Pig MTSS1 gene distal enhancer, sgRNA and application thereof

Abstract

This invention relates to the fields of molecular biology and animal genetic improvement, specifically to a pig MTSS1 Distal enhancers, sgRNAs, and their applications. This invention utilizes CUT&Tag technology to construct histone modification maps in porcine skeletal muscle and screen for... MTSS1 Upstream potential enhancers were identified; subsequently, their enhancer activity was verified using a dual-luciferase reporter system; further, 3C-qPCR was used to confirm the interaction between this enhancer and... MTSS1 The promoter exhibits chromatin spatial interactions; and this enhancer sequence was knocked out in primary fibroblasts using CRISPR / Cas9, with qRT-PCR used to detect its effects on chromatin. MTSS1 The effect on expression. The results show that this enhancer has a significant regulatory effect. This invention provides a basis for analysis. MTSS1 It provides technical support for regulatory mechanisms and skeletal muscle breeding.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and animal genetic improvement, specifically to a pig MTSS1 Distal enhancers of genes, sgRNA and their applications. Background Technology

[0002] Skeletal muscle development in pigs has a decisive impact on their growth performance, feed utilization efficiency, and meat quality traits, making it a key research direction in pig breeding. Identifying and analyzing the key genes controlling skeletal muscle development and their regulatory mechanisms is of great significance for promoting the selection of high-quality pig breeds and achieving molecular breeding.

[0003] MTSS1 Metastasis Suppressor 1 (MSS) is a key cytoskeleton regulator that is highly expressed in tissues such as skeletal muscle. It contains an IMD (IRSp53 / MIM homology domain) that can directly bind to actin, promoting its polymerization and thus mediating membrane protrusion formation and cell structural stability. MTSS1 It may participate in growth, development, and metabolic regulation by modulating the Wnt signaling pathway, and is also closely related to traits such as muscle development and fat deposition. Furthermore, studies in mice have shown that its overexpression can lead to the disappearance of actin stress fibers and abnormalities in actin filament structure. Previous studies have provided preliminary insights into its function in areas such as tumor biology and osteogenic differentiation; however, its expression regulation mechanism in porcine skeletal muscle tissue, particularly the existence and specific function of its upstream distal cis-acting elements (such as enhancers), has not yet been publicly reported.

[0004] Traditional 3C-qPCR technology can be used to identify spatial interactions between gene promoters and distal DNA regions, but it cannot confirm whether the region has enhancer activity. While the CRISPR / Cas9 system can be used for gene knockout, it relies on known functional target regions. In the field of enhancer function identification, a systematic, precise, and verifiable methodology is still lacking.

[0005] In recent years, CUT&Tag (Cleavage Under Targets and Tagmentation) has emerged as a new chromatin state analysis technique, capable of mapping active enhancers in specific tissues or cell types at high resolution, with particular advantages in low-cell-count samples. However, no publicly available studies have yet applied CUT&Tag to porcine skeletal muscle tissue. MTSS1 Systematic screening, functional verification, and regulatory analysis of potential enhancer regions of genes.

[0006] Therefore, a comprehensive research method integrating CUT&Tag chromatin modification analysis, dual-luciferase reporter system functional verification, 3C-qPCR spatial interaction detection, and CRISPR / Cas9 gene editing is of great significance for systematically elucidating the expression regulatory mechanism of the porcine MTSS1 gene, especially in identifying and verifying the existence and regulatory role of its functional enhancer sequences. Constructing a regulatory model of the MTSS1 gene will provide crucial theoretical support for molecular regulation research on skeletal muscle development and offer a practical and scalable technical tool for molecular breeding of pigs. Summary of the Invention

[0007] To overcome the shortcomings and disadvantages of the existing technology, the primary objective of this invention is to provide a pig MTSS1 Distal enhancer of the gene.

[0008] Another object of the present invention is to provide the above-mentioned pig MTSS1 Applications of gene distal enhancers.

[0009] Another object of the present invention is to provide a targeted pig MTSS1 sgRNA of distal enhancer of a gene.

[0010] A fourth objective of this invention is to provide applications of the aforementioned sgRNA.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A type of pig MTSS1 The distal enhancer of the gene has the nucleotide sequence shown in SEQ ID NO: 1;

[0013] The aforementioned MTSS1 Distal enhancers in pigs MTSS1 Applications in the field of gene regulation;

[0014] The aforementioned MTSS1 Application of distal enhancers in pig genetic breeding;

[0015] The aforementioned MTSS1 Distal enhancers in the preparation of prevention MTSS1 Applications in products related to gene-related diseases;

[0016] A type of targeted pig MTSS1 The sgRNAs of the gene distal enhancers, including sgRNA1 and sgRNA2, have the following nucleotide sequences:

[0017] sgRNA1: 5'-GACGTAGTAGCAAATGCTCT-3';

[0018] sgRNA2: 5'-GGTGGCCACTAGTTCACGGA-3';

[0019] A type of pig containing target pigs MTSS1 Recombinant vectors of sgRNAs from distal enhancers of genes, derived from the aforementioned targeted pigs MTSS1 The sgRNA of the distal enhancer of the gene was obtained by ligating it to a vector.

[0020] The preferred carrier is pSpCas9(BB)-2A-Puro (PX459);

[0021] 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:

[0022] (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.

[0023] (2) Anneal the single-stranded DNA oligo synthesized in step (1) to form double-stranded oligonucleotides;

[0024] (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;

[0025] A gene-editing tool comprising the aforementioned targeted pig MTSS1 sgRNA or recombinant vector of distal enhancer of gene;

[0026] The gene editing tool preferably further includes an electroporation buffer;

[0027] 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;

[0028] 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 repression models;

[0029] 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;

[0030] The recombinant vector contains a target pig. MTSS1 Recombinant vectors containing the distal enhancer sgRNA1 of the gene and targeting pigs MTSS1 The recombinant vector of sgRNA2, the distal enhancer of the gene, has a mass ratio of 1:1.

[0031] The pig MTSS1 Distal enhancer knockout or MTSS1 The method for constructing a gene-suppressed cell model includes the following steps:

[0032] Porcine fibroblasts were resuspended in electroporation buffer and then supplemented with a solution containing the target porcine fibroblast. MTSS1 Recombinant vectors containing the distal enhancer sgRNA1 of the gene and 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;

[0033] The preferred content of puromycin is 2 µg / mL;

[0034] The cells mentioned can be porcine fibroblasts;

[0035] The target pig MTSS1 Application of sgRNA, recombinant vectors, or gene editing tools as distal enhancers in pig genetic breeding;

[0036] 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;

[0037] The principle of this invention:

[0038] (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.

[0039] (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.

[0040] (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.

[0041] (4) Construct a specific sgRNA targeting this enhancer sequence, and knock out the target enhancer region in porcine primary fibroblasts using the CRISPR / Cas9 gene editing system; 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. MTSS1 Regulation of transcriptional activity ( Figure 1 ).

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] (1) The present invention combines CUT&Tag, dual-luciferase reporter system, 3C-qPCR and CRISPR / Cas9 gene editing technology to clearly identify a segment located in pigs MTSS1 The upstream functional enhancer sequence of the gene exhibits typical H3K27ac modification characteristics at the epigenetic level, and is associated with... MTSS1 Promoters exhibit spatial interactions, which can significantly enhance their functionality. MTSS1 The expression demonstrates its role as a distal cis-regulatory element.

[0044] (2) This invention proposes and establishes for the first time an enhancer analysis system integrating H3K27ac modification map construction, spatial interaction detection, and functional verification, for systematically analyzing the distal regulatory mechanisms of key genes in porcine skeletal muscle. This system overcomes the limitations of existing technologies in enhancer identification and functional confirmation, and has significant novelty and practicality.

[0045] (3) This invention is not only for MTSS1 The study of gene expression regulation mechanisms provides a theoretical basis and technical means, and also provides a key reference for the development of molecular markers related to pig skeletal muscle development and genetic breeding, with broad application prospects. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the enhancer analysis system of the present invention.

[0047] Figure 2 yes MTSS1 A map of chromatin modifications in regions adjacent to a gene.

[0048] Figure 3 This is a result analysis diagram of the enhancer activity of the enhancer sequence verified by the dual-luciferase reporter system. The left figure is a schematic diagram of the plasmid vector construction, and the right figure is the relative fluorescence intensity of different plasmid vectors.

[0049] Figure 4 This is a schematic diagram of 3C-qPCR site selection and primer design.

[0050] Figure 5 This is a graph showing the results of 3C-qPCR detection of chromatin interaction intensity.

[0051] Figure 6 This is a graph showing the results of invalid sgRNA editing.

[0052] Figure 7 This is a diagram showing the effective sgRNA editing sites and the editing results.

[0053] Figure 8 Before and after enhancer knockout MTSS1 A graph showing the results of gene expression level analysis. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0055] In this embodiment, all pigs were Berkshire × Ganxi Two-Ended Black crossbreeds, sourced from a pig breeding company in Pingxiang, Jiangxi, China.

[0056] In the examples, all restriction endonucleases and T4 DNA ligases were purchased from New England Biolabs (Massachusetts, USA); gel recovery, plasmid extraction, and other kits were purchased from IGEBiotechnology (Guangzhou, China).

[0057] Example 1: Determined based on CUT & Tag sequencing MTSS1 Potential enhancer sequence location of the gene

[0058] 1. Collection of porcine skeletal muscle samples

[0059] Pigs were raised in a uniform feeding environment and under standardized feed for 100 days before being slaughtered. The carcasses were then divided, and samples were taken from the longissimus dorsi muscle at the 6th to 7th ribs of the right half of the carcass for testing.

[0060] 2. CUT & Tag Sequencing

[0061] The CUT&Tag assay was performed using the NovoNGS® CUT&Tag 2.0 high-sensitivity kit (NovoProtein, catalog number: N259-YH01, RRID: AB_2886259). The specific procedure is as follows:

[0062] (1) Cell nuclei were extracted from the longissimus dorsi muscle tissue of pigs and bound to pre-coated Concanavalin A (ConA) magnetic beads for 10 min according to the kit instructions to facilitate subsequent antibody recognition and solid-phase operation.

[0063] (2) The cell nucleus system bound to ConA magnetic beads in step (1) was left to stand and the supernatant was discarded. Then it was incubated with specific primary antibodies, including H3K4me3 (CST, 9751T) and H3K27ac (CST, 8173T). The incubation conditions were 4°C and incubation for 2 h.

[0064] (3) After incubation, let stand and discard the supernatant, add secondary antibody (Abcam, ab6702) and continue incubation for 1 h to form target protein-antibody complex.

[0065] (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.

[0066] (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.

[0067] (6) The library was purified using AMPure magnetic beads and the library quality was evaluated using the Agilent Bioanalyzer 2100 system.

[0068] (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.

[0069] 3. Data Processing

[0070] (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.

[0071] (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.

[0072] (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.

[0073] (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.

[0074] CUT&Tag results are as follows Figure 2 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.

[0075] 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.

[0076] The nucleotide sequence of the candidate enhancer (SEQ ID NO:1) is shown below:

[0077]

[0078] Example 2: Dual-luciferase assay to verify enhancer activity of candidate enhancer sequences

[0079] 1. Culture of primary porcine fibroblasts

[0080] Pig embryonic fibroblasts were obtained from 30-day-old fetuses, and the specific steps are as follows:

[0081] (1) Berkshire × Ganxi Two-headed Black sows were euthanized by intravenous injection of potassium chloride solution (100 mg / kg) under anesthesia. The anesthetics used were intramuscular injection of Zoltil® 50 (5 mg / kg, Virbac France) and xylazine (1.0 mg / kg, Jilin Huamu Animal Health Products Co., Ltd., China).

[0082] (2) Fetuses were obtained using conventional methods, and porcine primary fibroblasts were isolated from the fetuses using type IV collagenase (Sigma-Aldrich, USA) at a concentration of 200 U / mL.

[0083] (3) The porcine primary fibroblasts obtained in step (2) were seeded into Dulbecco 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, catalog number 25200056). To ensure that the cells were free from mycoplasma contamination during cell culture, the MycoAlert™ Mycoplasma Detection Kit (Lonza, Switzerland) was routinely used for detection.

[0084] 2. Carrier Construction

[0085] (1) Artificial synthesis commissioned to a synthetic company MTSS1 The nucleotide sequence of the gene promoter region (SEQ ID NO: 2) was introduced at the 5' and 3' ends, respectively. Hind III and Xho I. Restriction endonuclease cleavage sites; the nucleotide sequence of the synthesized promoter region and the reporter vector pGL3-basic (Promega, USA) were respectively cleaved using restriction endonuclease. Hind III and XhoI was subjected to enzyme digestion, and the digested products were recovered by gel electrophoresis. Ligation was performed using T4 DNA ligase according to standard methods. The ligated product was transformed into *E. coli* DH5α competent cells and cultured on plates. Single colonies were picked and expanded, and plasmids were extracted and identified to obtain the recombinant vector pGL3-Pro( MTSS1 ).

[0086] MTSS1 Gene promoter region (SEQ ID NO:2):

[0087]

[0088] (2) The nucleotide sequence of the candidate enhancer region in Example 1 (shown as SEQ ID NO:1) was artificially synthesized by a synthetic company, and introduced into the 5' and 3' ends respectively. Bam HI and Sal I. Restriction endonuclease cleavage site, referring to step 2, via... Bam HI and Sal The nucleotide sequence of the candidate enhancer region was cloned into the recombinant vector pGL3-Pro-En obtained in step 2 by the I restriction site. MTSS1 In ), the recombinant vector pGL3-Pro-En was obtained. MTSS1 ).

[0089] 3. Dual-luciferase assay

[0090] (1) To detect candidate enhancer pairs MTSS1 To assess gene expression regulation activity, porcine embryonic fibroblasts from step 1 were seeded into 24-well plates, with 1 × 10⁶ cells seeded per well. 5 One cell was cultured for one day for subsequent transfection.

[0091] (2) The next day, co-transfection was performed using Lipofectamine 3000 (Invitrogen) according to the instructions. 900 ng of pGL3-basic and pGL3-Pro were added to each well. MTSS1 ) or pGL3-Pro-En ( MTSS1 ), and 100 ng pRLTK as an internal control, the untransfected group as a negative control, and the empty vector pGL3-basic as a control group ( Figure 3 ).

[0092] (3) Forty-eight hours after transfection, luciferase activity was detected using a dual-luciferase reporter system (Promega, E1910) and a multi-functional microplate reader (Biotek, USA). The final relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renida luciferase activity. All data were obtained from three biological replicates, and statistical analysis was performed using a two-tailed t-test for significance testing.

[0093] The results of the dual-luciferase assay are as follows: Figure 3 As shown, compared to the pGL3-basic empty carrier, pGL3-Pro ( MTSS1 It can significantly increase luciferase activity. p =0.00245), while after the addition of the enhancer sequence, pGL3-Pro-En( MTSS1 The luciferase activity in the group was further significantly increased. p =0.00341 andp =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.

[0094] Example 3: Based on 3C-qPCR MTSS1 Enhancer-promoter interaction detection

[0095] 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).

[0096] 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. MTSS1 site, using Eco RI Hind 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.

[0097] 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.

[0098] 4. qPCR detection

[0099] (1) Primer design

[0100] qPCR primers were designed using the Primer3 online tool (https: / / primer3.ut.ee / ). The primers targeted predefined promoter and enhancer regions; specific sequences are shown in Table 1, and specific target locations are detailed in [Table 1]. Figure 4 .

[0101] Table 1 qPCR primer information

[0102]

[0103] (2) qPCR amplification

[0104] Using the DNA digested in step (2) or the 3C library obtained in step (3) as templates, qPCR was performed using the SYBR Green I fluorescent dye system. Two sets of controls were set up for qPCR: a positive control and a negative control. The effectiveness of the system was verified by using GAPDH promoter primers. The DNA digested in step (2) or the 3C library obtained in step (3) was amplified using primers 77R1-F, 77R1-R, 93R1-R, 93R2-F and 93R2-R. The DNA digested in step (2) was not ligated with T4 DNA ligase and was used as a background noise control (Table 2). The reaction system and procedure for qPCR are shown in Tables 3 and 4.

[0105] Table 2 qPCR grouping

[0106]

[0107] Table 3 qPCR reaction system

[0108]

[0109] Table 4 qPCR reaction procedure

[0110]

[0111] (3) Data Analysis

[0112] The Ct values ​​of all qPCR reactions were normalized to the IgG group signal. The qPCR signals of each primer pair were normalized to the IgG magnetic bead control group. All data are the average of three biological replicates.

[0113] The results are as follows Figure 5 No obvious amplification signal was detected in experimental group 1 without T4 DNA ligase, while the relative signal intensity of experimental group 2 (enzyme digestion + T4 DNA ligase) was significantly higher than that of experimental group 1, indicating that there is indeed physical contact between the two fragments in three-dimensional space, thus verifying the existence of their chromatin long-range interaction relationship.

[0114] Example 4: CRISPR / Cas9 Construction and Gene Expression Validation Method Targeting Enhancer Sequences

[0115] 1. sgRNA design

[0116] Four specific sgRNAs (Table 5) were designed and screened using the CRISPOR online tool (http: / / crispor.tefor.net) targeting the enhancer (SEQ ID NO:1). The sgRNA selection criterion was an MIT specificity score greater than 95 to minimize off-target effects. Primer pairs were designed and synthesized for each sgRNA in the format of "5'-caccg+sgRNA sequence" for the forward primer and "5'-aaac+sgRNA reverse complementary sequence+c" for the reverse primer.

[0117] Table 5 Target recognition sequences of sgRNA

[0118]

[0119] 2. Carrier Construction

[0120] This invention employs Golden Gate cloning and assembly technology to clone sgRNA targeting enhancer sequences into... Bbs The linearized pSpCas9(BB)-2A-Puro vector (PX459 V2.0, addgene, #62988) was used. The specific steps are as follows:

[0121] (1) Linearization of PX459 V2.0 plasmid vector: using Bbs I linearized PX459 V2.0 by enzyme digestion. The linearization reaction system was: 1 μg of PX459 V2.0 plasmid vector, 3 μL of 10×Cutsmart buffer. Bbs I 1 μL, ddH2O added to bring the total to 30 μL; linearization reaction conditions: 37℃ for 2 h, 85℃ for 15 min, and storage at 4℃. Subsequently, the digested fragments were separated by 2% agarose gel electrophoresis, and the target band was recovered by gel extraction and purified using a kit.

[0122] (2) Annealing of sgRNA: Prepare an annealing system, specifically: 2 μL of 10 μM sgRNA forward primer, 2 μL of 10 μM sgRNA reverse primer, 2.5 μL of 10×T4 DNA ligase buffer, and 18.5 μL of ddH2O. Place the prepared annealing system in boiling water and react for 2 min. After natural cooling, add 75 μL of double-distilled water to a final concentration of 2 μM to obtain the sgRNA annealing product.

[0123] (3) Ligation: The annealed product of sgRNA was ligated with the PX459 V2.0 linearized plasmid vector using T4 ligase. The reaction system was as follows: 30 ng of PX459 V2.0 linearized plasmid, 3 μL of annealed product of sgRNA, 0.5 μL of T4 DNA ligase, 1 μL of 10×T4 DNA ligase buffer, and ddH2O to a final volume of 10 μL. The prepared system was placed in a PCR instrument at 16℃ for 1 h, 85℃ for 20 min, and stored at 4℃.

[0124] 3. Transfection, screening, and detection

[0125] (1) Porcine primary fibroblasts were cultured according to the method described in Example 2 and cultured to 70-80% confluence before electroporation.

[0126] (2) After digesting the cells from step (1) with 0.25% trypsin-EDTA (Gibco, USA), centrifuge at 200×g for 5 min, resuspend in 100 µL Transter-E buffer, and add a total of 10 µg of sgRNA plasmid mixture (5 µg of each sgRNA plasmid); electrotransfection was performed in Lonza Nucleocuvette™ electrotransfer tubes using a double-pulse program (FF113+CA137); after electrotransfection, the cells were transferred to 60 mm culture dishes containing 5 mL of preheated complete medium for culture.

[0127] (3) After electroporation for 48 h, the cells were replaced with a complete culture medium containing 2 µg / mL puromycin for screening. The screening was continued for 2 days and the screened cells were collected.

[0128] (4) Extract the genomic DNA from the cells screened in step (3), perform PCR amplification on the target region, and verify whether the enhancer was successfully knocked out by gel electrophoresis. The PCR primers are shown in Table 6. The PCR reaction system is as follows: 15.0 μL of 2×Taq PCRMaster Mix, 1.0 μL of genomic DNA (~20 ng), 2.0 μL of upstream primer (concentration 5 pmol / μL), 2.0 μL of downstream primer (concentration 5 pmol / μL), and 10.0 μL of ddH2O. The PCR reaction program is 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, for a total of 35 cycles; 72°C final extension for 5 min.

[0129] (5) Perform qRT-PCR experiments to detect the effect of enhancer knockout on MTSS1 The effect on gene expression levels was investigated. The qRT-PCR primers are shown in Table 7. The qRT-PCR reaction system (20 μL) consisted of: 10 μL of 2×ChamO Universal SYBR qPCR MasterMix, 1 μL of cDNA, 0.4 μL of upstream primer, 0.4 μL of downstream primer, and 8.2 μL of RNase-free ddH2O. The PCR reaction program was: 95°C pre-denaturation for 10 min; 95°C denaturation for 10 s, 60°C annealing for 30 s, and 72°C extension for 30 s, for a total of 40 cycles.

[0130] Table 6 PCR Primers

[0131]

[0132] Table 7 qRT-PCR primers

[0133]

[0134] Because enhancer nucleotide sequences are relatively long, effective knockout of enhancers using a single sgRNA is difficult. Therefore, this invention selects different sgRNA combinations to knock out enhancer regions, with three replicates for each combination. PCR results of knockout of the target fragment using different sgRNA combinations are shown below. Figure 6 and Figure 7 .from Figure 6 As can be seen, sgRNA1+sgRNA3, sgRNA1+sgRNA4, and sgRNA2+sgRNA3 all failed to effectively knock out the enhancer region, while the combined knockout of sgRNA1+sgRNA2 resulted in... MTSS1 -F1 and MTSS1 - The fragment length obtained by R1 amplification matches the expected length of the remaining fragment after knockout and there is specific amplification. Figure 7 This indicates that the combination of sgRNA1 and sgRNA2 effectively knocked out the enhancer region.

[0135] Further analysis of the sgRNA1+sgRNA2 combined knockout enhancer region MTSS1 The expression level, the results are shown in Figure 8 .from Figure 8 As can be seen, compared with the non-knockout control group, the CRISPR / Cas9 knockout mediated by the sgRNA1+sgRNA2 combination significantly reduced MTSS1 The gene expression level (approximately 70%) further validates that this enhancer has a regulatory function. MTSS1 The function of gene expression.

[0136] In summary, this invention combines CUT&Tag, dual-luciferase reporter system, 3C-qPCR, and CRISPR / Cas9 gene editing technologies to study and confirm the effects of pig gene editing. MTSS1 Distal enhancer pairs MTSS1 Spatial regulatory functions of genes and their promoters. MTSS1 The gene is a key regulator of the porcine cytoskeleton, highly expressed in tissues such as skeletal muscle, and closely linked to traits related to muscle development and fat deposition. The porcine gene provided by this invention... MTSS1 Distal enhancers can be applied to the genetic breeding of traits such as muscle development and fat deposition in pigs, as well as to the prevention and control of diseases in pigs. MTSS1 In addition to gene-related diseases, the sgRNA combination and the constructed pig sgRNA provided by this invention... MTSS1 The cell model regulated by distal enhancers can be further used to construct corresponding animal models, which can then be used for research. MTSS1 It provides ideas and directions for gene function, molecular breeding of livestock such as pigs, and drug screening.

[0137] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

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

1.

2. 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'.

3. A method containing targeted pigs MTSS1 Recombinant vectors of sgRNAs of distal enhancers of genes, characterized by The targeted pig as described in claim 2 MTSS1 The sgRNA of the distal enhancer of the gene was obtained by ligating it to the vector.

4. The method of claim 3 comprising a target pig 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.

5. A gene editing tool, characterized in that... Includes the targeted pig of claim 2 MTSS1 The sgRNA of the distal enhancer of the gene or the recombinant vector as described in claim 3.

6. The targeted pig as described in claim 2 MTSS1 The sgRNA of the distal enhancer of the gene, the recombinant vector of claim 3, or the gene editing tool of claim 5 in constructing pigs MTSS1 Distal enhancer knockout or MTSS1 Application in gene suppression models.

7. A type of pig MTSS1 Distal enhancer knockout or MTSS1 Gene-suppressed cell model, characterized by: The target pig as described in claim 3 MTSS1 The sgRNA of the distal enhancer of the gene was obtained by transfecting a recombinant vector into porcine cells; The cells mentioned are porcine fibroblasts.

8. The targeted pig as described in claim 2 MTSS1 The application of sgRNA of gene distal enhancer, the recombinant vector of claim 3, or the gene editing tool of claim 5 in pig genetic breeding.

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