Pig MTSS1 gene far-end enhancer, sgRNA and application of pig MTSS1 gene far-end enhancer and sgRNA

Abstract

The invention relates to the technical field of molecular biology and animal genetic improvement, in particular to a pig MTSS1 gene distal enhancer, sgRNA and application of the pig MTSS1 gene distal enhancer. According to the method, CUTamp is utilized; the method comprises the following steps: constructing a histone modification map in pig skeletal muscle by a Tag technology, and screening out MTSS1 upstream potential enhancers; then, verifying the enhancer activity through a dual-luciferase report system; 3C-qPCR is further adopted to confirm that chromatin space interaction exists between the enhancer and an MTSS1 promoter; the enhancer sequence is knocked out in primary fibroblasts by using CRISPR / Cas9, and the influence of the enhancer sequence on the expression of the MTSS1 is detected by qRT-PCR (quantitative real-time polymerase chain reaction). Results show that the enhancer has a significant regulation effect. Technical support is provided for analysis of an MTSS1 regulation mechanism and skeletal muscle breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and animal genetic improvement technology, and particularly relates to a pig MTSS1 gene distal enhancer, sgRNA and application thereof. BACKGROUND

[0002] Skeletal muscle development of pigs has a decisive influence on their growth performance, feed utilization efficiency and meat quality traits, and is a key research direction in pig breeding. Mining and analyzing key genes and their regulatory mechanisms that control skeletal muscle development is of great significance for promoting high-quality pig breeding and realizing molecular breeding.

[0003] MTSS1 (Metastasis Suppressor 1) is a key cytoskeleton regulatory factor that is highly expressed in tissues such as skeletal muscle, and contains an IMD (IRSp53 / MIM homology domain) domain that can directly bind to actin and promote its polymerization, thereby mediating membrane protrusion formation and cell structure stability. MTSS1 It may be involved in growth and development and metabolic regulation related processes by regulating the Wnt signaling pathway, and is closely related to muscle development and fat deposition related traits. In addition, studies in mice have shown that its overexpression can lead to the disappearance of actin stress fibers and abnormal actin filament structure. Its function has been preliminarily revealed in the fields of tumor biology and osteogenic differentiation, but its expression regulatory mechanism in pig skeletal muscle tissue, especially whether its upstream distal cis-acting elements (such as enhancers) exist and their specific functions, have not been reported.

[0004] Traditional 3C-qPCR technology can be used to identify whether there is spatial interaction between the promoter of a gene and the distal DNA region, but it cannot confirm whether the region has enhancer activity; while the CRISPR / Cas9 system can be used for gene knockout, but it depends on known functional target regions. In the field of enhancer function identification, there is still a lack of a systematic, precise and verifiable method system.

[0005] In recent years, CUT&Tag (Cleavage Under Targets and Tagmentation) as a new chromatin state analysis technology can map the active enhancers in a specific tissue or cell type with high resolution, and has an advantage in low cell number samples. However, there is no public research on the application of CUT&Tag in the systematic screening, functional verification and regulatory analysis of potential enhancer regions of genes in pig skeletal muscle tissue. MTSS1 gene potential enhancer region.

[0006] Therefore, the integrated research method of CUT&Tag chromatin modification analysis, dual luciferase reporter system function verification, 3C-qPCR spatial interaction detection and CRISPR / Cas9 gene editing, systematic analysis of the expression regulation mechanism of the pig MTSS1 gene, especially in identifying and verifying the existence and regulation of the functional enhancer sequence, has important significance. By constructing the regulation model of the MTSS1 gene, key theoretical support for the molecular regulation research of skeletal muscle development is provided, and practical technical tools that can be operated and popularized for pig molecular breeding are provided. SUMMARY

[0007] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present application is to provide a pig MTSS1 gene distal enhancer.

[0008] Another purpose of the present application is to provide the application of the above-mentioned pig MTSS1 gene distal enhancer.

[0009] Still another purpose of the present application is to provide an sgRNA targeting the pig MTSS1 gene distal enhancer.

[0010] The fourth purpose of the present application is to provide the application of the above-mentioned sgRNA.

[0011] The purposes of the present application are achieved by the following technical solutions: A pig MTSS1 gene distal enhancer, the nucleotide sequence of which is shown in SEQ ID NO: 1. The MTSS1 gene distal enhancer is used in the field of pig MTSS1 gene regulation. The MTSS1 gene distal enhancer is used in pig genetic breeding. The MTSS1 gene distal enhancer is used in the preparation of products for preventing and treating MTSS1 gene related diseases. An sgRNA targeting the pig MTSS1 gene distal enhancer, comprising sgRNA1 and sgRNA2, the nucleotide sequences of which are as follows: sgRNA1: 5'-GACGTAGTAGCAAATGCTCT-3'; sgRNA2: 5'-GGTGGCCACTAGTTCACGGA-3'; A recombinant vector comprising an sgRNA targeting the pig MTSS1 gene distal enhancer, comprising the above-mentioned sgRNA targeting the pig MTSS1The sgRNA of the distal enhancer of the gene was obtained by ligating it to a vector. The preferred carrier is pSpCas9(BB)-2A-Puro (PX459); The aforementioned includes targeted pigs MTSS1 The method for constructing a recombinant vector of sgRNA from a distal enhancer of a gene includes the following steps: (1) Based on the target pig MTSS1 Two complementary single-stranded oligonucleotides were designed and synthesized for the sgRNA of the gene distal enhancer, one of which contained the sgRNA sequence and the other served as its complementary strand. (2) Anneal the single-stranded DNA oligo synthesized in step (1) to form double-stranded oligonucleotides; (3) After linearizing the CAS9 backbone vector by enzyme digestion, it is ligated with the double-stranded oligonucleotides obtained in step (2) to obtain a target porcine oligonucleotide. MTSS1 Recombinant vectors containing the distal enhancer sgRNA1 of the gene, containing targeted pig MTSS1 Recombinant vector of sgRNA2, a distal enhancer of a gene; A gene-editing tool comprising the aforementioned targeted pig MTSS1 sgRNA or recombinant vector of distal enhancer of gene; The gene editing tool preferably further includes an electroporation buffer; The target pig MTSS1 sgRNAs, recombinant vectors, or gene editing tools of distal enhancers in pigs MTSS1 Distal enhancers of genes or MTSS1 Applications in gene expression; The target pig MTSS1 sgRNAs of distal enhancers, recombinant vectors, or gene editing tools are used in the construction of pigs. MTSS1 Distal enhancer knockout or MTSS1 Applications in gene suppression models; A type of pig MTSS1 Distal enhancer knockout or MTSS1 The gene-suppressing cell model involves using the aforementioned targeted pigs. MTSS1 The sgRNA of the distal enhancer of the gene was obtained by transfecting a recombinant vector into porcine cells; The recombinant vector contains a target pig MTSS1 Recombinant vectors containing sgRNA1, a distal enhancer of the gene, and vectors targeting pigs MTSS1 The recombinant vector of sgRNA2, the distal enhancer of the gene, has a mass ratio of 1:1. The pig MTSS1 Distal enhancer knockout or MTSS1 The method for constructing a gene-suppressed cell model includes the following steps: Porcine fibroblasts were resuspended in electroporation buffer and then supplemented with a solution containing the target porcine fibroblast. MTSS1 Recombinant vectors containing sgRNA1, a distal enhancer of the gene, and vectors targeting pigs MTSS1 A mixture of recombinant vectors containing the distal enhancer sgRNA2 was used for electroporation; after electroporation, cells were transferred to complete culture medium; after 48 h of electroporation, complete culture medium containing puromycin was added for selection, and selection was continued for 2-3 days to obtain porcine cells. MTSS1 Distal enhancer knockout or MTSS1 Gene suppression cell model; The preferred content of puromycin is 2 µg / mL; The cells mentioned can be porcine fibroblasts; The target pig MTSS1 Applications of sgRNAs, recombinant vectors, or gene editing tools as distal enhancers in pig genetic breeding; The target pig MTSS1 sgRNAs of distal enhancers, recombinant vectors, or gene editing tools are used in the preparation of prevention and treatment methods. MTSS1 Products or preparations for screening and prevention of gene-related diseases MTSS1 Applications in products related to gene-related diseases; The principle of this invention: (1) This invention uses porcine longissimus dorsi muscle tissue as the research object, and combines CUT&Tag technology to construct the model. MTSS1 Chromatin modification maps of regions adjacent to the gene were used to screen for potential enhancer sequences using H3K27ac as a marker.

[0012] (2) The selected candidate enhancer sequences were cloned into a dual-luciferase reporter system and transfected into porcine primary fibroblasts to verify whether they had enhancing effects. MTSS1 Function of promoter transcriptional activity.

[0013] (3) Based on the functional validation results, chromatin conformation capture (3C-qPCR) technology was further used to detect the screened enhancers and MTSS1 Spatial interactions between promoters were investigated, and their localization relationships in the three-dimensional chromatin structure were analyzed.

[0014] (4) Construct a specific sgRNA targeting this enhancer sequence, and use the CRISPR / Cas9 gene editing system to knock out the target enhancer region in porcine primary fibroblasts; detect the enhancer knockout before and after the knockout by qRT-PCR. MTSS1 Changes in gene expression levels validate the effect of this enhancer pair at the functional level. Figure 1 Regulation of transcriptional activity ( MTSS1 ).

[0015] The present application has the following advantages and effects relative to the prior art: (1) The present application combines the comprehensive method of CUT&Tag, dual-luciferase reporter system, 3C-qPCR and CRISPR / Cas9 gene editing technology, and clearly identifies a functional enhancer sequence located upstream of the pig MTSS1 gene. The sequence has typical H3K27ac modification characteristics at the epigenetic level, and has a spatial interaction relationship with the MTSS1 promoter. Functionally, it can significantly enhance the expression of MTSS1 , proving its role as a long-distance cis-regulatory element.

[0016] (2) The present application first proposes and establishes an enhancer analysis system integrating H3K27ac modification map construction, spatial interaction detection and function verification, which is used to systematically analyze the long-distance regulation mechanism of key genes in pig skeletal muscle. The system breaks through the limitations of the prior art in enhancer identification and function confirmation, and has significant novelty and practicality.

[0017] (3) The present application not only provides a theoretical basis and technical means for the expression regulation mechanism research of Figure 1 gene, but also provides a key reference for the development of molecular markers related to pig skeletal muscle development and genetic breeding, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 2 is a schematic diagram of the enhancer analysis system of the present application.

[0019] MTSS1 is a chromatin modification map of the Figure 3 gene adjacent region.

[0020] Figure 4 is a result analysis diagram of the enhancer activity of the enhancer sequence verified by the dual-luciferase reporter system, wherein the left diagram is a schematic diagram of plasmid vector construction, and the right diagram is the relative fluorescence intensity of different plasmid vectors.

[0021] Figure 5 is a schematic diagram of 3C-qPCR site selection and primer design.

[0022] Figure 6 is a result analysis diagram of chromatin interaction intensity detected by 3C-qPCR.

[0023] Figure 7 is an ineffective sgRNA editing result analysis diagram.

[0024] Figure 8 is an effective sgRNA editing site and editing result analysis diagram.

[0025] MTSS1Figure 1 is a graph showing the results of analysis of gene expression levels before and after enhancer knockout. MTSS1 Figure 2 is a graph showing the results of analysis of gene expression levels before and after enhancer knockout. DETAILED DESCRIPTION

[0026] The application will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments of the application are not limited thereto.

[0027] In the embodiments, all pig individuals are a crossbreed of Berkshire and Ganzixilang, and are from a pig breeding company in Pingxiang, Jiangxi, China.

[0028] In the embodiments, all restriction enzymes and T4 DNA ligase are purchased from New England Biolabs (Massachusetts, USA); and all reagent kits for gel recovery and plasmid extraction are purchased from IGEBiotechnology (Guangzhou, China).

[0029] Example 1 Determination of potential enhancer sequence positions of genes based on CUT&Tag sequencing Figure 2 Example 1 Determination of potential enhancer sequence positions of genes based on CUT&Tag sequencing 1. Collection of pig skeletal muscle samples

[0030] After the pigs are raised for 100 days under uniform feeding environment and standard feed, the carcasses are divided, and the longissimus dorsi muscle at the 6-7 rib of the right half carcass is sampled and determined.

[0031] 2. CUT&Tag sequencing

[0032] The CUT&Tag experiment is performed using a Novo CUT&Tag 2.0 high-sensitivity kit (Novo Protein, Catalog No.: N259-YH01, RRID: AB_2886259), and the specific operation is as follows: (1) The nuclei are lysed and extracted from the pig longissimus dorsi muscle tissue, and combined with pre-coated Concanavalin A (ConA) magnetic beads for 10 min according to the kit instructions, to facilitate subsequent antibody recognition and solid-phase operation.

[0033] (2) The nuclei system combined with ConA magnetic beads in step (1) is allowed to stand, and the supernatant is discarded, and then incubated with specific primary antibodies, including H3K4me3 (CST, 9751T) and H3K27ac (CST, 8173T), at 4°C for 2 h.

[0034] (3) After incubation, the supernatant is discarded, and secondary antibodies (Abcam, ab6702) are added for further incubation for 1 h to form a target protein-antibody complex.

[0035] (4) After incubation, let stand, discard the supernatant and wash, then add pA-Tn5 transposase pre-bound sequencing adapter and incubate for 1 h. The enzyme complex is precisely located in the DNA region near the target protein and activates the targeted transposition reaction (tagmentation), inserting adapter sequences at both ends of the DNA (read1: 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3', read2: 5'-CTGTCTCTTATACACATCTGACGCTGCCGACGA-3') to facilitate subsequent PCR amplification and construction of sequencing libraries. After incubation, let stand, discard the supernatant and wash.

[0036] (5) After the transposition reaction, the antibody and target protein were digested with proteinase K to release and purify the DNA; then the recovered DNA fragments were amplified into a library, and PCR amplification was set to 11 cycles.

[0037] (6) The library was purified using AMPure magnetic beads and the library quality was evaluated using the Agilent Bioanalyzer 2100 system.

[0038] (7) The samples with index tags were clustered on the cBot Cluster Generation System (Illumina) using the TruSeq PE Cluster Kit v3-cBot-HS kit, strictly following the manufacturer's instructions. Finally, the library was sequenced at 150 bp paired ends on the Illumina NovaSeq platform.

[0039] 3. Data Processing

[0040] (1) The raw sequencing data of CUT&Tag obtained in step 2 are first aligned to the pig reference genome Sus scrofa 11.1 using the BWA-MEM algorithm (version v0.7.17) to generate SAM / BAM format files.

[0041] (2) Then, the PCR repeat sequences were labeled using the Picard tool (v2.20, https: / / broadinstitute.github.io / picard / ) and the uniquely aligned reads were further screened using SAMtools (v1.10). High-quality alignment results were retained and output as BAM format files for subsequent analysis.

[0042] (3) The enrichment region identification of histone modifications (such as H3K4me3, H3K27ac, etc.) and transcription factor CTCF is based on peak call using MACS2 software (v2.1.1). This step takes the BAM file processed in step (2) as input and the call command is set to: -f BAMPE -q 0.05 --nomodel --shift -0 --keep-dup all, where the input is specified as paired end sequencing (BAMPE) and all repetitive reads are retained to adapt to the characteristics of CUT&Tag data.

[0043] (4) Finally, the narrow peaks from different histones were merged using the merge function in BEDTools software (v2.25.0) to obtain a set of histone modification consistent peaks at the diploid level.

[0044] CUT&Tag results are as follows MTSS1 As shown in the figure. It can be seen from the figure that in MTSS1 Significant H3K27ac signal enrichment was observed in the upstream region of chr4: 14.775–14.800 Mb (marked by blue shading in the figure), suggesting that this region has typical active enhancer characteristics; at the same time, no H3K4me3 signal was observed in this region, ruling out the possibility that it is a promoter region.

[0045] To verify the conservation of this region, multi-species sequence alignment was performed on the DNA sequence corresponding to the blue-marked region. The results showed low sequence homology between pigs and other animals, suggesting it is a pig-specific or low-conserved regulatory element. This low-conservation characteristic indicates that it may have evolved into a pig-specific regulatory function, possessing clear novelty and species specificity. Therefore, this region was selected as a candidate enhancer for subsequent spatial interaction detection, functional verification, and gene editing experiments to systematically analyze its role in... MTSS1 Its role in gene regulation.

[0046] The nucleotide sequence of the candidate enhancer (SEQ ID NO:1) is shown below: Example 2 Dual-luciferase assay to verify enhancer activity of candidate enhancer sequences

[0047] 1. Culture of porcine primary fibroblasts

[0048] Porcine embryonic fibroblasts were obtained from 30-day-old fetuses, with the following specific steps: (1) Bucy × Ganzixiang Muyin sows were euthanized by intravenous injection of potassium chloride solution (100 mg / kg) under anesthesia, and the anesthetic used was intramuscular injection of Zoletil® 50 (5 mg / kg, France VAK Company) and xylazine (1.0 mg / kg, Jilin Huamuyu Animal Health Products Co., Ltd., China).

[0049] (2) Fetuses were obtained according to the conventional method, and porcine primary fibroblasts were isolated from the fetuses using 200 U / mL of collagenase type IV (Sigma-Aldrich, USA).

[0050] (3) The porcine primary fibroblasts obtained in step (2) were inoculated into Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) containing 12% (v / v) fetal bovine serum (ExCellBio, Australia), 100 IU / mL penicillin and 100 µg / mL streptomycin, and cultured in a humidified incubator containing 5% CO2 at 37°C; when the cells reached 70-80% confluence, they were passaged using 0.25% trypsin-EDTA (Gibco, Cat. No. 25200056). To ensure that there was no mycoplasma contamination during cell culture, the MycoAlert™ Mycoplasma Detection Kit (Lonza, Switzerland) was routinely used for detection.

[0051] 2. Vector construction (1) Artificial synthesis by a commissioned synthesis company Hind The nucleotide sequence of the gene promoter region (SEQ ID NO: 2) was artificially synthesized, and the Xho III and Hind I restriction enzyme cleavage sites were introduced at the 5' and 3' ends, respectively; the synthesized nucleotide sequence of the promoter region and the reporter vector pGL3-basic (Promega Corporation, USA) were subjected to enzyme cleavage using Xho III and MTSS1 I, respectively, and the enzyme-cleaved products were subjected to gel recovery, and T4 DNA ligase was used for ligation according to the conventional method, and the ligated product was transformed into E. coli DH5α competent cells and plated for culture; after single colony expansion, the plasmid was extracted, identified, and the recombinant vector pGL3-Pro( MTSS1 ) was obtained.

[0052] Bam Gene promoter region (SEQ ID NO: 2): (2) The nucleotide sequence of the candidate enhancer region (shown in SEQ ID NO: 1) in Example 1 was artificially synthesized by a synthesis company, and the restriction enzyme cleavage sites of Sal H I and Bam I were introduced at the 5' and 3' ends, respectively, and the nucleotide sequence of the candidate enhancer region was cloned into the recombinant vector pGL3-Pro-En( Sal ) obtained in Step 2 by referring to the method of Step 2, to obtain the recombinant vector pGL3-Pro-En( MTSS1 ). MTSS1 ) obtained in Step 2 by referring to the method of Step 2, to obtain the recombinant vector pGL3-Pro-En( MTSS1 ).

[0053] 3. Dual-luciferase assay (1) To detect the activity of the candidate enhancer in regulating the expression of MTSS1 gene, the porcine embryonic fibroblasts in Step 1 were inoculated into a 24-well plate at 1 x 10 5 cells per well, and cultured for 1 day for subsequent transfection.

[0054] (2) The next day, co-transfection was performed using Lipofectamine 3000 (Invitrogen) according to the instructions, 900 ng of pGL3-basic, pGL3-Pro( MTSS1 ) or pGL3-Pro-En( Figure 3 ) was added to each well, and 100 ng of pRLTK was used as an internal reference. The untransfected group was used as a negative control, and the empty vector pGL3-basic was used as a control group. Figure 3 ).

[0055] (3) After 48 h of transfection, the luciferase activity was detected using the dual-luciferase reporter system (Promega, E1910) and the multifunctional enzyme labeler (Biotek, USA). The final relative luciferase activity was calculated by the ratio of firefly luciferase activity to Renilla luciferase activity. All data were from three biological replicates, and statistical analysis was performed using two-tailed T test for significance test.

[0056] The results of the dual-luciferase assay are shown in MTSS1 Compared with the pGL3-basic empty vector, pGL3-Pro( MTSS1 ) can significantly increase the luciferase activity ( p = 0.00245), and after the addition of the enhancer sequence, the luciferase activity of the pGL3-Pro-En( MTSS1 ) group is further significantly increased ( p = 0.00341 and p=0.00453), exhibiting an activation capacity of approximately 10-fold or more. This result clearly demonstrates that this enhancer sequence (chr4:14,774,391–14,775,525) can significantly enhance cellular activity at the cellular level. MTSS1 Promoter-mediated transcriptional activity, with typical enhancer function, further supports its key role in transcriptional regulation.

[0057] Example 3: Based on 3C-qPCR MTSS1 Enhancer-promoter interaction detection 1. Cell nucleus purification: Following Example 1, approximately 2g of porcine longissimus dorsi muscle tissue was placed in PBS buffer containing 2% (v / v) paraformaldehyde and crosslinked and fixed in a vacuum environment under ice bath conditions for 30min. Then, 2M glycine (final concentration 0.125M) was added to terminate the crosslinking reaction, and the mixture was incubated on ice for another 5min. After crosslinking, the cell nuclei were extracted and purified using a self-made nuclear extraction buffer (10mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, with protease inhibitors added before use).

[0058] 2. Enzyme digestion: To perform restriction endonuclease digestion, the purified cell nuclei from step (1) were resuspended in 1.2×NEBuffer 2.1 buffer (New England Biolabs), and treated with 0.2% (v / v) SDS at 37°C for 20 min to open the chromatin structure. Then, 2% (v / v) Triton X-100 was added to neutralize the SDS, and incubation continued for 30 min. Eco site, using Hind RI Figure 4 The three restriction endonucleases, III and Sau96I, were digested in the same reaction system (400 U of each enzyme was added), incubated overnight at 37°C, and then the reaction was terminated by incubation at 65°C for 20 min.

[0059] 3. Add T4 DNA ligase to the system after enzyme digestion in step (2) to a final concentration of 100U, ligate at 16℃ for 5 h, and continue to incubate at room temperature for 45 min to complete the ligation reaction; after ligation, use proteinase K to perform anti-crosslinking treatment overnight at 65℃, and then recover DNA by phenol / chloroform extraction and ethanol precipitation, which is the 3C library.

[0060] 4. qPCR detection (1) Primer design qPCR primer design was based on the online tool Primer3 (https: / / primer3.ut.ee / ). The primers used were targeted to the preset promoter and enhancer regions. The specific sequences are shown in Table 1, and the specific targeting positions are shown in Figure 5 .

[0061] Table 1 qPCR primer information

[0062] (2) qPCR amplification qPCR was performed using SYBR Green I fluorescent dye system with the DNA after step (2) enzyme digestion or the 3C library obtained in step (3) as the template. Two groups of controls were set up for qPCR, namely positive control and negative control, and GAPDH promoter primers were used for detection to verify the effectiveness of the system. The primers 77R1-F, 77R1-R, 93R1-R, 93R2-F and 93R2-R were used for corresponding amplification of the DNA after step (2) enzyme digestion or the 3C library obtained in step (3), wherein the DNA after step (2) enzyme digestion was not subjected to subsequent T4 DNA ligase connection, and it was used as a background noise control (Table 2). The reaction system and program of qPCR are shown in Tables 3 and 4.

[0063] Table 2 qPCR grouping

[0064] Table 3 qPCR reaction system

[0065] Table 4 qPCR reaction program

[0066] (3) Data analysis The Ct values of all qPCR reactions were normalized, and the normalization standard was the signal of the IgG group. The qPCR signals of each primer pair were normalized to the IgG magnetic bead control group, and all data were the average values of three biological replicates.

[0067] The results are shown in Bbs No significant amplification signal was detected in test group 1 without T4 DNA ligase, while the relative signal intensity of test group 2 (enzyme digestion + T4 DNA ligase) was significantly higher than that of test group 1, indicating that there was indeed physical contact between the two groups of fragments in three-dimensional space, thereby verifying the existence of chromatin long-range interaction relationship.

[0068] Example 4 CRISPR / Cas9 construction targeting enhancer sequence and gene expression verification method

[0069] 1. sgRNA design Four specific sgRNAs were designed by using the CRISPOR online tool (http: / / crispor.tefor.net) for the enhancer (SEQ ID NO: 1) (Table 5), and the sgRNA screening standard was MIT specificity score greater than 95 to reduce off-target effects as much as possible. The corresponding primer pair was designed and artificially synthesized for each sgRNA according to the format of "5'-caccg + sgRNA sequence" for the forward primer and "5'-aaac + sgRNA reverse complementary sequence + c" for the reverse primer.

[0070] Table 5 Purpose recognition sequence of sgRNA

[0071] 2. Vector construction The sgRNA targeting the enhancer sequence was cloned into the linearized pSpCas9(BB)-2A-Puro vector (PX459 V2.0, addgene, #62988) by using the Golden Gate cloning assembly technology. Bbs I The specific steps are as follows: (1) Linearization of PX459 V2.0 plasmid vector: PX459 V2.0 was linearized by using Bbsl, and the linearization reaction system was: PX459 V2.0 plasmid vector 1 μg, 10×Cutsmart buffer 3 μL, Bbsl 1 μL, and ddH2O was added to 30 μL; the enzyme linearization reaction conditions were: 37°C for 2 h, 85°C for 15 min, and 4°C storage. Subsequently, the enzyme cutting fragments were separated by using a 2% agarose gel electrophoresis, the target band was cut and recovered, and the kit was used for purification. Bbs I 1 μL, and ddH2O was added to 30 μL; the enzyme linearization reaction conditions were: 37°C for 2 h, 85°C for 15 min, and 4°C storage. Subsequently, the enzyme cutting fragments were separated by using a 2% agarose gel electrophoresis, the target band was cut and recovered, and the kit was used for purification. MTSS1

[0072] (2) Annealing of sgRNA: The annealing system was prepared, and the specific system was: 10 μM sgRNA forward primer 2 μL, 10 μM sgRNA reverse primer 2 μL, 10×T4 DNA ligase buffer 2.5 μL, and ddH2O 18.5 μL; the prepared annealing system was placed in boiling water for 2 min, and then naturally cooled, 75 μL of double distilled water was added to a final concentration of 2 μM, and the sgRNA annealing product was obtained.

[0073] ​(3) Ligation: The sgRNA annealing product was ligated with the PX459 V2.0 linearized plasmid vector using T4 ligase, and the reaction system was as follows: PX459 V2.0 linearized plasmid 30 ng, sgRNA annealing product 3 μL, T4 DNA ligase 0.5 μL, 10×T4 DNA ligase buffer 1 μL, and ddH2O was added to 10 μL; the added system was placed in a PCR instrument at 16°C for 1 h, 85°C for 20 min, and 4°C for preservation.

[0074] 3. Transfection, screening and detection (1) The porcine primary fibroblasts were cultured according to the method described in Reference Example 2, and were cultured to 70-80% confluence before electroporation.

[0075] (2) After the cells in step (1) were digested with trypsin-EDTA (Gibco, USA) with a mass fraction of 0.25%, centrifuged at 200×g for 5 min, resuspended in 100 μL Transter-E buffer, and a total of 10 μg of sgRNA plasmid mixture (5 μg of each sgRNA plasmid) was added; electroporation was completed in Lonza Nucleocuvette™ electroporation tubes using a double pulse program (FF113+CA137); after electroporation, the cells were transferred to a 60 mm culture dish containing 5 mL of preheated complete medium for culture.

[0076] (3) After 48 h of electroporation, the complete medium containing 2 μg / mL puromycin was replaced for screening, and the screened cells were collected after 2 days of continuous screening.

[0077] (4) The genomic DNA of the cells screened in step (3) was extracted, the target region was PCR amplified, and whether the enhancer was successfully knocked out was verified by gel electrophoresis, wherein the PCR primers are shown in Table 6, and the PCR reaction system is as follows: 2×Taq PCR Master Mix 15.0 μL, genomic DNA (~20 ng) 1.0 μL, upstream primer (concentration 5 pmol / μL) 2.0 μL, downstream primer (concentration 5 pmol / μL) 2.0 μL, and ddH2O 10.0 μL; the PCR reaction program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles; and 72°C final extension for 5 min.

[0078] (5) qRT-PCR test was performed to detect the effect of enhancer knockout on Figure 6The influence on the gene expression level, wherein the qRT-PCR primer is shown in Table 7, the qRT-PCR reaction system (20 μL) is as follows: 2x ChamO Universal SYBR qPCR MasterMix 10 μL, cDNA 1 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, RNase Free ddH2O 8.2 μL; the PCR reaction procedure is as follows: 95 °C pre-denaturation 10 min; 95 °C denaturation 10 s, 60 °C annealing 30 s, 72 °C extension 30 s, a total of 40 cycles.

[0079] Table 6 PCR primer

[0080] Table 7 qRT-PCR primer

[0081] Based on the fact that the enhancer nucleotide sequence is long and it is difficult to realize the effective knockout of the enhancer by one sgRNA, the present application selects different sgRNA combinations to knockout the enhancer region, and each combination is repeated three times, and the PCR results of the knockout of the target fragment by different sgRNA combinations are shown in Figure 7 and Figure 6 It can be seen from MTSS1 that the sgRNA1+sgRNA3, sgRNA1+sgRNA4 and sgRNA2+sgRNA3 do not realize the effective knockout of the enhancer region, while after the knockout by the sgRNA1+sgRNA2 combination, the fragment length obtained by amplification by MTSS1 -F1 and Figure 7 -R1 is consistent with the expected length of the remaining fragment after knockout and specific amplification exists MTSS1 , which indicates that the sgRNA1+sgRNA2 combination realizes the effective knockout of the enhancer region.

[0082] The expression level of Figure 8 after the knockout of the enhancer region by the sgRNA1+sgRNA2 combination is further analyzed, and the results are shown in Figure 8 . It can be seen from MTSS1 that compared with the unknocked control group, the CRISPR / Cas9 knockout mediated by the sgRNA1+sgRNA2 combination significantly reduces the expression level of the MTSS1 gene (about 70%), which further verifies that the enhancer has the function of regulating the expression of the MTSS1 gene.

[0083] In summary, the comprehensive method combining CUT&Tag, a dual-luciferase reporter system, 3C-qPCR and CRISPR / Cas9 gene editing technology is used to research and confirm that the pigMTSS1 Distal enhancer of a gene MTSS1 Spatial regulatory function of a gene and its promoter. MTSS1 The gene is a key porcine cytoskeleton regulator, which is highly expressed in tissues such as skeletal muscle, and is closely related to muscle development and fat deposition related traits. The porcine MTSS1 The distal enhancer of the gene can be applied to the genetic breeding of porcine muscle development and fat deposition and the prevention and treatment of porcine MTSS1 Gene related diseases, in addition, the sgRNA combination provided by the present application and the constructed porcine MTSS1 The distal enhancer of the gene regulates the cell model, which can further construct the corresponding animal model, and then can provide ideas and directions for the research ​ Gene function, molecular breeding of pigs and other domestic animals, and drug screening.

[0084] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application.

Claims

1. A type of pig MTSS1 Distal enhancers of genes, characterized by Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The claim 1 MTSS1 Distal enhancers in pigs MTSS1 Applications in the field of gene regulation.

3. The claim 1 MTSS1 Application of distal enhancers in pig genetic breeding.

4. A targeted pig MTSS1 The sgRNA of a gene distal enhancer is characterized by It contains sgRNA1 and sgRNA2, whose nucleotide sequences are shown below: sgRNA1: 5'-GACGTAGTAGCAAATGCTCT-3'; sgRNA2: 5'-GGTGGCCACTAGTTCACGGA-3'.

5. A method comprising targeted pigs MTSS1 Recombinant vectors of sgRNAs of distal enhancers of genes, characterized by The targeted pig as described in claim 4 MTSS1 The sgRNA of the distal enhancer of the gene was obtained by ligating it to the vector.

6. The method of claim 5 comprising targeting pigs MTSS1 A method for constructing a recombinant vector of sgRNA from a gene distal enhancer, characterized in that... It includes the following steps: (1) Based on the target pig MTSS1 Two complementary single-stranded oligonucleotides were designed and synthesized for the sgRNA of the gene distal enhancer, one of which contained the sgRNA sequence and the other served as its complementary strand. (2) Anneal the single-stranded DNA oligo synthesized in step (1) to form double-stranded oligonucleotides; (3) After linearizing the CAS9 backbone vector by enzyme digestion, it is ligated with the double-stranded oligonucleotides obtained in step (2) to obtain a target porcine oligonucleotide. MTSS1 Recombinant vectors for sgRNAs of distal enhancers of genes.

7. A gene-editing tool, characterized in that... Includes the targeted pig as described in claim 4 MTSS1 The sgRNA of the distal enhancer of the gene or the recombinant vector as described in claim 5.

8. The targeted pig as described in claim 4 MTSS1 The sgRNA of the distal enhancer of the gene, the recombinant vector of claim 5, or the gene editing tool of claim 7 in inhibiting pigs MTSS1 Distal enhancers of genes or MTSS1 In gene expression, constructing pigs MTSS1 Distal enhancer knockout or MTSS1 Application in gene suppression models.

9. A type of pig MTSS1 Distal enhancer knockout or MTSS1 Gene-suppressed cell model, characterized by: The targeted pig as described in claim 5 MTSS1 The sgRNA of the distal enhancer of the gene was obtained by transfecting it into pig cells.

10. The targeted pig according to claim 4 MTSS1 The application of sgRNA of gene distal enhancer, the recombinant vector of claim 5, or the gene editing tool of claim 7 in pig genetic breeding.

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