Four transfection conditions were set, as detailed in Table 2. The transfection process was as follows: After collecting the cell pellet, the cells were washed three times with Opti-MEM. Then, 20 μg of the Cas9 / gRNA co-expression vector plasmid was added to the cell pellet, and the volume was adjusted to 100 μL with Opti-MEM. After mixing, the mixture was transferred to electroporation cuvettes, and electroporation was performed using the four transfection conditions set above. After electroporation, the cells were transferred to 100 mL dishes for normal culture. Forty-eight hours after transfection, the electroporation effect and cell viability were observed using an inverted fluorescence microscope with randomly selected fields of view. The fluorescence intensity was analyzed using ImageJ. It was found that the transfection efficiency at 250 V–2.5 ms was superior to other conditions. Therefore, it is recommended to use 250 V–2.5 ms for transfecting high molecular weight plasmid vectors (>10000 bp). The transfection effect and fluorescence intensity are shown in Table 2. Figure 9 and Figure 10 As shown.

[0058] Table 2 Transfection conditions

[0059]

[0060] (3) Obtaining positive cells

[0061] After collecting the cell pellet, the cells were washed three times with Opti-MEM. Then, 20 μg of Cas9 / gRNA-Rosa26-1 and Cas9 / gRNA-Rosa26-2 plasmids were added to the cell pellet, and the volume was adjusted to 100 μL with Opti-MEM. After mixing, the cells were transferred to electroporation cuvettes and electroporated at 250 V for 2.5 ms. After electroporation, the cells were transferred to 100 mm dishes and cultured normally. After 48 hours, positive cells were enriched by flow cytometry using fluorescent selection labels. The results are shown below. Figure 11 As shown.

[0062] Example 4: Verification of sgRNA Targeting Efficiency

[0063] This invention validates the targeting efficiency of two sgRNAs (sgRNA1 and sgRNA2). The target amplification primers were designed using NCBI-primer and synthesized by BGI Genomics. The specific primer sequences are shown in Table 3.

[0064] Table 3 Primers for mutation detection

[0065]

[0066] The previously enriched positive cells and untransfected wild-type cells were treated with trypsin, washed once with PBS, and the cell pellet was collected. Genomic DNA was then extracted using the Tiangen DNA Extraction Kit, and PCR amplification was performed using mutation detection primers (Table 3). The PCR system consisted of: PrimeSTAR HS DNA Polymerase 0.5 μL, dNTP Mixture (2.5 μM) 4.0 μL, and 2×PrimeSTAR GC Buffer (Mg2+). 2+ 25 μL of (plus) Primer-F, 2.0 μL of Primer-R, 1.5 μL of DNA, 15.0 μL of H2O, total volume 50 μL. Amplification program: 94℃ for 2 min, 98℃ for 10 s, 55℃ for 15 s, 68℃ for 42 s, 30 cycles, 68℃ for 10 min, 4℃ for 60 min.

[0067] PCR purification: (1) Collect the gel strip containing the target DNA using a portable gel cutter and place it in a clean 1.5ml centrifuge tube; (2) Weigh it; (3) Add an equal ratio (1g:1ml) of Binding Buffer, heat at 50-60℃ for 10min until completely melted; (4) Place the adsorption column in a clean centrifuge tube, then transfer the melted liquid into the adsorption column, centrifuge at 10000rpm for 1min, and discard the filtrate; (5) Add 700ml Wash Buffer to the adsorption column, centrifuge at 10000rpm for 1min, discard the filtrate, and repeat this step once; (6) Centrifuge at 10000rpm for 1min; (7) Take out the adsorption column and place it at room temperature for 3-5min to allow the ethanol to evaporate completely; (8) Place the adsorption column in a new centrifuge tube, add 30μL of enzyme-free water, place for 2min, and centrifuge at 12000rpm for 2min; (9) Measure the concentration and store at -20℃.

[0068] DNA hybridization: Positive cell PCR products and wild-type cell PCR products were annealed and hybridized at a 1:1 ratio. The system consisted of 2.5 μL of positive cell PCR product, 2.5 μL of wild-type cell PCR product, 1.1 μL of Solution I, and 4.4 μL of H2O, for a total volume of 10.5 μL. The program was: 95℃ for 5 min, 94℃ for 2 s, 75℃ for 1 s, and 16℃ for 2 min, with a cooling rate of 0.1℃ / s from 95℃ to 16℃. The hybridized DNA fragments were ligated into PMD19-T cells, transformed, plated, and single clones were picked, cultured, and sequenced. Sequence alignment analysis was used to determine the editing efficiency of Cas9 / gRNA-Rosa26-1 and Cas9 / gRNA-Rosa26-2.

[0069] The results showed that for Cas9 / gRNA-Rosa26-1, 15 clones were selected. Comparison of sequencing results with the reference genome and wild-type genome revealed that 6 clones had sequence mutations in the Cas9 / gRNA-Rosa26-1 cleavage region, resulting in a targeting efficiency of 40.00% for Rosa26-sgRNA1. For Cas9 / gRNA-Rosa26-2, 15 clones were selected, and 4 clones had sequence mutations in the Cas9 / gRNA-Rosa26-2 cleavage region, resulting in a targeting efficiency of 26.67% for Rosa26-sgRNA2. Specific results are as follows: Figure 12 As shown above, the results indicate that both sgRNAs (RNA sequences such as SEQ ID NO2 and SEQ ID NO.3) provided by this invention can effectively recognize the Rosa26 site in cashmere goats and perform efficient site-specific cleavage of this site using the Cas9 enzyme. In particular, sgRNA1 has a higher cleavage efficiency, reaching 40.00%.

[0070] Off-target effect analysis: Although the cleavage efficiency of sgRNA1 is greater than 40%, there is still a possibility of off-target cleavage. Therefore, we used the off-target site prediction website (HTTP: / / CRISPOR.TEFOR.NET / ) to select five potential off-target sites for off-target effect detection. These five sites are: ①Chr2: NC_030809.1:22159187-22159209; ②Chr16: NC_030823.1:75835081-75835103; ③Chr26: NC_030833.1:7859711-7859733; ④Chr28: NC_030835.1:29593283-29593305; ⑤Chr15: NC_030822.1:1293585-1293607. The detection primers are shown in Table 4.

[0071] Table 4 Primers for Off-Target Effect Detection

[0072]

[0073] The PCR products of each potential off-target site were sequenced and compared with the wild-type PCR products. The results are as follows: Figure 13A As shown in Figure -E, the sequence homology was 100%, and no off-target events occurred at any of the five potential off-target sites. These results indicate that sgRNA1 is not only highly efficient but also has a low off-target rate, making it a good choice for site-specific integration of exogenous genes.

[0074] Example 5: Integration of the exogenous gene EGFP into the Rosa26 site using the CRISPR / Cas9 system

[0075] Construction of homologous integration vector: (1) Based on the TA clonal mutation detection results, approximately 1000 bp upstream and downstream of the sgRNA-1 target site at the Rosa26 site were selected as upstream and downstream homologous arms, and the upstream and downstream homologous arms were amplified by PCR. The primer sequences are shown in Table 5, and the electrophoresis results are as follows. Figure 14 As shown; (2) The upstream and downstream homologous arms recovered from the gel were preserved in PMD19-T and sent for sequencing. Through sequence alignment analysis, the homology was as high as 99.0% or more, and they were named PMD-Rosa26-ZT and PMD-Rosa26-YT, respectively; (3) pEGFP-C1 and PMD-Rosa26-ZT were double-digested with PciI and AseI, respectively. The vector backbone (pEGFP-C1) was ligated with Rosa26-ZT using T4 ligase and named Rosa26-EGFP-ZT. Rosa26-EGFP-ZT and PMD-Rosa26-YT were single-digested with MluI, respectively. Rosa26-EGFP-ZT was ligated with Rosa26-YT using T4 ligase to obtain the complete Rosa26 site EGFP targeting vector, named Rosa26-EGFP. The vector diagram and targeting diagram are shown in the figure. Figure 15 and Figure 16 As shown.

[0076] Table 5. Homologous arm primers

[0077]

[0078] Cell transfection: Cas9 / gRNA-Rosa26-1 and Rosa26-EGFP plasmid vectors were extracted using the PureYield Plasmid Midiprep System. Goat fetal fibroblasts were revived in cell culture medium containing 10% FBS. The electroporation apparatus was set to transfection conditions of 250V / 2.5ms, and 20 μg of Cas9 / gRNA-Rosa26-1 and 10 μg of Rosa26-EGFP were simultaneously transfected into the cells. After transfection, the cells were carefully transferred to 100 mm dishes for normal culture. To increase integration efficiency, 6 μM RS-1 was added to the cell culture medium after transfection.

[0079] Screening of positive monoclonal cells: 48 hours after transfection, cells were treated with trypsin, the cell pellet was collected, and the cells were washed three times with PBS, then resuspended in 500 μL of PBS. Single cells exhibiting green fluorescence after transfection were seeded into 96-well plates using flow cytometry. The 96-well plates should be pre-filled with culture medium containing 20% ​​FBS. On day 8, cells with greater than 50% confluence and green fluorescence in the 96-well plates were passaged into 24-well plates for further culture. When the cell confluence in the 24-well plates reached approximately 90%, cells were collected. Half of the cells were passaged for further culture, and the other half were used to extract genomic DNA for identification. Using the extracted monoclonal cell genome as a template, primers targeting specific sites were designed for PCR identification. The PCR products were then analyzed by 1.5% agarose gel electrophoresis. The primer sequences are shown in Table 6, and the electrophoresis results are as follows. Figure 17 As shown. PCR identification revealed that 12 out of 26 monoclonal cell lines obtained from the Rosa26 site underwent site-directed integration of the EGFP gene, with an integration efficiency of 46.15%. Three of these cell lines showed only a single 2056 bp band, classifying them as double-knockout cell lines, with a double-knockout rate of 25%. Positive cells are shown below. Figure 18 As shown.

[0080] Table 6 Primers for positive cell identification

[0081]

[0082] In summary, the DNA fragment encoding the sgRNA provided by this invention can be used to specifically modify (knock out or knock in) the Rosa26 site at the cellular, embryonic, or even individual level by constructing Cas9 / gRNA expression vectors and homologous integration vectors. This allows for the study of the expression of specific genes and provides technical support for the breeding of new goat breeds. At the same time, the sgRNA provided by this invention can effectively reduce off-target phenomena in the CRISPR / Cas9 system, thereby reducing mutations in non-target gene sequences caused by non-specific cleavage.

[0083] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A sgRNA specifically targeting the Rosa26 safe site of Cashmere Goat, the RNA sequence of which is shown in SEQ ID NO. 2 or SEQ ID NO. 3; the Rosa26 safe site of Cashmere Goat is located on chromosome 22 of Cashmere Goat, and the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The sgRNA of claim 1, wherein, The DNA sequence for transcribing the sgRNA is shown in SEQ ID NO. 4 or SEQ ID NO.

5.

3. A CRISPR / Cas9 targeting vector comprising the sgRNA of any one of claims 1-2, characterized in that, The vector contains a fluorescent marker gene EGFP and a drug screening marker Puro.

4. A homologous integration vector specifically targeting the Rosa26 safe site of the goat, characterized in that, The homologous integration vector contains the sgRNA according to any one of claims 1-2, and the upstream and downstream homologous arm sequences of the homologous integration vector are shown in SEQ ID NO. 6 or SEQ ID NO.

7.

5. A homologous integration vector as claimed in any one of claims 4, characterised in that, Uniqueness of the upstream and downstream homologous arms.

6. The sgRNA of any one of claims 1-2 or the CRISPR / Cas9 targeting vector of claim 3 or the homologous integration vector of any one of claims 4-5 for use in specifically recognizing and targeting modification of the safe site Rosa26 in Cashmere goats, characterized in that, According to the predicted sequence of the Rosa26 site, the corresponding sgRNA is designed, the Cas9 / gRNA co-expression vector and the homologous integration vector based on the CRISPR / Cas9 system are constructed, and the efficient Cas9 / gRNA co-expression vector and the homologous integration vector are transfected into Cashmere Goat fetal fibroblasts to obtain a cell strain with the Rosa26 site site-specific integration of the EGFP gene.

7. Use according to claim 6, wherein Site-specific knockout, site-specific integration of exogenous genes or site-specific overexpression of endogenous genes.

8. Use of the sgRNA according to any one of claims 1-2 or the CRISPR / Cas9 targeting vector according to claim 3 or the homologous integration vector according to any one of claims 4-5 in the preparation of transgenic animals or the improvement of production performance and disease resistance of livestock.

9. Use of the sgRNA according to any one of claims 1-2 or the CRISPR / Cas9 targeting vector according to claim 3 or the homologous integration vector according to any one of claims 4-5 in the breeding of new varieties of gene-edited Cashmere Goats.

10. The content of any of claims 1-9, wherein, The Cashmere Goats include Albas Cashmere Goats.

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