A system, preparation method and application for MSTN / IGF2 dual gene editing

Abstract

This application relates to a system, preparation method, and application for MSTN / IGF2 dual gene editing. By designing and screening highly efficient spacer sequences targeting the MSTN and IGF2 genes, dual gene editing was performed using the Cas12f system, successfully obtaining a cell line with MSTN and IGF2 dual gene editing. Further, cloned embryos were prepared and transplanted using somatic cell nuclear transfer technology, ultimately yielding phenotypically significant dual-gene-edited calves. Experimental results show that the edited calves achieved a birth weight of 55 kg, significantly higher than the wild type (31 kg), demonstrating that this dual-gene editing strategy has a significant effect on promoting the growth and weight of beef cattle, providing new genetic materials and technical pathways for beef cattle breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic breeding, specifically relating to a system, preparation method and application for MSTN / IGF2 dual gene editing. Background Technology

[0002] In CRISPR gene editing systems, the spacer is a 20-base sequence that guides the front end of the sgRNA. Through complementary base pairing, it determines the CRISPR system's ability to precisely identify and cleave genomic target sites, acting as the "navigation head" of the entire editing process. Theoretically, a corresponding spacer sequence can be designed for any gene. However, the editing efficiency of a spacer is highly dependent on its own sequence characteristics (such as GC content and secondary structure) and its compatibility with specific Cas proteins (such as Cas12f). Existing universal spacer design rules have limited predictive accuracy across different species and gene loci, resulting in many designed spacers exhibiting low editing efficiency or instability in practical applications, making them difficult to directly use for efficient genetic improvement.

[0003] In multi-gene collaborative breeding of important economic animals like beef cattle, more stringent requirements are placed on the design and screening of spacers. Taking the creation of new beef cattle breeds possessing both "double-muscular rump" and "rapid growth" traits as an example, it is essential to obtain highly efficient spacers targeting both the MSTN and IGF2 genes. This requires not only that each spacer possesses high individual activity but also that they work synergistically without interfering with each other during co-transfection. Currently, there is a severe lack of experimentally validated, specific spacer sequences capable of efficiently editing bovine MSTN and IGF2 genes, especially spacers compatible with the compact Cas12f system. This missing key component has become a major obstacle to the rapid creation of new beef cattle breeding materials using gene editing technology.

[0004] Therefore, there is an urgent need in this field to provide a set of specific and highly efficient spacer sequences for bovine MSTN and IGF2 genes that have undergone rigorous functional validation to fill the aforementioned gap. Through independent design and systematic screening, multiple highly efficient spacers have been provided, and their editing efficiencies of up to 50% and 38.14% have been confirmed by T7E1 restriction enzyme digestion and TA cloning sequencing. The acquisition of these specific spacer sequences provides an indispensable key tool and material basis for the successful construction of dual-gene-edited cell lines and the eventual birth of phenotypically significant cloned beef cattle. Summary of the Invention

[0005] The purpose of this invention is to provide a system, preparation method and application for MSTN / IGF2 dual gene editing. By synergistically editing the above genes and combining somatic cell nuclear transfer technology, a new beef cattle breeding material with significant double-muscular hip and rapid growth phenotype has been successfully created, so as to solve the technical bottlenecks of long breeding cycle, low efficiency and limited improvement of traits by single gene editing in traditional breeding.

[0006] On the one hand, the present invention provides a system for MSTN / IGF2 dual gene editing, which adopts the following technical solution: A system for MSTN / IGF2 dual gene editing, comprising MSTN-sgRNA1, MSTN-sgRNA2, and IGF2-sgRNA; MSTN-sgRNA1 includes TAM1 and MSTN-spacer1; MSTN-sgRNA2 includes TAM2 and MSTN-spacer2; IGF2-sgRNA includes TAM3 and IGF2-spacer.

[0007] Preferably, the nucleotide sequence of the target site recognized by MSTN-sgRNA1 is SEQ ID NO.4; The nucleotide sequence of the target site recognized by MSTN-sgRNA2 is SEQ ID NO.5; The nucleotide sequence of the target site recognized by IGF2-sgRNA is SEQ ID NO.6.

[0008] Preferably, the nucleotide sequence of MSTN-spacer1 is SEQ ID NO.1; The nucleotide sequence of MSTN-spacer2 is SEQ ID NO.2; The nucleotide sequence of IGF2-spacer is SEQ ID NO.3.

[0009] Preferably, the nucleotide sequence of TAM1 is ATTC; The nucleotide sequence of TAM2 is CTTA; The nucleotide sequence of TAM3 is CTTC.

[0010] On the other hand, the present invention also provides a method for preparing a system for MSTN / IGF2 dual gene editing, comprising the following steps: S1. Obtain information on MSTN and IGF2 genes in domestic cattle from the database; S2. Target prediction for key regulatory regions of MSTN and IGF2 genes; S3. Design and synthesize sgRNA sequences targeting specific sites.

[0011] Preferably, the MSTN gene has the sequence number NC 037329 and a sequence length of 6628 bp; the IGF2 gene has the sequence number NC 037356 and a sequence length of 27320 bp. Both the MSTN and IGF2 genes are derived from West China cattle.

[0012] Preferably, the principle for target prediction in step S2 is as follows: The target sequence is 24 bp, and its 5' end must contain the TAM sequence required for recognition by the Cas12f system; Prioritize target sites located at natural mutation sites in the gene coding region that are common across all transcripts; Sequences highly homologous to other regions of the bovine genome were excluded.

[0013] This invention also provides an application of a system for MSTN / IGF2 dual gene editing, which can be used to prepare MSTN / IGF2 dual gene edited cell lines from West China cattle.

[0014] Preferably, the cell line can be used for the breeding of West China cattle.

[0015] In summary, the beneficial effects of the present invention are as follows: This invention provides a system for MSTN / IGF2 dual gene editing, its preparation method, and its applications, successfully achieving efficient and specific editing of bovine MSTN and IGF2 genes. Verification via T7E1 and TA cloning showed that the sgRNA editing efficiency for the MSTN gene reached up to 50%, and the sgRNA editing efficiency for the IGF2 gene reached 38.14%, significantly ensuring the success rate of dual-gene synergistic editing. Based on this, this invention successfully obtained multiple positive cell lines for MSTN / IGF2 dual gene editing, laying a reliable cellular material foundation for the subsequent mass production of gene-edited beef cattle using somatic cell nuclear transfer technology.

[0016] At the individual level, the dual-gene editing effect of this invention exhibits extremely significant phenotypic improvement. Live calves born after embryo transfer achieved a birth weight of 55 kg, approximately 1.8 times that of the control wild-type cattle (31 kg). This result fully demonstrates the significant synergistic effect between MSTN gene editing (increasing muscle mass) and IGF2 gene editing (promoting growth and development), successfully creating a new beef cattle breeding material with excellent growth rate and meat production potential, providing strong technical support for breaking through traditional breeding bottlenecks and achieving efficient and precise breeding. Attached Figure Description

[0017] Figure 1The diagram shows the recognition sites for the three types of spacers; Figure 2 Electrophoresis image for screening efficient sgRNAs for T7E1 binding to TA clones; Figure 3 Sequence diagram for screening efficient sgRNAs by T7E1 binding to TA clones; Figure 4 Genotyping statistics for homozygous edited monoclonal cell lines; Figure 5 Genotyping of homozygous edited cloned cattle using two genes; Figure 6 Cloned cattle that have not undergone gene editing; Figure 7 For cloned cattle that have undergone MSTN / IGF2 double gene editing. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0019] Table 1. Instruments used in the experiment

[0020] Table 2. Reagents used in the experiment

[0021] Example Example 1 S1. Retrieve domestic cattle data from the NCBI database. Bos taurus The complete genomic DNA sequence and exon structure information of the MSTN gene (Accession: NC 037329) and IGF2 gene (Accession: NC037356) in domestic cattle.

[0022] S2. Benchling software was used to predict crRNA targets in the early functional domain (Exon2) of the MSTN gene and the key regulatory region of the IGF2 gene.

[0023] The screening criteria include: ① The target sequence is 24 bp, and its 5' end must contain the TAM sequence required for recognition by the Cas12f system.

[0024] ② Prioritize target sites located at natural mutation sites in the gene coding region that are common across all transcripts to ensure that editing can effectively disrupt gene function.

[0025] ③ Assess the potential off-target effects of the target and exclude sequences that are highly homologous to other regions of the bovine genome.

[0026] S3. Design and synthesize multiple candidate sgRNA oligonucleotide chains with restriction enzyme sites for each gene. Using the Tiangen Genomic DNA Extraction Kit, add 5 μL of 10× buffer to a clean 0.2 mL PCR tube, then add 1 μg of Cas12f plasmid and 1 μL of BsaI enzyme, and add ddH2O to make up to 50 μL. Mix thoroughly. Perform the restriction enzyme reaction at 37℃ for 4 h, and perform column recovery according to the kit instructions. Dissolve the primers with an appropriate amount of ddH2O according to the primer synthesis report to a final concentration of 100 μM. Add 5 μL of 10× PCR Buffer, 22.5 μL each of sgRNA upstream and downstream to the PCR tube, mix thoroughly, and anneal at 99℃ for 10 min to form double strands. Add 5 μL of Solution I, 1 μL of the BsaI-digested Cas12f vector backbone, and 4 μL of sgRNA to the PCR tube, mix well, and ligate at 16℃ for 4 h. Thaw competent cells on ice. Add 33 μL of the target DNA fragment, mix gently, and incubate on crushed ice for 30 min. Then heat shock in a 42°C water bath for 45 s. Immediately transfer to crushed ice, incubate for 2 min, and plate. Pick single colonies, streak, and send to the company for sequencing. Extract plasmids according to the kit instructions.

[0027] S4. Observe the morphology and confluence of bovine fibroblasts in 6-well plates. When the cell confluence reaches about 80%, remove the culture medium, wash with PBS buffer, add 1 mL of 0.25% trypsin, digest at 37℃ for 2 min, add 2 mL of DMEM containing 10% FBS to stop digestion, pipette and transfer to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 3 min to remove as much culture medium as possible, add 100 μL of electroporation buffer to resuspend the cells, add to the bottom of the electroporation cuvette, gently tap the bottom of the cuvette to make it uniform, set the electroporation program to CZ-167 and perform electroporation; use the matching pipette to aspirate all the liquid in the electroporation cuvette and add it to one well of a 6-well plate containing 2 mL of MSCs, shake well. Fibroblasts were electroporated with Cas12f-sgRNA. The electroporation mortality rate and electroporation efficiency were observed under a fluorescence microscope 24 h after electroporation. If the electroporation mortality rate was less than 50% and the electroporation efficiency was greater than 30%, it could be used for the next step of the experiment. 48 h after electroporation, the culture medium was removed, 1 mL of PBS was added, the PBS was removed, 1 mL of 0.25% trypsin was added, and the mixture was incubated at 37°C for 2 min. 2 mL of fibroblast culture medium was added to stop the digestion. The mixture was centrifuged at 1200 rpm for 3 min, the supernatant was removed, and the cell pellet was resuspended in 300 μL of DMEM resuspension medium containing 1% FBS.

[0028] S5. Lyse cells in a 40 μL system. Add 1 μL template product, 1 μL outer upstream primer, 1 μL outer downstream primer, 12.5 μL KOD enzyme, and 9.5 μL ddH2O to a 200 μL microcentrifuge tube for the first round of nested PCR. Add 2 μL of the first round PCR product, 2 μL outer upstream primer, 2 μL outer downstream primer, 25 μL KOD enzyme, and 19 μL ddH2O to a 200 μL microcentrifuge tube for the second round of nested PCR. Digest with T7E1 enzyme in a 20 μL system. Use the nested PCR second round product as the control group and the T7E1 digested product as the experimental group for gel electrophoresis. The agarose gel concentration is 2.5%, and electrophoresis is performed at 120V for 30 min. Observe the electrophoresis pattern and take pictures.

[0029] S6. Clone the above PCR product into the T vector, and randomly select multiple single clones for Sanger sequencing. By comparing the sequencing results with the wild-type sequence, accurately calculate the insertion / deletion mutation ratio to obtain the most accurate editing efficiency. The calculation formula is: Editing efficiency = (number of clones with mutations / total number of sequenced clones) × 100%.

[0030] Table 3. Three sgRNAs and their corresponding spacer sequences

[0031] Reference Figure 1 The identification site diagram clearly marks the complementary pairing regions of each spacer with the target DNA sequence and their specific locations in the gene structure, providing an intuitive visual basis for understanding the targeting role of the spacers in CRISPR / Cas12f system-mediated dual gene editing.

[0032] Reference Figure 2-3 The results of agarose gel electrophoresis (AGE1) and T7E1 clearly show significant cleavage effects of sgRNA1, sgRNA2, and IGF2-sgRNA1. Highly efficient sgRNAs were screened and verified using TA cloning and sequencing.

[0033] This experiment successfully screened highly efficient sgRNAs targeting the bovine MSTN and IGF2 genes. TA cloning and sequencing confirmed the presence of an MSTN gene sgRNA with editing efficiencies of approximately 50% and 23%, respectively, and an IGF2 gene sgRNA with an editing efficiency of 38.14%. These functionally validated sgRNAs will be directly used for the subsequent construction of dual-gene editing cell lines.

[0034] Example 2 S1. Using the electroporation-mediated DNA obtained in step S5 of Example 1, bovine fibroblasts were transfected. The resuspended cells were then sorted by flow cytometry, with a positive rate of 30%–40%. Single cells expressing green fluorescence were collected and injected into individual wells of a 96-well plate. The cells were continuously cultured in an incubator, observed periodically, and wells confirmed to be of monoclonal origin were labeled. Once the monoclonal cell population had grown to a sufficient number, it was sequentially expanded to 24-well plates, 6-well plates, and culture dishes.

[0035] S2. High-quality genomic DNA was extracted from each monoclonal cell line. Specific primers were designed to amplify the target regions of the MSTN and IGF2 genes by PCR. The PCR products were rapidly screened using the T7E1 restriction enzyme method to preliminarily identify the cell lines with edited genes. The PCR products of the preliminarily screened positive cell lines were subjected to TA cloning and sequencing. By analyzing the sequences of multiple sequenced clones, their genotypes (such as homozygous mutations, heterozygous mutations, and biallelic mutations) were accurately determined, and finally, dual-gene positive cell lines with ideal editing of both MSTN and IGF2 were screened.

[0036] This experimental protocol aims to successfully obtain at least three MSTN / IGF2 double-gene-edited bovine fetal fibroblast cell lines with clear genetic backgrounds, well-defined genotypes, and optimal cell condition. These cell lines will serve as key new breeding materials, directly used in subsequent somatic cell nuclear transfer experiments to produce gene-edited calves with a prominent double-muscular rump and rapid growth phenotype.

[0037] Reference Figure 4 This study presents the statistical results of genotyping and amino acid changes in MSTN / IGF2 dual-gene-edited cell lines obtained after flow cytometry sorting and single-clone culture. The results, obtained through TA cloning and sequencing analysis, confirmed the successful acquisition of homozygous cell lines with efficient editing at both the MSTN and IGF2 gene loci.

[0038] Example 3 S1. The MSTN / IGF2 double gene-edited cell line, which was frozen in liquid nitrogen and confirmed by genotype identification, was used as the experimental group, and the homologous wild-type cell line was used as the control group. The cells were revived and cultured to a good state of confluence of 70%-80% for nuclear transfer.

[0039] S2. Collect bovine ovaries from the slaughterhouse and extract the cumulus-oocyte complex. After in vitro maturation culture, select mature oocytes that have extruded the first polar body. Using micromanipulation techniques, remove the nucleus (first polar body and the metaphase chromosomes below) from the oocytes using a capillary glass tube to form cytoplasmic bodies.

[0040] S3. Using a micromanipulator, inject a complete donor cell (from the experimental or control group) into the periovarian space of the enucleated cytoplasm. Place the constructed oocyte-donor cell complex in a fusion solution, and induce cell fusion (allowing the donor cell nucleus to enter the cytoplasm) through electrical stimulation, simultaneously activating the reconstructed embryo and initiating embryonic development.

[0041] S4. The successfully activated reconstructed embryos were cultured in vitro, and their development was continuously monitored. After reaching the blastocyst stage, the blastocyst development rate of the two groups of embryos was counted and recorded to assess whether gene editing affected the developmental potential of early embryos.

[0042] S5. Select healthy recipient cows with synchronized physiological cycles. Cloned embryos developed to the blastocyst stage (experimental and control groups) are transferred to the uterine horns of the recipient cows using a non-surgical method. The pregnancy status of the recipient cows is monitored regularly by ultrasound, and pregnancy and abortion rates are recorded.

[0043] S6. After the calves are born, ear tissue or blood samples are collected, DNA is extracted, and the MSTN and IGF2 genotypes are verified again by PCR and sequencing to confirm that they are cloned offspring of the donor cells and that the editing sites are correct. The birth weight of all newborn calves is accurately measured and recorded, and body dimensions such as height, body length, and chest circumference are measured.

[0044] Reference Figure 5 Genome sequencing using PCR identified the cloned cattle as homozygous double-gene edited clones. Statistical analysis of their body size revealed that the cloned cattle were larger than wild-type cattle in all tests. This indicates that the double-gene-edited cloned cattle possess more pronounced productive traits.

[0045] Reference Figure 6-7 The experimental group will produce cloned calves with a significantly higher birth weight than the control group. Based on preliminary data, the expected birth weight of the experimental group is approximately 55 kg, while the control group (wild-type cloned cattle) has a birth weight of approximately 31 kg. The experimental group calves should exhibit stronger muscle deposition and faster weight gain in subsequent growth. The establishment of the control group (wild-type cloned cattle) effectively eliminates potential variations caused by the cloning technology itself, confirming that the significant phenotype of a 55 kg birth weight is entirely due to MSTN / IGF2 dual gene editing.

[0046] Table 4. Body size data of homozygous edited cloned cattle and control group.

[0047] The success of this experiment will demonstrate that by utilizing the screened, highly efficient sgRNA-binding somatic cell nuclear transfer technology, it is possible to efficiently and reproducibly create new beef cattle germplasm with groundbreaking economic traits.

[0048] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A system for MSTN / IGF2 dual gene editing, characterized in that, The system includes Cas12f plasmid, MSTN-sgRNA1, MSTN-sgRNA2, and IGF2-sgRNA; The MSTN-sgRNA1 includes TAM1 and MSTN-spacer1; The MSTN-sgRNA2 includes TAM2 and MSTN-spacer2; The IGF2-sgRNA includes TAM3 and IGF2-spacer; The nucleotide sequence of the MSTN-spacer1 is SEQ ID NO.1; The nucleotide sequence of the MSTN-spacer2 is SEQ ID NO.2; The nucleotide sequence of the IGF2-spacer is SEQ ID NO.

3.

2. The system for MSTN / IGF2 dual gene editing according to claim 1, characterized in that: The nucleotide sequence of the target site recognized by MSTN-sgRNA1 is SEQ ID NO.4; The nucleotide sequence of the target site recognized by MSTN-sgRNA2 is SEQ ID NO.5; The nucleotide sequence of the target site recognized by the IGF2-sgRNA is SEQ ID NO.

6.

3. The system for MSTN / IGF2 dual gene editing according to claim 1, characterized in that: The nucleotide sequence of TAM1 is ATTC; The nucleotide sequence of TAM2 is CTTA; The nucleotide sequence of TAM3 is CTTC.

4. A method for preparing a system for MSTN / IGF2 dual gene editing as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Obtain information on the MSTN and IGF2 genes of domestic cattle from the database; S2. Target prediction for key regulatory regions of MSTN and IGF2 genes; S3. Design and synthesize sgRNA sequences targeting specific sites.

5. A method for preparing a system for MSTN / IGF2 dual gene editing according to claim 4, characterized in that, The MSTN gene has the sequence number NC 037329 and a sequence length of 6628 bp; the IGF2 gene has the sequence number NC037356 and a sequence length of 27320 bp. Both the MSTN gene and the IGF2 gene were derived from West China cattle.

6. A method for preparing a system for MSTN / IGF2 dual gene editing according to claim 4, characterized in that, The principle for target prediction in step S2 is as follows: The target sequence is 24 bp, and its 5' end must contain the TAM sequence required for recognition by the Cas12f system; Select target sites located at natural mutation sites in the gene coding region that are common to all transcripts; Sequences highly homologous to other regions of the bovine genome were excluded.

7. The application of the system for MSTN / IGF2 dual gene editing as described in any one of claims 1-3, characterized in that, The system can be used to prepare MSTN / IGF2 dual-gene edited cell lines from Huaxi cattle.

8. The application of the system for MSTN / IGF2 dual gene editing according to claim 7, characterized in that, The cell line can be used for breeding and propagation of West China cattle.

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