Myostatin (MSTN) protein mutant, and preparation method therefor and use thereof

Abstract

The present invention relates to the fields of genetic engineering and animal husbandry genetic breeding. Disclosed are a myostatin (MSTN) protein mutant, and a preparation method therefor and a use thereof. The amino acid sequence of the mutant is any one of sequences set forth in SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 20. According to the present invention, under the guidance of a specially designed sgRNA, by means of adenine base editing, a mutant where cysteine at position 281 and / or 282 of a sheep MSTN full-length protein containing a signal peptide is mutated into arginine is obtained. The nucleotide sequence of the sgRNA is as set forth in SEQ ID NO: 5. A sheep carrying the MSTN mutant of the present invention has a significantly increased myofiber cross-sectional area and exhibits a remarkable double-muscling phenotype, thus leading to an increased meat yield. The present invention provides technical support for efficient preparation and variety breeding of the double-muscling phenotype sheep.

Description

A myostatin MSTN protein mutant and its preparation method and application Technical Field

[0001] The present invention relates to the fields of genetic engineering and animal husbandry genetic breeding, and in particular to a myostatin (MSTN) protein mutant and a preparation method and application thereof. Background Art

[0002] Myostatin (MSTN), a member of the transforming growth factor-β (TGF-β) superfamily, is primarily expressed in skeletal muscle and acts as a negative regulator of muscle growth and development. Overexpression of the mouse MSTN gene inhibits skeletal muscle proliferation and differentiation. Knockout of the MSTN gene results in single muscle cells growing to approximately twice their normal size, with a significant increase in the number of muscle fibers, resulting in a double-muscle phenomenon, and a significant increase in body weight. The ovine MSTN gene consists of three exons and two introns, with a CDS region of 1128 nucleotides, which translates into a protein of 375 amino acid residues. The MSTN protein has a typical TGF-β superfamily structure, consisting of a 24-amino acid signal peptide, an N-terminal pro-peptide, and a C-terminal mature peptide containing nine Cys residues. The maturation of the MSTN protein involves three enzymatic cleavage steps: signal peptide excision, cleavage by the serine kinase Furin, and cleavage by the BMP-1 / TLD metalloproteinase. The MSTN protein signal peptide is hydrolyzed to form pro-MSTN, which then forms a homodimer through intermolecular disulfide bonds. The serine kinase furin recognizes and cleaves the arginine-serine-arginine-arginine site (amino acid residues 263-266), forming a 27.7 kDa precursor peptide and a 12.5 kDa mature peptide, which are released into the extracellular space and exert their effects through endocrine or paracrine pathways. Most of the extracellular MSTN is released as latent-MSTN, in which the propeptide and mature peptide are non-covalently bound together. After latent-MSTN is transported to the target site, the BMP-1 / TLD metalloprotease cleaves the MSTN precursor (amino acid residue 98), releasing the active mature MSTN peptide dimer. The mature MSTN peptide contains nine Cys residues and is formed through four intramolecular disulfide bonds and one intermolecular disulfide bond, forming the high-order structure of active MSTN.

[0003] MSTN is highly conserved across mammals. Nucleotide mutations or deletions can inactivate or reduce MSTN protein activity, leading to blockage of MSTN downstream signaling pathways, activation, proliferation, and differentiation of skeletal muscle satellite cells, and increased myofibril protein synthesis, ultimately manifesting as muscle fiber hypertrophy or hyperplasia. Deletion mutations in the MSTN gene coding region of Belgian Blue cattle cause premature translation termination, resulting in a truncated MSTN protein and a double-muscled buttocks phenotype. Texel sheep harboring the SNP c.*1232G>A mutation in the MSTN 3'-UTR show no change in MSTN mRNA levels, but blood MSTN protein levels decrease by 66%, indicating a pronounced double-muscled phenotype. The Whippet dog, a greyhound breed, harbors the MSTN c.939(del2) mutation and exhibits a typical double-muscled phenotype.

[0004] In light of this, researchers have attempted to create livestock with a double-muscled phenotype by knocking out the MSTN gene, thereby providing germplasm resources for livestock breeding. Using CRISPR / Cas9 gene editing technology, mutations can be generated in the gene coding region, resulting in amino acid additions, deletions, or substitutions, thereby achieving gene function knockout or reduction. For example, Guo R et al. used CRISPR / Cas9 to edit the MSTN gene in Haimen goats, generating goat individuals with a double-muscled phenotype; Wang X et al. used CRISPR / Cas9 to edit the MSTN gene in Tan sheep, also generating sheep individuals with a double-muscled phenotype; and Lian Zhengxing et al.'s patent (CN 111793123 A) used CRISPR / Cas9 to generate a mutant of the sheep myostatin MSTN, in which the cysteine ​​at position 339 of the full-length MSTN protein containing the signal peptide is deleted. Animals carrying this mutation exhibit a pronounced double-muscled hip trait. In the above examples, sheep with double-muscle phenotypes were created using CRISPR / Cas9 to edit the Indels produced by MSTN. Due to the high randomness of the Indel types produced by CRISPR / Cas9 editing and the complex genotype, the Indel types used to achieve the purpose of knocking out or reducing gene function are only a part or even a small part of them. Therefore, although the editing efficiency of CRISPR / Cas9 has greatly improved compared with previous editing methods, the efficiency of gene function knockout or knockdown is still uncertain. Considering the high cost and long cycle of large animals, gene-edited animal varieties need to obtain the homozygous genotype of the edited gene. It is still very necessary to use more precise gene editing methods to efficiently create mutants with double-muscle phenotypes. Summary of the Invention

[0005] The purpose of the present invention is to provide a myostatin MSTN protein mutant and its preparation method and application to solve the problems existing in the above-mentioned prior art. Under the guidance of a specially designed sgRNA, the present invention uses ABE editing to mutate the T in positions 841, 844, and 846 of the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide to C, that is, to form a mutant in which the cysteine ​​at positions 281 and / or 282 of the MSTN full-length protein (NP_001009428.1) containing a signal peptide is mutated to arginine, destroying the disulfide bond within the MSTN molecule, making it difficult to form an active MSTN with a specific higher-order structure, thereby achieving the purpose of inactivating MSTN. Animals carrying this mutant have obvious double-muscled buttocks traits, providing technical support for the efficient preparation and variety breeding of double-muscled buttocks phenotype sheep.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a myostatin MSTN protein mutant, the amino acid sequence of which is any one of the sequences shown in SEQ ID NO.15, SEQ ID NO.17 or SEQ ID NO.20.

[0008] The present invention also provides a gene encoding the above-mentioned MSTN protein mutant.

[0009] Further, when the amino acid sequence of the MSTN protein mutant is SEQ ID NO.15, the nucleotide sequence of the gene is shown as SEQ ID NO.13; when the amino acid sequence of the MSTN protein mutant is SEQ ID NO.17, the nucleotide sequence of the gene is shown as SEQ ID NO.16 or SEQ ID NO.18; when the amino acid sequence of the MSTN protein mutant is SEQ ID NO.20, the nucleotide sequence of the gene is shown as SEQ ID NO.19 or SEQ ID NO.21.

[0010] The present invention also provides a method for preparing a genome containing the above-mentioned biological material, comprising the steps of obtaining the biological material by adenine base editing technology under the guidance of sgRNA;

[0011] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.

[0012] Furthermore, the biological materials include cells, embryos and animals.

[0013] The present invention also provides an sgRNA for editing the MSTN gene, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.

[0014] The present invention also provides an expression vector or expression cassette for expressing the sgRNA.

[0015] The present invention also provides a method for increasing animal muscle content or promoting animal muscle development, comprising using the sgRNA or the expression vector or expression cassette to mutate the MSTN encoding gene in the animal genome into the above-mentioned gene through adenine base editing technology, wherein the animal is a sheep.

[0016] The present invention also provides a method for increasing meat production in animals, comprising using the sgRNA or the expression vector or expression cassette to mutate the MSTN encoding gene in the animal genome into the above-mentioned gene through adenine base editing technology, wherein the animal is a sheep.

[0017] The present invention also provides a genetic breeding method for a double-muscled-hipped animal, comprising mutating the MSTN encoding gene in the genome of the animal into the above-mentioned gene, wherein the animal is a sheep.

[0018] Those skilled in the art should understand that, based on the conservation and homology of MSTN proteins of different animals, the above-mentioned effects of MSTN protein mutants of other animals and their encoding genes having the same function obtained by mutating the cysteine ​​at position 281 of the MSTN protein shown in SEQ ID NO.15 to arginine and / or mutating the cysteine ​​at position 282 of the MSTN protein shown in SEQ ID NO.17 to arginine are also within the scope of protection of the present invention.

[0019] The present invention discloses the following technical effects:

[0020] The present invention obtains a mutant of the sheep MSTN full-length protein containing a signal peptide, in which the cysteine ​​at position 281 and / or 282 is mutated to arginine through adenine base editing (ABE). This mutant can promote the growth and development of sheep muscles. Sheep carrying the MSTN mutant described in the present invention have increased muscle fiber cross-sectional area, exhibiting a significant double-muscled buttocks phenotype, thereby increasing meat production.

[0021] The present invention provides a sgRNA that efficiently targets the MSTN protein-coding gene sequence. Under the guidance of this sgRNA, through the ABE editing technology, a mutant of the sheep MSTN full-length protein containing a signal peptide in which the 281st and / or 282nd cysteine ​​is mutated to arginine can be successfully obtained, with an editing efficiency exceeding 70%. Under the guidance of this sgRNA, using the ABE editing technology, an animal model with a double-muscled buttocks phenotype can be efficiently prepared or genetic breeding of animals with double-muscled buttocks traits can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 is a genotype sequencing analysis of sheep embryos carrying the 281st and / or 282nd mutant coding genes of the MSTN full-length protein prepared by ABE editing; wherein WT is the wild type, MT1 is a mutant in which the 841st T in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide is partially mutated into C (chimeric), forming a mutant in which the 281st cysteine ​​in the MSTN full-length protein (NP_001009428.1) containing a signal peptide is partially mutated into arginine, MT2 is a mutant in which the 844th T in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide is partially mutated into C (chimeric), forming a mutant in which the 282nd cysteine ​​in the MSTN full-length protein (NP_001009428.1) containing a signal peptide is partially mutated into arginine, and MT3 is a mutant in which the 844th T in the coding region of the MSTN full-length gene (Gene ID: The 844th and 846th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) were partially mutated into C (chimeric), forming a mutant in which the 282nd cysteine ​​of the MSTN full-length protein (NP_001009428.1) containing a signal peptide was partially mutated into arginine; MT4 is a mutant in which the 844th and 846th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide were partially mutated into C, forming a mutant in which the 282nd cysteine ​​of the MSTN full-length protein (NP_001009428.1) containing a signal peptide was mutated into arginine; MT5 is a mutant in which the 844th and 846th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide was partially mutated into C, forming a mutant in which the 282nd cysteine ​​of the MSTN full-length protein (NP_001009428.1) containing a signal peptide was partially mutated into arginine; The 841st and 844th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) were mutated to C, forming a mutant in which the 281st and 282nd cysteines of the MSTN full-length protein (NP_001009428.1) containing a signal peptide were mutated to arginine; MT6 is a mutant in which the 841st, 844th and 846th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide were mutated to C, forming a mutant in which the 281st and 282nd cysteines of the MSTN full-length protein (NP_001009428.1) containing a signal peptide were mutated to arginine; MT7 is a mutant in which the 841st, 844th and 846th T positions in the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide were mutated to C, forming a mutant in which the 281st and 282nd cysteines of the MSTN full-length protein (NP_001009428.1) containing a signal peptide were mutated to arginine; ID: 443449) in the coding region, T at position 844 was mutated to C, and T at positions 841 and 846 were partially mutated to C, forming a mutant of the full-length MSTN protein (NP_001009428.1) containing a signal peptide, in which cysteine ​​at position 282 was mutated to arginine, and cysteine ​​at position 281 was partially mutated to arginine;

[0024] Figure 2 shows the genotype sequencing analysis of Hu sheep carrying the gene encoding the mutant at position 281 and / or 282 of the MSTN full-length protein prepared by ABE editing; wherein WT is the wild-type Hu sheep, MT1 is the Hu sheep in which the cysteine ​​at position 282 of the MSTN full-length protein is mutated to arginine; MT2 is the Hu sheep in which the cysteine ​​at position 281 of the MSTN full-length protein is mutated to arginine; MT3 and MT4 are Hu sheep in which the cysteine ​​at positions 281 and 282 of the MSTN full-length protein are mutated to arginine;

[0025] Figure 3 shows the growth phenotype analysis of Hu sheep carrying MSTN protein mutants; the mutant lambs are Hu sheep carrying MSTN protein mutants, and wild-type Hu sheep serve as the control group;

[0026] Figure 4 is a phenotypic comparison of Hu sheep individuals (MT1, MT2 and MT3) carrying MSTN full-length protein mutants and Hu sheep individuals with wild-type MSTN genes (WT); wherein A is a frontal view of Hu sheep individual MT1 carrying the 282nd mutant of MSTN full-length protein and a wild-type Hu sheep individual; B is a side view of Hu sheep individual MT1 carrying the 282nd mutant of MSTN full-length protein and a wild-type Hu sheep individual; C is a side view of Hu sheep individual MT2 carrying the 281st mutant of MSTN full-length protein; D is a side view of Hu sheep individual MT3 carrying the 281st and 282nd mutants of MSTN full-length protein;

[0027] Figure 5 is a comparison of muscle fibers of Hu sheep MT1 carrying the 282nd mutant of MSTN full-length protein, Hu sheep MT2 carrying the 281st mutant of MSTN full-length protein, Hu sheep MT3 carrying the 281st and 282nd mutants of MSTN full-length protein, and Hu sheep individuals with wild-type MSTN gene (WT); among them, A is the comparison of HE staining results of muscle tissue sections (cross-sections), and B is the comparison of muscle fiber cross-sectional areas. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1 ABE editing to produce sheep embryos carrying the gene encoding the 281st and / or 282nd mutants of the full-length MSTN protein

[0034] 1. Construction of sgRNA expression vector

[0035] Oligonucleotide DNA sequences were designed based on the sheep MSTN gene sequence as the target sequence and sent to a commercial primer synthesis company for synthesis (about 1 OD per strand, and the purification method was PAGE). The specific sequences are as follows:

[0036] sg-F: 5'-CCGGACGACAGCATCGAGATTCTG-3', SEQ ID NO.1;

[0037] sg-R: 5'-AAACCAGAATCTCGATGCTGTCGT-3', SEQ ID NO. 2.

[0038] The two oligo DNAs were dissolved in ultrapure water, mixed and annealed to form a double-stranded DNA fragment sg-FR with a sticky end containing a BsaⅠ restriction site.

[0039] The sg-FR was ligated with the linearized plasmid pGL3-U6-sgRNA-PGK-puromycin (provided by Addgene) after digestion with Bsa Ⅰ to obtain the recombinant vector pGL3-sg, which was used as the expression vector for sgRNA. The inserted sg-FR fragment was fused with the fragment on the vector to express sgRNA.

[0040] 2. In vitro transcription of sgRNA and ABE mRNA

[0041] Using the recombinant vector pGL3-sg as a template, the primers sgRNA-TF (sequence: 5'-TAATACGACTCACTATAGGACGACAGCATCGAGATTCTG-3', SEQ ID NO.3) and sgRNA-TR (sequence: 5'-AAAAGCACCGACTCGGTGCCA-3', SEQ ID NO.4) with T7 promoter at the 5' end were designed and synthesized. PCR amplification and gel recovery and purification were performed to obtain the sgRNA in vitro transcription template containing T7 promoter. 200 ng of the recovered in vitro transcription template was taken and transcribed according to the in vitro transcription kit (Ambion, MEGAshortscript TM In vitro transcription of sgRNA was performed according to the instructions of the T7 Transcription Kit to obtain sgRNA. The nucleotide sequence of sgRNA is as follows:

[0042] sgRNA (SEQ ID NO. 5):

[0043] The target sequence is (5'-acgacagcatcgagattctg-3', SEQ ID NO. 6), which is the antisense strand of the 83-102 nt sequence of the third exon of the sheep MSTN gene (Gene ID: 443449).

[0044] Using ABE8e (provided by Addgene) as a template, primers ABE-F (sequence: 5'-CTAATACGACTCACTATAGGGAGAG-3', SEQ ID NO. 7) and ABE-R (sequence: 5'-AAGGCACAGTCGAGGCTG-3', SEQ ID NO. 8) with a T7 promoter at the 5' end were designed and synthesized for PCR amplification and gel recovery and purification to obtain an ABE in vitro transcription template containing a T7 promoter. 1000 ng of the recovered and purified in vitro transcription template was taken and in vitro transcription and tailing (Vazyme, E. coli Poly(A) Polymerase, DD4111-PC-01) were performed according to the instructions of the in vitro transcription kit (Vazyme, T7 High Yield RNA Transcription Kit, DD4201-01) to obtain ABE mRNA. The mRNA sequence encoding the ABE protein is shown in SEQ ID NO. 9:

[0045] SEQ ID NO.9:

[0046] 3. Sheep embryo microinjection

[0047] Sheep fertilized eggs were taken for cytoplasmic microinjection, and the sgRNA prepared above and ABE mRNA were mixed to obtain a mixed system (sgRNA + ABE mRNA); the final concentration of sgRNA in the mixed system was 50 ng / μL, and the final concentration of ABE mRNA was 100 ng / μL; the mixed system was injected at a volume of 5 pL per fertilized egg and cultured in vitro to blastocysts.

[0048] 4. Amplification of the whole genome of a single blastocyst

[0049] The single sheep blastocyst obtained above was placed in a PCR tube, supplemented with PBS to 4 μL, and whole genome indiscriminate amplification was performed according to the instructions of the single cell whole genome DNA amplification kit (Vazyme, Discover-sc Single Cell WGA Kit, N603-01).

[0050] 5. PCR amplification detection of target region

[0051] Randomly select 52 whole genome DNA amplification products from the above single blastocysts, take 0.5-1 μL of each as template, and perform PCR reaction with the following primer pairs to amplify the region of the MSTN gene in the embryo that is expected to be edited, with an expected amplification length of about 495 bp.

[0052] Primer pairs for PCR reaction:

[0053] MSTN-F: 5'-TTCCTATGGTTCCTGTAGACTTT-3', SEQ ID NO.10;

[0054] MSTN-R: 5'-TTAATTGGAGACATCTTTGTAGG-3', SEQ ID NO. 11.

[0055] The PCR amplification products from these 52 embryos were bidirectionally sequenced using MSTN-F and MSTN-R, and compared with the wild-type MSTN gene sequence to confirm the editing status. Analysis of the sequencing results showed that 39 embryos had target site editing, with an effective target site editing efficiency of 75%. Among them, the MSTN of 4 embryos contained a partial T>C mutation at position 841 of the coding region (SEQ ID NO.12) of the MSTN full-length gene (GeneID:443449) containing a signal peptide (chimeric, MT1 in Figure 1 ), the sequence after mutation was shown in SEQ ID NO.13, and the cysteine ​​at position 281 of the MSTN full-length protein (SEQ ID NO.14) was mutated to arginine, as shown in SEQ ID NO.15; the MSTN of 2 embryos contained a partial T>C mutation at position 844 of the coding region (GeneID:443449) containing a signal peptide (chimeric, MT2 in Figure 1 ), the sequence after mutation was shown in SEQ ID NO.16, and the cysteine ​​at position 282 of the MSTN full-length protein (SEQ ID NO.14) was mutated to arginine, as shown in SEQ ID NO.17; the MSTN of 3 embryos contained a full-length MSTN gene (GeneID:443449) containing a signal peptide The 844th and 846th positions of the coding region of the MSTN full-length gene (Gene ID: 443449) were partially T>C mutations (chimeric, MT3 in FIG1 ), and the sequence after mutation is shown in SEQ ID NO. 18. The cysteine ​​at position 282 of the MSTN full-length protein (SEQ ID NO. 14) was mutated to arginine, as shown in SEQ ID NO. 17. The MSTN of 5 embryos was T>C mutations at positions 844 and 846 of the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide (MT4 in FIG1 ), and the sequence after mutation is shown in SEQ ID NO. 18. The cysteine ​​at position 282 of the MSTN full-length protein (SEQ ID NO. 14) was mutated to arginine, as shown in SEQ ID NO. 17. The MSTN of 3 embryos was T>C mutations at positions 841 and 844 of the coding region of the MSTN full-length gene (Gene ID: 443449) containing a signal peptide (MT5 in FIG1 ), and the sequence after mutation is shown in SEQ ID As shown in SEQ ID NO.19, the cysteine ​​residues at positions 281 and 282 of the MSTN full-length protein (SEQ ID NO.14) were mutated to arginine residues, as shown in SEQ ID NO.20. The MSTN residues of 19 embryos were T>C mutations at positions 841, 844, and 846 of the coding region of the MSTN full-length gene (GeneID:443449) containing the signal peptide (MT6 in Figure 1). The mutated sequence is shown in SEQ ID NO.21. The cysteine ​​residues at positions 281 and 282 of the MSTN full-length protein (SEQ ID NO.14) were mutated to arginine residues, as shown in SEQ ID NO.20; the MSTN of the three embryos showed partial T>C mutations at positions 841, 844, and 846 of the coding region of the full-length MSTN gene (Gene ID: 443449), including the signal peptide (MT7 in Figure 1). The mutated sequence is shown in SEQ ID NO. 21. The cysteine ​​residues at positions 281 and 282 of the full-length MSTN protein (SEQ ID NO. 14) were mutated to arginine residues, as shown in SEQ ID NO. 20.

[0056] SEQ ID NO.12:

[0057] SEQ ID NO.13:

[0058] SEQ ID NO.14:

[0059] SEQ ID NO.15:

[0060] SEQ ID NO.16:

[0061] SEQ ID NO.17:

[0062] SEQ ID NO.18:

[0063] SEQ ID NO.19:

[0064] SEQ ID NO.20:

[0065] SEQ ID NO.21:

[0066] Example 2 ABE editing to produce Hu sheep carrying the gene encoding the MSTN mutant

[0067] A total of 93 fertilized eggs were obtained from 8 donor ewes (Huyang), and the mixed system (sgRNA + ABE mRNA) obtained in Part 3 of Example 1 was injected into the above fertilized eggs in the form of cytoplasmic microinjection. The injection volume of each fertilized egg was 5 pL, and the final concentration of sgRNA in the mixture was 50 ng / μL, and the final concentration of ABE mRNA was 100 ng / μL; 72 fertilized eggs in good condition after injection were transplanted to 14 recipient ewes, and a total of 11 lambs were born. Lamb ear tissue samples were taken to extract genomic DNA, and PCR amplification was performed using the MSTN-F+MSTN-R detection primer pair in Example 1. The PCR products were sent to a sequencing company for bidirectional sequencing using MSTN-F and MSTN-R primers. The results showed that two lambs had a T>C mutation at position 841 in the coding region of the full-length gene containing a signal peptide (Gene ID: 443449), and a cysteine ​​mutation at position 281 of the MSTN full-length protein was converted to arginine (MT2 in Figure 2); two lambs had a T>C mutation at positions 844 and 846 in the coding region of the full-length gene containing a signal peptide (Gene ID: 443449), and a cysteine ​​mutation at position 282 of the MSTN full-length protein was converted to arginine (MT1 in Figure 2); and one lamb had a full-length gene containing a signal peptide (Gene ID: 443449). The 841st and 844th T>C mutations and the 846th T>C partial mutation (chimeric) in the coding region of the MSTN full-length gene (Gene ID: 443449) were generated, and the 281st and 282nd cysteines in the MSTN full-length protein were mutated to arginine (MT3 in Figure 2); 3 lambs produced the full-length gene of MSTN containing the signal peptide (Gene ID: 443449) with the 841st, 844th and 846th T>C mutations in the coding region, and the 281st and 282nd cysteines in the MSTN full-length protein were mutated to arginine (MT4 in Figure 2), with a gene editing positive rate of 72.7%.

[0068] Example 3 Phenotypic Analysis of Hu Sheep with MSTN Gene Mutants

[0069] The MSTN mutant lambs and wild-type Hu sheep obtained in Example 2 were raised and phenotypically observed, and it was found that the weight of the MSTN protein mutant lambs at 3 months, 4 months, and 6 months of age was significantly higher than that of the wild-type Hu sheep (Figure 3). Comparison of the 7-month-old MSTN protein mutant Hu sheep with the 7-month-old wild-type Hu sheep raised in the same manner showed that the hind and forequarter muscles of the MSTN protein mutant Hu sheep were well-developed and plump (as shown in Figure 4), showing an obvious double-muscled buttocks phenotype. The hind gluteal muscles of the MSTN protein mutant Hu sheep and the wild-type Hu sheep were surgically collected for fixation, tissue sectioning, and HE staining. The results showed that the cross-sectional area of ​​the muscle fibers in the hind gluteal muscles of the MSTN protein mutant Hu sheep (MT1 to MT3 in Figure 5) was significantly increased compared with the wild-type Hu sheep (WT in Figure 5).

[0070] Example 4 ABE editing to prepare sheep fibroblasts carrying the gene encoding the MSTN protein mutant

[0071] The recombinant vector pGL3-sg and ABE8e vector were co-transfected into sheep fibroblasts. Puromycin was added for selection 48 hours later for 7 days. Monoclonal cell screening and propagation were performed, and a total of 18 monoclonal strains were obtained. DNA was extracted from the monoclonal cells and PCR amplified using the MSTN-F+MSTN-R primer pair. The amplified products were bidirectionally sequenced using MSTN-F and MSTN-R, respectively, and compared with the wild-type MSTN gene sequence. It was found that 13 monoclonal cell strains were effectively edited. Among them, the MSTN of two clones had a T>C mutation at position 841 in the coding region of the full-length MSTN gene (Gene ID: 443449) containing a signal peptide, and the sequence after mutation was shown in SEQ ID NO.13. The cysteine ​​at position 281 of the MSTN full-length protein was mutated to arginine, as shown in SEQ ID NO.15; the MSTN of two clones had a T>C mutation at positions 844 and 846 in the coding region of the full-length MSTN gene (Gene ID: 443449) containing a signal peptide, and the sequence after mutation was shown in SEQ ID NO. As shown in SEQ ID NO.18, the cysteine ​​at position 282 of the MSTN full-length protein was mutated to arginine, as shown in SEQ ID NO.17; the 9 cloned MSTNs had T>C mutations at positions 841, 844 and 846 of the coding region of the MSTN full-length gene (Gene ID:443449) containing the signal peptide, and the sequence after mutation was shown in SEQ ID NO.21, and the cysteine ​​at positions 281 and 282 of the MSTN full-length protein were mutated to arginine, as shown in SEQ ID NO.20.

[0072] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A myostatin MSTN protein mutant, characterized in that: The amino acid sequence is any one of the sequences shown in SEQ ID NO.15, SEQ ID NO.17 or SEQ ID NO.

20.

2. A gene encoding the MSTN protein mutant according to claim 1.

3. The gene according to claim 2, characterized in that When the amino acid sequence of the MSTN protein mutant is SEQ ID NO.15, the nucleotide sequence of the gene is shown as SEQ ID NO.13; when the amino acid sequence of the MSTN protein mutant is SEQ ID NO.17, the nucleotide sequence of the gene is shown as SEQ ID NO.16 or SEQ ID NO.18; when the amino acid sequence of the MSTN protein mutant is SEQ ID NO.20, the nucleotide sequence of the gene is shown as SEQ ID NO.19 or SEQ ID NO.

21.

4. A method for preparing a biological material whose genome contains the gene according to claim 2 or 3, characterized in that: The method comprises the steps of obtaining the biological material by adenine base editing technology under the guidance of sgRNA; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

5.

5. The method according to claim 4, characterized in that The biological materials include cells, embryos and animals.

6. A sgRNA for editing the MSTN gene, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

5.

7. An expression vector or expression cassette expressing the sgRNA according to claim 6.

8. A method for increasing the muscle content of an animal or promoting the muscle development of an animal, characterized in that: The method comprises using the sgRNA described in claim 6 or the expression vector or expression cassette described in claim 7 to mutate the MSTN encoding gene in the animal genome into the gene described in claim 2 or 3 through adenine base editing technology.

9. A method for increasing meat production of animals, characterized in that: The method comprises using the sgRNA described in claim 6 or the expression vector or expression cassette described in claim 7 to mutate the MSTN encoding gene in the animal genome into the gene described in claim 2 or 3 through adenine base editing technology.

10. A genetic breeding method for double-muscled animals, characterized in that: The method comprises using the sgRNA described in claim 6 or the expression vector or expression cassette described in claim 7 to mutate the MSTN encoding gene in the animal genome into the gene described in claim 2 or 3.

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