Transgenic animals with modified myostatin genes

Abstract

We provide an animal having a myostatin gene in which 12 base pairs of exon 2 are deleted. [Solution] A transgenic animal is provided having an artificially modified myostatin gene in its genome, wherein the artificial modification is located in the second exon of the myostatin gene, and the artificial modification is a deletion of 12 base pairs in the second exon that encodes an amino acid sequence in the order of leucine, tryptophan, isoleucine, and tyrosine, compared to the amino acid sequence of myostatin in wild-type animals, and the transgenic animal expresses less myostatin gene mRNA than wild-type animals and expresses a mature myostatin protein with the same sequence as wild-type animals.

Description

Technical Field

[0001] The present application relates to transgenic animals or cells having artificially modified genes. The transgenic animals or cells have a myostatin gene having a 12-base pair deletion in the second exon.

[0002] The present application relates to technologies related to the production of transgenic animals or cells.

Background Art

[0003] "Belgian Blue" is well known as an excellent breed of cattle with well-developed muscles. This breed was accidentally created by crossbreeding by Belgian breeders in the 19th century. They do not consume as much feed as compared to the wild type, but they are characterized by a high proliferation rate of muscle cells due to genetic reasons, and as a result, the common feed becomes low-fat and high-protein. It has been reported that these characteristics are caused by the modification of the myostatin gene (McPherron AC, Lawler AM, Lee SJ (1997) Nature 387:83-90).

[0004] Myostatin, from its name, indicates the meaning of "muscle (myo) + inhibitor (statin)", and during the ongoing research, it has already been known that myostatin protein inhibits muscle differentiation and growth. Myostatin has been investigated in various animal models to control muscle cell differentiation and proliferation by gene regulation using gene editing tools.

[0005] Furthermore, in the case of myostatin transgenic large animals, high-quality meat can be obtained, thereby increasing its utilization. However, the deletion of the myostatin gene still has technical limitations accompanied by several side effects such as cardiac hypertrophy, increased blood pressure, and short lifespan.

[0006] As a result of the aforementioned technical limitations, myostatin transgenic animals are not yet available for industrial use.

[0007] Accordingly, the applicant attempted to obtain a healthy myostatin transgenic animal with increased muscle mass. As a result, this disclosure is completed by confirming that the transgenic bovine animals of the application have a specific variant in which myostatin is modified and are transgenic animals without the conventional side effects. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] CN104531705A [Patent Document 2] CN107034221A [Non-patent literature]

[0009] [Non-Patent Document 1] Am J Physiol Endocrinol Metab.2017 Mar 1;312(3):E150-E160, Myostatin propeptide mutation of hyper muscular compact mice decreases formation of myostatin and improves insulin sensitivity. [Overview of the project] [Problems that the invention aims to solve]

[0010] One object of this application is to provide an animal having an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted.

[0011] Another object of this application is to provide an embryo having an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted.

[0012] Another object of this application is to provide a composition for deleting 12 base pairs in the second exon of the myostatin gene.

[0013] Another object of this application is to provide a use for the composition to induce muscle growth in the muscles of animals. [Means for solving the problem]

[0014] To solve the aforementioned problems, this specification provides transgenic animals having a myostatin gene in which a specific portion has been artificially modified.

[0015] Modifications may occur in the second exon of the myostatin gene.

[0016] The aforementioned modification involves the deletion of 12 base pairs in the second exon, corresponding to the region encoding the amino acid sequences of leucine, tryptophan, isoleucine, and tyrosine, compared to the myostatin gene sequence in wild-type animals.

[0017] A nucleic acid encoding the amino acid sequence leucine, tryptophan, isoleucine, and tyrosine may have one or more sequences encoding each amino acid.

[0018] In other words, the sequence encoding leucine may be one selected from 5'-CTT-3', 5'-CTC-3', 5'-CTA-3', or 5'-CTG-3', the sequence encoding tryptophan may be one selected from 5'-TGG-3', the sequence encoding isoleucine may be one selected from 5'-ATT-3', 5'-ATC-3', or 5'-ATA-3', and the sequence encoding tyrosine may be one selected from 5'-TAT-3' or 5'-TAC-3'.

[0019] The amount of myostatin mRNA expression in transgenic animals is lower than that in wild-type animals.

[0020] In addition, transgenic animals can express a mature myostatin protein having the same amino acid sequence as that of wild-type animals.

[0021] Transgenic animals may have an increased amount of muscle visually compared to wild-type animals.

[0022] Transgenic animals include mammals.

[0023] Mammals include ungulates.

[0024] Ungulates include artiodactyls. Artiodactyls may include, but are not limited to, pigs, deer, cows, sheep, and goats.

[0025] Mammals may include rodents. Rodents may include, but are not limited to, mice and rats.

[0026] Preferably, the transgenic animal in the present application is a bovine subfamily animal. The bovine subfamily animal expresses a promyostatin protein containing the amino acid sequence represented by SEQ ID NO: 30.

[0027] Furthermore, the present application provides an engineered cell having an artificially modified myostatin gene with specific portions modified.

[0028] Transformation of the engineered cell may occur in the second exon of the myostatin gene.

[0029] The above modification is a deletion of 12 base pairs in the second exon corresponding to the region encoding the amino acid sequences of leucine, tryptophan, isoleucine, and tyrosine as compared to the myostatin gene sequence in wild-type cells.

[0030] A nucleic acid encoding the amino acid sequence leucine, tryptophan, isoleucine, and tyrosine may have one or more sequences encoding each amino acid.

[0031] In other words, the sequence encoding leucine may be one of 5'-CTT-3', 5'-CTC-3', 5'-CTA-3', or 5'-CTG-3', the sequence encoding tryptophan may be one of 5'-TGG-3', the sequence encoding isoleucine may be one of 5'-ATT-3', 5'-ATC-3', or 5'-ATA-3', and the sequence encoding tyrosine may be one of 5'-TAT-3' or 5'-TAC-3'.

[0032] The manipulated cells expressed less myostatin mRNA than wild-type cells.

[0033] In addition, the manipulated cells can express mature myostatin protein with the same amino acid sequence as wild-type animals.

[0034] The cells may be embryonic cells, somatic cells, or stem cells.

[0035] Examples of cells include, but are not limited to, oocytes, epithelial cells, fibroblasts, nerve cells, keratinizing cells, hematopoietic cells, melanin-forming cells, chondrocytes, macrophages, monocytes, muscle cells, and B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, placental cells, and germ cells. Furthermore, adult stem cells derived from various tissues of origin, such as adipose tissue, uterine tissue, bone marrow, muscle tissue, placenta, umbilical cord blood, or skin (epithelium), can be used. Non-human host embryos can generally be embryos including 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, aborted embryo, or blastocyst.

[0036] Cells can be obtained from mammals.

[0037] Preferably, the cells of the present invention can be obtained from animals of the Bovidae subfamily.

[0038] The manipulated cells may express a promyostatin protein having the amino acid sequence represented by sequence identification number:30.

[0039] This application provides a composition for modifying the myostatin gene.

[0040] To modify the myostatin gene, the composition is A guide RNA containing a guide sequence that forms a complementary bond with the target sequence, or DNA encoding the guide RNA; and the nucleic acid sequence encoding the Cas protein or guide RNA. It may include.

[0041] The target sequence may include one or more sequences selected from sequence identification numbers 38 to 60.

[0042] The guide sequence may include one or more sequences selected from sequence identification numbers 62 to 84.

[0043] The Cas protein may be one selected from the group consisting of Streptococcus pyogenes-derived Cas9 protein, Staphylococcus aureus-derived Cas9 protein, or Cas12a protein (CPF1: conventionally known as Prevotella and Francisella 1). The nucleic acid encoding the Cas protein may be any one selected from the group consisting of nucleic acids encoding the Cas9 protein from Streptococcus pyogenes, the Cas9 protein from Staphylococcus aureus, or the Cas12a protein (conventional CPF1: Prevotella and Francisella 1) protein.

[0044] The composition may be present in the plasmid vector in the form of guide RNA and DNA encoding the Cas protein.

[0045] The composition may be present in the viral vector in the form of guide RNA and DNA encoding the Cas protein.

[0046] In this case, the viral vector may be one or more selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, poxvirus vectors, and herpes simplex virus vectors.

[0047] The genetically modified composition may also be in the form of a complex (RNP: ribonucleoprotein) containing guide RNA and Cas protein.

[0048] Furthermore, the present application provides a method for preparing cells or embryos having an artificially modified myostatin gene modified with the above composition.

[0049] The present invention's method for producing myostatin-modified cells or embryos may include a step of contacting the cells or embryos with the composition. The contact step may be performed in vivo or ex vivo.

[0050] The contact step may be carried out by one or more methods selected from microinjection, electroporation, liposomes, plasmids, viral vectors, nanoparticles, and protein translation domain (PTD) fusion protein methods.

[0051] Furthermore, this application provides a method for preparing animals having an artificially modified myostatin gene.

[0052] The method for producing myostatin transgenic animals according to this disclosure may include the steps of producing a transgenic embryo having an artificially modified gene by contacting an embryo with a composition as described above, and transferring the transgenic embryo to a surrogate mother.

[0053] Animals produced by the above manufacturing method express less myostatin mRNA than wild-type animals.

[0054] The animal may be a mammal other than a human.

[0055] Favorable effects

[0056] The myostatin transgenic animals of this invention can increase muscle mass compared to wild-type animals due to the low expression level of myostatin mRNA.

[0057] As a result, it is possible to provide high-quality meat with a low fat content and a high protein content.

[0058] Conventional myostatin-mutated transgenic animals have various side effects, such as a short lifespan. However, this invention can provide healthy myostatin-mutated transgenic animals that do not have such various side effects.

[0059] Furthermore, the compositions provided by this application are capable of modifying the myostatin gene and may be capable of increasing muscle mass when injected into animal tissue. [Brief explanation of the drawing]

[0060] [Figure 1] A diagram showing the location of modifications in the myostatin gene is provided, and the protospacer sequences used in one example of this disclosure are listed. [Figure 2] This is a schematic diagram of a method for producing transgenic embryos containing an artificially modified myostatin gene in which 12 base pairs of exon 2 are deleted. [Figure 3] This shows confirmation of myostatin modification in manipulated embryos containing a myostatin gene with 12 base pairs deleted in exon 2, using the T7E1 assay. [Figure 4]This shows Sanger sequencing of myostatin-modified embryos containing a guide RNA that includes the protospacer sequence of the myostatin gene and a sequence that binds to its complementary target sequence, and it shows multiple variants. [Figure 5] The following shows different amounts of guide RNA or CAS9 mRNA used to drive the deletion of 12 base pairs in the second exon of the myostatin gene, in order to determine the most appropriate amount of guide RNA and CAS9 mRNA to advance this invention. [Figure 6] This image shows a cow with a 12-base pair myostatin gene deletion in exon 2, photographed once a month for external examination during the first 1 to 4 months after birth. [Figure 7] This shows confirmation of myostatin modification in cattle with a myostatin gene containing 12 base pairs deleted in the second exon, using the T7E1 assay. [Figure 8] To confirm the off-target effects that can be generated by CRISPR / Cas9, five sequences relating to potential off-target sites identified by the T7E1 assay are shown. It was confirmed that neither heterozygous nor homozygous knockout occurred for any of the five off-target sites, with or without mixing with wild-type DNA. [Figure 9] The deep sequencing results of 17 cows born after embryos generated by the method shown in Figure 2 were implanted in the uterus of a surrogate mother are shown, revealing the deletion of 12 base pairs in the myostatin gene. [Figure 10] The results of deep sequencing of wild-type cattle as a negative control for cattle carrying a myostatin gene with a 12-base pair deletion in exon 2 are listed below. [Figure 11] The results of deep sequencing for cattle No. 6, which has a myostatin gene with a 12-base pair deletion in exon 2, are listed below. [Figure 12]The results of deep sequencing for cattle No. 14, which has a myostatin gene with a deletion of 12 base pairs in exon 2, are listed below. [Figure 13] The results of deep sequencing for cattle No. 17, which has a myostatin gene with a deletion of 12 base pairs in exon 2, are listed below. [Figure 14] This shows the amount of myostatin mRNA expression in cattle 14 and 17, which have a myostatin gene with a 12-base pair deletion in exon 2. [Figure 15] This paper demonstrates the transfer of germline cells from MSTN-mutated females, the production of MSTN-mutated blastocysts from MSTN-mutated bovine oocytes, and presents representative images of pregnancy diagnosis using ultrasound at day 30. [Figure 16] This is an image of somatic cells derived from follicular fluid obtained during OPU (Oral Pulse Processing). [Figure 17] The results of the T7E1 assay and sequencing data from MSTN mutant female blastocysts are shown. [Figure 18] The results and sequencing data of the T7E1 assay from somatic cells in follicular fluid are shown. [Figure 19] This section presents an overview of semen from the MSTN male founder, based on Computer-Assisted Semen Analysis. [Figure 20] This image shows a representative blastocyst resulting from verified germline transfer from MSTN mutant bovine. [Figure 21] This shows the mutation rate of the MSTN gene in blastocysts derived from the semen of MSTN mutant bovine fertilized in vitro. [Modes for carrying out the invention]

[0061] To illustrate the contents disclosed herein, several terms are defined herein. In addition to these terms, other terms may be defined elsewhere in this specification where necessary. Unless expressly defined otherwise herein, industry terms used herein have their meanings as recognized in those fields. In case of any conflict, the definition herein prevails.

[0062] Definitions of general terms

[0063] Conserved region of the myostatin gene

[0064] The conserved region of the myostatin gene refers to the nucleic acid sequence that encodes a commonly conserved region in the amino acid sequence of myostatin that remains unchanged throughout evolution and across species.

[0065] In this application, the term "species-conserved region of the myostatin gene" includes nucleic acid sequences that encode amino acids in the order of leucine, tryptophan, isoleucine, and tyrosine within the myostatin conservation region (see Table 3).

[0066] The conserved region of myostatin across species may have the same amino acid sequence, but may also have several base codons for the amino acid sequence depending on the species. In other words, the nucleic acid sequence encoding leucine may be one of 5'-CTT-3', 5'-CTC-3', 5'-CTA-3', or 5'-CTG-3'; the nucleic acid sequence encoding tryptophan may be one of 5'-TGG-3'; the nucleic acid sequence encoding isoleucine may be one of 5'-ATT-3', 5'-ATC-3', or 5'-ATA-3'; and the nucleic acid sequence encoding tyrosine may be one of 5'-TAT-3' or 5'-TAC-3'.

[0067] Therefore, the nucleic acid sequence of the "conserved region of the myostatin gene" in this application may differ from species to species. In this specification, the "conserved region of the myostatin gene" may be abbreviated as the "conserved region".

[0068] Transgenic animals

[0069] In this application, the term "transgenic animal" means an animal that possesses an artificially modified myostatin gene.

[0070] In this application, a "transgenic animal" has an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted, and expresses a mature myostatin protein having the same sequence as wild-type animals.

[0071] The trait of the artificially modified myostatin gene in the transgenic animals of this invention is inherited by offspring.

[0072] As first-generation animals, F0 possesses an artificially modified myostatin gene. F0 can produce progeny F1. The myostatin gene contained in F1 and sub-F1 progeny has the same nucleotide sequence as the artificially modified myostatin gene. The term “transgenic animal” in this application includes F0, F1, and sub-F1 progeny. In other words, if an animal F1 has a modified myostatin gene, it is a transgenic animal in this application even if no direct artificial manipulation for transformation is applied during the production process of animal F1 or after animal F1 has been produced.

[0073] animal

[0074] The animals referred to in this application include non-human animals.

[0075] Animals include mammals.

[0076] Mammals include ungulates.

[0077] Ungulates include even-toed ungulates. Even-toed ungulates may include, but are not limited to, pigs, deer, cattle, sheep, and goats.

[0078] Mammals may include rodents. Rodents may include, but are not limited to, mice and rats.

[0079] target area

[0080] In this application, the term "target region" refers to a region in the wild-type genome whose genes will be artificially manipulated in order to produce a transgenic animal, and which includes a protospacer sequence and a target sequence, as shown below.

[0081] Protospacer Array

[0082] In this application, the term "protospacer sequence" refers to 20 sequences adjacent to the PAM sequence, based on the location of the PAM sequence in the target region of this application. The protospacer sequence and the target sequence are complementary sequences. In other words, this means the same sequence as the guide sequence that binds complementaryly to the target sequence. However, the guide sequence may have a sequence in which T (thymine) is substituted with U (uracil) in the protospacer sequence.

[0083] target sequence

[0084] The term "target sequence" in this application refers to a sequence included in the target region of this application that complementarily binds to a protospacer sequence. The target sequence may also bind complementarily to a guide sequence.

[0085] The meanings of A, T, C, G, and U

[0086] As used herein, the symbols A, T, C, G, and U are to be interpreted as having meanings understood by those skilled in the art. Each of these symbols may be appropriately interpreted as a base, a nucleoside, or a nucleotide on DNA or RNA, depending on the context and the art. For example, if each symbol means a base, then A, T, C, G, and U may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), respectively. If each symbol means a nucleoside, then A, T, C, G, and U may be interpreted as adenosine (A), thymine (T), cytidine (C), guanosine (G), or uridine (U), respectively. If they mean a nucleotide in a sequence, then A, T, C, G, and U each represent a nucleotide containing a nucleoside.

[0087] The present application will be explained in detail below.

[0088] This application relates to a transgenic animal having an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted.

[0089] Myostatin

[0090] The transgenic animal of this invention is characterized by containing an artificially modified myostatin gene.

[0091] The structure and function of myostatin are described in detail below.

[0092] Structure of myostatin

[0093] Myostatin genes in higher organisms known today are characterized by having three exons and two introns. Myostatin genes are known to be mostly found in muscle tissue.

[0094] Myostatin mRNA generates myostatin protein, which consists of approximately 375 amino acids and is divided into three parts: the signal peptide region, the propeptide (prodomain) region (28 kDa, N-terminus), and the maturation region (12 kDa, C-terminus).

[0095] The structure of the precursor protein, promyostatin, consists of two identical subunits, and the maturation regions form disulfide bonds with each other, thereby maintaining the homodimeric protein morphology.

[0096] Function and signaling pathways of myostatin maturation proteins

[0097] Regarding the signaling pathway of myostatin protein, after the first cleavage of the precursor protein, promyostatin, by the enzyme furin, it is divided into a propeptide region and a maturation region. After cleavage, in the latent complex, the propeptide region binds to the maturation region via non-covalent bonds. Subsequently, after a second cleavage by BMP / Tolloid, the myostatin maturation region is phosphorylated by binding to the activin type II receptor as it is secreted from the cell. The signal is again transmitted to the activin type I receptor, and then to the receptor regulatory proteins, Smad2 and Smad3, which, in combination with co-Smad4, regulate the transcription of target genes. As a result of this signaling pathway, the mature myostatin protein is expressed.

[0098] Conventional modifications of the myostatin gene

[0099] Previous studies related to the myostatin gene have focused on subjects that do not express mature myostatin protein. Studies have been conducted to prepare animals with increased muscle mass and reduced fat by suppressing the expression of mature myostatin protein. Furthermore, through these studies of subjects that do not express mature myostatin protein, research is underway on the myostatin signaling pathway, with the aim of utilizing it in diseases where muscle mass rapidly decreases, such as in terminal cancer patients, and for muscle fiber regeneration.

[0100] In many myostatin transgenic animals, the myostatin gene is modified so that the myostatin protein, which inhibits muscle growth, is not expressed to a significant degree in somatic cells. In other words, this is achieved by modifying the cleavage region in the signaling pathway of mature myostatin protein, thereby suppressing the expression of mature myostatin protein. Animals cloned by nuclear transfer of somatic cells have twice the muscle mass, and their muscle mass is increased compared to wild-type animals.

[0101] However, transgenic animals with myostatin gene modifications obtained by the conventional methods described above have short lifespans. Therefore, they have drawbacks, particularly in large animals, in that they cause reproductive problems and potentially fatal health side effects.

[0102] This application emphasizes the advantages of myostatin gene modification in relation to transgenic animals that minimize the side effects caused by conventional myostatin gene modification.

[0103] Specifically, by deleting 12 base pairs targeting a specific region of exon 2 that is not a cleavage region in the signaling pathway of mature myostatin protein, the present invention provides animals in which the expression of mature myostatin protein is suppressed compared to the wild type, rather than being completely absent.

[0104] Myostatin transgenic animals

[0105] One embodiment of the present invention is a myostatin transgenic animal having an artificially modified myostatin gene. In one embodiment, it may be an ungulate, for example, a bovine.

[0106] In the following, this disclosure will be explained in detail using a bovine animal (cattle) possessing the artificially modified myostatin gene of the present application as an example.

[0107] Feature 1 - Genetic modification of the genome of transgenic animals

[0108] The transgenic animals of this application may have a myostatin gene composition that differs from that of wild-type animals with respect to the myostatin gene composition.

[0109] The gene modification described in this application refers to the deletion of a nucleic acid sequence encoding four specific amino acids (leucine, tryptophan, isoleucine, and tyrosine in that order) within a particular conserved region of the myostatin protein's amino acid sequence.

[0110] The transgenic animal of this invention has a myostatin gene in which 12 base pairs of the second exon, which is a nucleic acid sequence encoding the amino acid sequence of the conserved region, are deleted.

[0111] If the transgenic animal is a bovine, pig, or human, the deletion of 12 base pairs may be the deletion of a base pair (sequenced as 5'-3') at positions 93-104 of the sequence encoding the second exon of the wild-type myostatin gene.

[0112] If the transgenic animal is a mouse, the deletion of 12 base pairs may be a deletion of base pairs at positions 94-105 of the sequence encoding the second exon of the wild-type myostatin gene.

[0113] Feature 2 - Changes in mRNA composition and myostatin gene expression levels in transgenic animals

[0114] The transgenic animals of this invention may have myostatin mRNA configurations that differ from those of wild-type animals. The transgenic animals of this invention have myostatin mRNA in which 12 bases are deleted.

[0115] In one embodiment of the present invention, the amount of myostatin mRNA expression in transgenic animals can be measured.

[0116] In certain embodiments, the level of myostatin mRNA expression in the transgenic animals of the present invention is at least 60% lower than that in wild-type animals. Preferably, the level of myostatin mRNA expression in the transgenic animals of the present invention is lower than that in wild-type animals, but this does not mean that expression is absent.

[0117] Feature 3 - Changes in the protein composition of the myostatin gene and the expression of mature myostatin protein in transgenic animals

[0118] The 12 deleted base pairs in the transgenic animals of this invention are nucleic acids encoding a conserved amino acid sequence that has not undergone species-specific amino acid sequence modifications during the evolution of the myostatin gene. The conserved amino acid sequence is the amino acid sequence in the order of leucine, tryptophan, isoleucine, and tyrosine.

[0119] Therefore, the transgenic animals of this invention express a myostatin protein that lacks four amino acids in the sequence of leucine, tryptophan, isoleucine, and tyrosine compared to the wild-type promyostatin protein.

[0120] A promyostatin protein lacking four amino acids may be one of sequence identification numbers 30-33.

[0121] The promyostatin protein of transgenic animals may have modifications in some sequences, but may have 90% or higher homology to one of sequence identification numbers 30-33.

[0122] For example, if the transgenic animal is a bovine (cattle), a promyostatin protein with sequence identification number 30, which has four amino acids deleted, may be expressed.

[0123] For example, if the transgenic animal is a pig, a promyostatin protein with sequence identification number 31, which has four amino acids deleted, may be expressed.

[0124] For example, if the transgenic animal is a mouse, a promyostatin protein with sequence identification number 32, which has four amino acids deleted, may be expressed.

[0125] For example, if the transgenic animal is human, a promyostatin protein with sequence identification number 33, which has four amino acids deleted, may be expressed.

[0126] [Table 1]

[0127] The four amino acids that will be deleted do not overlap with the regions of the promyostatin protein that are cleaved during the process of mature myostatin protein formation.

[0128] Since the amino acid deletion site is not located in the region where cleavage occurs, the promyostatin protein matures into a mature myostatin protein via the normal signaling process. In other words, the deletion of the specific amino acid in this application does not affect the normal process of mature myostatin protein formation.

[0129] Therefore, the mature myostatin protein expressed by the myostatin transgenic animals of this invention is identical to that of the wild type. In other words, it is characterized by having the same amino acid sequence as the wild-type mature myostatin protein.

[0130] In one embodiment, the mature myostatin protein of the transgenic animal may be one of sequence identification numbers 34-37.

[0131] The mature myostatin protein of transgenic animals may have modifications in some sequences, but may have 90% or higher homology to one of sequence identification numbers 34-37.

[0132] For example, if the transgenic animal is a bovine (cattle), it may express a mature myostatin protein with sequence identification number 34, which is identical to that of a wild bovine.

[0133] For example, if the transgenic animal is a pig, it may express the mature myostatin protein with sequence identification number 35, which is identical to that of a wild-type pig.

[0134] For example, if the transgenic animal is a mouse, it may express a mature myostatin protein with sequence identification number 36, which is identical to that of a wild-type mouse.

[0135] For example, if the transgenic animal is human, it may express the mature myostatin protein with sequence identification number 37, which is identical to that of wild-type humans.

[0136] [Table 2]

[0137] Mature myostatin protein may exist in monomeric or dimeric form in the blood. The transgenic animals of this invention can express the same mature myostatin protein as the wild type. In other words, the mature myostatin protein of the transgenic animal of this application has the same amino acid sequence as the mature myostatin protein of wild-type animals.

[0138] In one embodiment of the present invention, the mature myostatin protein of a transgenic animal can be identified by mass spectrometry compared with wild-type mature myostatin protein.

[0139] In one embodiment of the present invention, the expression level of mature myostatin protein in the transgenic animal of the present invention may be lower than that of the wild-type animal. This result can also be seen from the fact that the amount of myostatin mRNA expression in the transgenic animal of the present invention was reduced compared to the amount of myostatin mRNA expression in the wild-type animal (see Figure 14).

[0140] Feature 4 - Increased muscle mass

[0141] The transgenic animals described herein exhibit a muscle hypertrophy phenotype due to reduced expression of myostatin mRNA and mature myostatin protein compared to wild-type animals. The muscle hypertrophy phenotype includes phenotypes such as increased muscle mass, increased muscle cell number, increased muscle cell size, and increased muscle cell differentiation rate.

[0142] In certain embodiments, the transgenic animals of the present disclosure may have a muscle mass increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to wild-type animals.

[0143] Feature 5 - No side effects such as shortened lifespan.

[0144] Conventional myostatin transgenic animals are known to be associated with shortened lifespan and health abnormalities.

[0145] Unlike conventional myostatin transgenic animals, the transgenic animals of this invention do not completely lack myostatin mRNA and mature myostatin protein expression. In other words, the expression of myostatin mRNA and mature myostatin protein is reduced compared to wild-type animals that do not express myostatin mRNA and mature myostatin protein.

[0146] Therefore, unlike the shortened lifespan and health abnormalities that may result from the non-expression of myostatin mRNA and mature myostatin protein, there may be no health abnormalities.

[0147] In certain embodiments, bovine animals (cattle) possessing the myostatin gene in which 12 base pairs of the present application are deleted were found to be healthy and free from health abnormalities.

[0148] In one embodiment, a bovine animal having the myostatin gene in which 12 base pairs of the present invention are deleted can reproduce and produce offspring.

[0149] Myostatin transgenic cells

[0150] Another aspect of the present invention is an engineered cell having an artificially modified myostatin gene.

[0151] The manipulated cells may be embryonic cells, somatic cells, or stem cells.

[0152] In certain embodiments, the cells include, but are not limited to, oocytes, epithelial cells, fibroblasts, nerve cells, keratinizing cells, hematopoietic cells, melaninizing cells, chondrocytes, macrophages, monocytes, muscle cells, and B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, placental cells, and germ cells. Furthermore, adult stem cells derived from various tissues of origin, such as adipose tissue, uterine tissue, bone marrow, muscle tissue, placenta, umbilical cord blood, or skin (epithelium), can be used. Non-human host embryos can generally be embryos including 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, aborted embryo, or blastocyst.

[0153] The characteristics of the manipulated cells in this invention are the same as those of the transgenic animals described in characteristics 1-3 above.

[0154] In summary, the manipulated cells have a myostatin gene in which 12 base pairs of the second exon are deleted.

[0155] Genetic modification in manipulated cells refers to the deletion of a nucleic acid sequence encoding four specific amino acids in a conserved region of the myostatin protein's amino acid sequence (leucine, tryptophan, isoleucine, and tyrosine in that order). Therefore, manipulated cells have a myostatin gene in which 12 base pairs of the second exon, the nucleic acid sequence encoding the conserved region's amino acid sequence, are deleted.

[0156] The manipulated cells may have a myostatin mRNA configuration different from that of wild-type cells. The manipulated cells of this invention have myostatin mRNA in which 12 bases are deleted.

[0157] The level of myostatin mRNA expression in manipulated cells is lower than that in wild-type animal cells.

[0158] Prepromyostatin proteins must undergo a cleavage step to become active, mature myostatin proteins.

[0159] Since the locations of the four deleted amino acids in transgenic cells are not within the region where cleavage occurs, the promyostatin protein matures into the mature myostatin protein via the normal signaling process. In other words, the deletion of the specific amino acids in this invention does not affect the normal process of mature myostatin protein formation.

[0160] In other words, the mature myostatin protein expressed by manipulated cells in which 12 base pairs of the myostatin gene of the present invention are deleted has the same amino acid sequence as the mature myostatin protein of wild-type cells.

[0161] Compositions for genetic engineering

[0162] According to another aspect of the disclosure provided herein, a composition for genetic engineering to modify the myostatin gene is provided.

[0163] To modify the myostatin gene, the composition for genetic engineering is A guide RNA containing a guide sequence that forms a complementary link with the target sequence of the myostatin gene, or DNA encoding the guide RNA; and It may also include a Cas protein or a nucleic acid sequence encoding a Cas protein.

[0164] The target sequence is a sequence that is complementary to the protospacer sequence targeted by the composition and is contained within the target region.

[0165] The target sequence is located in the second exon (exon 2) of the myostatin gene.

[0166] target sequence

[0167] The composition of this invention targets the myostatin gene in order to modify the myostatin gene.

[0168] The portion of a composition that can be targeted is called the target region.

[0169] The target region is located in the second exon (exon 2) of the myostatin gene.

[0170] The target region includes a target sequence and a protospacer sequence, and the sequence to which the guide sequence of the composition complementarily binds is called the target sequence.

[0171] In this application, because there are differences in genetic sequences between species, it is easy to target nucleic acids encoding the conserved region of the amino acid sequence of myostatin protein as the target region of the composition for genetic manipulation.

[0172] Accordingly, the target sequence is configured, depending on the species, to include some or all of the sequences that encode the conserved amino acid sequence of the myostatin protein, as described below.

[0173] The conserved amino acid sequences of promyostatin proteins by species are described in detail below. In one embodiment, the conserved amino acid sequence is described with respect to bovine animals relative to humans, pigs, or mice. Animals having the conserved amino acid sequence are not limited to these.

[0174] A comparison of the promyostatin protein sequences of bovine animals, humans, pigs, and mice is provided in part below (Table 3). The amino acid sequence at positions 156–159 of each promyostatin protein is conserved, and the conserved amino acid sequence is as follows: leucine, tryptophan, isoleucine, and tyrosine (see the bolded column in the table below).

[0175] [Table 3]

[0176] The site of the specific amino acid deletion in the promyostatin protein of this application is such a conserved amino acid sequence, namely the amino acid sequence from position 156 to 159 of the myostatin protein.

[0177] These amino acid sequences are located at positions 157-160 in the myostatin protein in mice, and the amino acid sequences in mice are the same as those of leucine, tryptophan, isoleucine, and tyrosine.

[0178] In this application, the region targeted by the composition may include some or all of the regions encoding a conserved amino acid sequence. The target sequence can be designed around one strand of a DNA double helix containing the conserved region.

[0179] The target sequence may include some or all of the following sequences encoding leucine in an amino acid sequence: 5'-CTT-3', 5'-CTC-3', 5'-CTA-3', or 5'-CTG-3'; 5'-TGG-3' encoding tryptophan in an amino acid sequence; 5'-ATT-3', 5'-ATC-3', or 5'-ATA-3' encoding isoleucine in an amino acid sequence; or 5'-TAT-3' or 5'-TAC-3' encoding tyrosine in an amino acid sequence; or some or all of the complementary sequences of such sequences. In one embodiment of the present application, the target sequence may include sequence identifier number: 28-5'-ATATATCCACAG-3'. In another embodiment of the present application, the target sequence may include sequence identifier number: 29-5'-CTGTGGATATAT-3'.

[0180] To design the target sequence, the PAM sequence in the target region should be considered. The PAM sequence may differ depending on the origin of the Cas protein.

[0181] The PAM sequence and adjacent sequences are called protospacer sequences. Apart from the PAM sequence, the protospacer sequence consists of 20 or fewer nucleotide sequences. The protospacer sequence and the target sequence are complementary sequences.

[0182] In one example, the target sequence of the myostatin gene may be selected from sequence identification numbers 38-60 [Table 4].

[0183] For example, sequence identification numbers 38-43 may be target sequences of the myostatin gene in bovine animals.

[0184] For example, sequence identification numbers 42, 43, and 45-48 may be target sequences of the porcine myostatin gene.

[0185] For example, sequence identification numbers 49-55 may be target sequences of the human myostatin gene.

[0186] For example, sequence identification numbers 56-60 may be target sequences of the mouse myostatin gene.

[0187] [Table 4]

[0188] In one embodiment, the composition of the present application comprises a guide RNA or DNA encoding a guide RNA, which includes a guide sequence complementary to a target sequence; and a Cas protein or a nucleic acid sequence encoding a Cas protein.

[0189] Guide RNA or DNA encoding guide RNA

[0190] The guide RNA of this invention includes a guide sequence complementary to the target sequence described above.

[0191] The guide RNA may include a first sequence, which is a guide sequence that can bind complementaryly to the target sequence, and a second sequence that is involved in forming a complex by interacting with the Cas protein.

[0192] The first sequence of the guide RNA in this application is the same as the protospacer sequence complementary to the designed target sequence, and is an RNA sequence composed of U (uracil) instead of T (thymine) in the protospacer sequence.

[0193] In another embodiment, the first sequence of the present application may be a portion of crRNA, and the second sequence may include another portion of crRNA and / or tracrRNA. For example, the guide RNA may be the first and second sequences composed solely of crRNA, or, as an alternative example, the guide RNA may be the first and second sequences containing both crRNA and tracrRNA.

[0194] In this case, the first sequence may be determined according to the target sequence, and part of the second sequence may be determined according to the type of microorganism from which the Cas protein originates.

[0195] For example, in the case of a guide RNA that binds to the Cas protein derived from Streptococcus pyogenes, the first sequence may be part of the crRNA sequence, and the second sequence may include tracrRNA.

[0196] In one embodiment, for a guide RNA that binds to Streptococcus pyogenes protein, the second sequence may include 5'-GUUUUAGUCCCUGAAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' (sequence identification number: 61).

[0197] On the other hand, the guide RNA of the present application may be in the form of a single sequence in which the first sequence and the second sequence are ligated together. Alternatively, the guide RNA may consist of two separate sequences, one containing the first sequence and the other containing a portion of the second sequence, which may be composed of crRNA and tracrRNA, respectively.

[0198] The following table shows examples of guide sequences that can be used in one embodiment of the present invention. The guide sequences listed in [Table 5] are RNA sequences that can bind complementarily to the target sequence of the myostatin gene.

[0199] Each of the guide sequences listed in Table 5 is a guide sequence that can target the sequences in Table 2.

[0200] The guide sequence of this application may be a sequence selected from sequence identification numbers 62 to 84.

[0201] For example, sequence identification numbers 62-68 are guide sequences that can bind complementarily to the target sequence of the myostatin gene in bovine animals.

[0202] For example, sequence identification numbers 66, 67, and 69 to 72 are guide sequences that can bind complementarily to the target sequence of the porcine myostatin gene.

[0203] For example, sequence identification numbers 73-79 are guide sequences that can bind complementaryly to the target sequence of the human myostatin gene. For example, sequence identification numbers 80-84 are guide sequences that can bind complementaryly to the target sequence of the mouse myostatin gene.

[0204] In one embodiment of this disclosure, a complex of guide RNA and Cas protein containing the above guide sequence (ribonucleoprotein particle: RNP) can be injected into cells or embryos.

[0205] [Table 5]

[0206] On the other hand, in another embodiment, the present application may provide DNA encoding guide RNA. In this case, the DNA sequence encoding the guide RNA includes a first sequence, a sequence encoding the guide sequence and being identical to the target sequences represented by sequence identification numbers 38 to 60, respectively; and a DNA sequence encoding the second sequence.

[0207] Cas protein or nucleic acid encoding Cas protein

[0208] The Cas protein of this application may be selected from the group consisting of Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Cas9 protein derived from Staphylococcus aureus, Cas9 protein derived from Neisseria meningitidis, and Cas12a (Cpf1) protein. In this application, the Cas protein may be in the form of a wild type or a variant thereof.

[0209] In this application, the Cas protein or the nucleic acid encoding the Cas protein may further include elements commonly used for delivery to the nucleus of eukaryotic cells, such as a nuclear localization sequence (NLS).

[0210] In one embodiment, the Cas protein may be a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Staphylococcus aureus, or a Cas12a(Cpf1) protein.

[0211] The PAM sequence may vary depending on the Cas protein. In one embodiment, SpCas9 has the NGG PAM sequence. In one embodiment, SaCas9 has the NNGRR or NNGRRT PAM sequence. In one embodiment, Cas12a(Cpf1) has the TTTN PAM sequence. N is one of A, T, G, or C. R is A or G.

[0212] Forms of compositions for genetic manipulation

[0213] The composition for genetic manipulation of myostatin according to the present invention may contain, independently or together, a guide RNA or nucleic acid encoding a guide RNA; and a Cas protein or nucleic acid encoding a Cas protein.

[0214] The guide RNA of this application may be delivered to cells in the form of RNA or DNA encoding the guide RNA. The guide RNA may also be in the form of an independent RNA, which is contained in a viral vector or encoded in the vector.

[0215] The Cas protein of this application can be delivered to cells in the form of RNA or DNA encoding RNA. The Cas protein may also be in the form of an independent RNA, which is contained in a viral vector or encoded in the vector.

[0216] In this case, the viral vector may be selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, poxvirus vectors, and herpes simplex virus vectors.

[0217] In one embodiment, the guide RNA and Cas protein may be composed of plasmid DNA containing sequences that encode each RNA and promoter, and the plasmid DNA contains sequences that encode the protein and promoter.

[0218] In another embodiment, the guide RNA and Cas protein may be configured in a form in which a sequence encoding RNA or protein and a promoter is included in a single plasmid DNA.

[0219] Alternatively, the guide RNA and Cas protein may be configured in the form of a viral vector instead of plasmid DNA.

[0220] In another embodiment, the guide RNA and Cas protein may be in the form of mRNA. In this case, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art.

[0221] The guide RNA and Cas protein of the present invention may preferably be configured in the form of a ribonucleoprotein (RNP) complex to which the guide RNA and Cas protein are bound.

[0222] In another embodiment, the guide RNA and Cas protein may be composed of various forms. For example, the guide RNA may be in the form of an independent RNA, and the Cas protein may be in the form of a vector containing a protein and a sequence encoding a promoter.

[0223] Furthermore, the composition may be composed in various forms. Therefore, there are no limitations, as those skilled in the art can appropriately use methods known in the art.

[0224] Method for producing engineered cells containing an artificially modified myostatin gene

[0225] Another aspect of the present disclosure provided herein relates to a method for producing engineered cells having a myostatin gene in which 12 base pairs of the second exon are deleted, using the above composition.

[0226] The cells may be embryonic cells, somatic cells, or stem cells.

[0227] In certain embodiments, the cells include, but are not limited to, oocytes, epithelial cells, fibroblasts, nerve cells, keratinizing cells, hematopoietic cells, melaninizing cells, chondrocytes, macrophages, monocytes, muscle cells, and B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, placental cells, and germ cells. Furthermore, adult stem cells derived from various tissues of origin, such as adipose tissue, uterine tissue, bone marrow, muscle tissue, placenta, umbilical cord blood, or skin (epithelium), can be used. Non-human host embryos can generally be embryos including 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, aborted embryo, or blastocyst.

[0228] Preferably, the cells may be embryonic cells.

[0229] The cells may be derived from animals.

[0230] Animals include mammals.

[0231] Mammals include ungulates.

[0232] Ungulates may include, but are not limited to, animals of the Bovidae subfamily and pigs.

[0233] Mammals include rodents.

[0234] Rodents may include, but are not limited to, mice.

[0235] The present invention's method for producing engineered cells containing an artificially modified myostatin gene may include a step of bringing the cells into contact with the composition. The contact step may be performed in vivo or ex vivo.

[0236] For example, but not limited to, the contact step may be introduced into the cells by transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells.

[0237] The introduced composition causes indels (insertions and deletions) to occur in the cell's genome.

[0238] An "indel" is a general term for a mutation in which several nucleotides are inserted or deleted in the middle of a DNA nucleotide sequence. Indels can be introduced into a target sequence during the process of cleaving and repairing nucleic acids (DNA, RNA) by the guide RNA-CRISPR complex of a composition.

[0239] As a result of the indel, the manipulated cells of this invention have a myostatin gene in which 12 base pairs in the second exon are deleted by the composition.

[0240] Furthermore, according to the method for producing engineered cells containing the artificially modified myostatin gene described above, the engineered cells of the present invention have a genetic modification that results in an in-frame deletion.

[0241] The effects of in-frame deletion are explained below.

[0242] In-frame deletion effect

[0243] Manipulated cells having a myostatin gene in which 12 base pairs of the second exon of the present invention are deleted have a genetic modification that results in an in-frame deletion.

[0244] An "in-frame deletion" requires at least three DNA bases, usually multiple deletions of three, to result in the loss of an entire codon, which can lead to the deletion of a corresponding amino acid in the resulting protein.

[0245] Since this invention is characterized by the deletion of 12 base pairs in the myostatin gene, the deletion is an in-frame deletion and does not result in frameshift alteration.

[0246] As a result of the in-frame deletion, the protein is in a form where four amino acids are deleted, and no translation to other amino acids or modification of stop codons occurs, which can occur in a typical frameshift alteration. In other words, the remaining amino acids, apart from the four missing ones, can be normally translated into the protein via transcription in the myostatin gene.

[0247] Method for producing transgenic animals containing artificially modified myostatin genes

[0248] Another aspect of the disclosure provided herein relates to a method for producing an animal using manipulated cells. Specifically, the present application relates to a method for producing an animal having a myostatin gene in which 12 base pairs of the second exon are deleted.

[0249] In any embodiment, the production method is carried out by transferring an embryonic cell having a myostatin gene in which 12 base pairs of the second exon are deleted into a surrogate mother to produce a transgenic animal having an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted.

[0250] In another embodiment, the manufacturing method relates to a method for producing an animal having transgenic tissue or organs by injecting a composition into the tissue or organs of an animal.

[0251] Animals include mammals.

[0252] Mammals include ungulates.

[0253] Ungulates may include, but are not limited to, animals of the Bovidae subfamily and pigs.

[0254] Mammals include rodents.

[0255] Rodents may include, but are not limited to, mice.

[0256] Method for producing transgenic animals using manipulated cells

[0257] Method for producing transgenic animals containing the artificially modified myostatin gene of the present invention.

[0258] In one embodiment, the method may include a step of transferring the engineered cells generated in the above step, which have an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted, into a surrogate mother.

[0259] The conventional explanations of each stage can be understood by referring to methods for producing transgenic animals known in the art.

[0260] In this application, embryonic cells can be produced by the method described in the above “Method for Producing Manipulated Cells Containing an Artificially Modified Myostatin Gene,” which is completed by using the step of bringing the cells into contact with the composition.

[0261] Embryonic cells may develop into blastocysts during the in vitro culture process.

[0262] Animals having a myostatin gene with a 12-base pair deletion in exon 2 can be produced at the stage of transferring blastocysts into a surrogate mother.

[0263] In one embodiment of the present disclosure, an embryo having an artificially modified myostatin gene with a 12-base pair deletion in exon 2 is transplanted to produce an animal having an artificially modified myostatin gene with a 12-base pair deletion in exon 2, preferably a bovine subfamily animal (cattle) having a myostatin gene with a 12-base pair deletion in exon 2.

[0264] The transgenic animal may be a chimeric or homologous transgenic animal.

[0265] A method for producing the transgenic animal of the present application containing an artificially modified myostatin gene is Regarding another specific example, obtaining the manipulated somatic cells containing the above-mentioned artificially modified myostatin gene; preparing an enucleated oocyte by removing the nucleus from an animal egg; microinjecting and fusing the nucleus of the manipulated somatic cells into the enucleated oocyte; activating the fused egg; and transferring the activated egg into a surrogate mother may be included.

[0266] The conventional description of each step can be understood by referring to the method for preparing a transgenic animal using the conventional somatic cell nuclear transfer technology known in the art.

[0267] The transgenic animal can be prepared by transplanting a somatic cell having an artificially modified myostatin gene or its nucleus into an enucleated oocyte according to the above method using the SCNT (somatic cell nuclear transfer) method. The transgenic animal may be a homologous transgenic animal.

[0268] In another embodiment, as a method for producing homologous transgenic animals, first transgenic animals having a myostatin gene in which 12 base pairs of the second exon are deleted may be crossed with each other to produce homologous transgenic animals.

[0269] The transgenic animal obtained by mating may contain the same myostatin gene as the one contained in the animal genome of the first transgenic animal, but with 12 base pairs deleted.

[0270] A method for producing animals in which some tissues are transgenic.

[0271] The transgenic animals of this invention may be animals that have an artificially modified myostatin gene in which 12 base pairs of the second exon are deleted in some tissue of the animal.

[0272] The tissue may be epithelial tissue, connective tissue, or muscle tissue, but preferably muscle tissue containing the myostatin gene.

[0273] The method may include a step of introducing the above-mentioned composition into the tissue of an animal.

[0274] When the composition is introduced into animal tissue, the animal may be tissue-specifically manipulated and possess a modified myostatin gene within the tissue.

[0275] Introduction may be carried out by injection, implantation, or transplantation.

[0276] Induction may be performed via the selected route of administration: subretinal, subcutaneous, intradermal, intraocular, intravitreous, intratumor, intralymph node, intramedullary, intramuscular, or intraperitoneal.

[0277] Use of myostatin transgenic animals in this application

[0278] Improved breeds of animals

[0279] Animals possessing a myostatin gene with a deletion of 12 base pairs in exon 2 can be used as improved breed animals. Improved breed animals may be, but are not limited to, bovine animals, pigs, mice, or rats with a deletion of 12 base pairs in the myostatin gene. Improved breed animals may also have more developed muscles compared to wild-type animals. Improved breed animals may also have reduced fat compared to wild-type animals.

[0280] Animals for disease model research

[0281] Animals possessing an artificially modified myostatin gene in which 12 base pairs of exon 2 are deleted can be used as disease model animals. These disease model animals may be, but are not limited to, bovine animals, pigs, mice, or rats in which 12 base pairs of the myostatin gene are deleted. The disease model may include, but is not limited to, studies involving muscle atrophy, sarcopenia, and myofibrils.

[0282] disease resistant animals

[0283] Animals possessing a myostatin gene with a deletion of 12 base pairs in exon 2 can be used as disease-resistant animals. Disease-resistant animals may be, but are not limited to, bovine animals, pigs, mice, or rats with a deletion of 12 base pairs in the myostatin gene. The diseases may include, but are not limited to, muscle atrophy, sarcopenia, and myofibrils.

[0284] Use of by-products

[0285] The meat, organs, skin, fur, and body fluids of transgenic animals having a myostatin gene with a 12-base pair deletion in exon 2 may be used, but are not limited thereto. The transgenic animal may be, but is not limited to, a bovine subfamily animal, pig, mouse, or rat having a 12-base pair deletion in the myostatin gene. The transgenic animal may have a low fat content and a high muscle content as compared to wild-type animals. Therefore, as a by-product of the animal, it is possible to obtain high-quality meat having a low fat content and a high muscle content.

[0286] Use of the composition for gene manipulation of the present application

[0287] Another aspect of the present disclosure provided herein relates to the use of the composition for gene manipulation of the present application.

[0288] The above-described compositions may be used, but are not limited thereto, for the purpose of increasing muscle mass.

[0289] At this time, the subject to which the composition can be administered may be a mammal including primates such as humans and monkeys, rodents such as mice and rats, and ungulates such as bovine subfamily animals, pigs, and horses. [[ID=???]]

[0290] Another aspect of the present disclosure provided herein may provide a method capable of increasing muscle in an administered tissue, including the step of administering the composition for gene manipulation of the present application.

[0291] The composition may be administered to a specific body part of the subject to which the composition is administered.

[0292] [[ID=???]] The specific body part may be around a tissue that requires muscle growth.

[0293] The specific body part may be around a tissue in which muscle has not developed in the infant state.

[0294] It seems there is a formatting issue with the "ID=???" tags which should likely be consecutive numbers. Please check and correct if needed for a more accurate translation.Administration may be carried out by infusion, blood transfusion, implantation, or transplantation.

[0295] Administration may be carried out via the selected route of administration: subcutaneous, intradermal, intramuscular, or intraperitoneal.

[0296] The single dose of the myostatin gene-modified composition (the effective amount to achieve a predetermined desired effect) is 10 per kilogram of the subject's body weight. 5 ~10 6 10 cells / kg (body weight), etc. 4 ~10 9 The number of cells may be selected from any integer value within the above range, but is not limited to these, and may be administered appropriately considering the age, health, and weight of the subject.

[0297] When the myostatin gene is artificially manipulated by methods or compositions of some embodiments disclosed herein, effects such as muscle growth may be obtained thereby.

[0298] The present invention will be described in more detail below by examples.

[0299] These embodiments are intended solely to illustrate the present application in more detail, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these embodiments.

[0300] [Examples]

[0301] [Example 1] Design of a single guide RNA (sgRNA)

[0302] Using CHOPCHOP software (https: / / chopchop.cbu.uib.no / ), sgRNAs containing sequences complementary to all single strands of the 12 base pairs of myostatin were designed. The sgRNAs contain the complementary binding sequences mentioned above and are used within the CRISPR / SpCas9, CRISPR / SaCas9, or CRISPR / Cpf1 PAM sequences related to the myostatin gene. The sgRNAs used in the experiments were designed to contain at least one of the guide sequences listed in Table 2.

[0303] Figure 1 shows one of the protospacer sequences of the myostatin gene.

[0304] The binding sequence of the guide RNA may be predicted in the sequence by binding the guide RNA to the complementary sequence (target sequence) of the sequence shown in Figure 1.

[0305] [Example 2] In vitro maturation of oocytes

[0306] Ovaries were collected from local slaughterhouses and sent to the laboratory within two hours. The ovaries transported from the slaughterhouses were aspirated using an 18-gauge needle syringe to obtain cumulus-oocyte complexes (COCs) derived from follicles with a diameter of 2–8 mm. COCs were classified as being surrounded by more than three layers of cumulus cells and uniformly distributed cytoplasm. During the in vitro maturation process, COCs were cultured in chemically defined TCM-199 medium containing 0.005 AU / mL FSH (Antrin, Teikoku, Cat. No. F2293), 1 μg / mL 17β-estradiol (Sigma-Aldrich, Cat. No. E4389), 100 μM cysteamine (Sigma-Aldrich, Cat. No. M6500), and 10% FBS (Gibco®, Cat. No. GIB-16000-044) in a humid atmosphere of 5% CO2 at 38.5°C.

[0307] [Example 3] Sperm purification, in vitro fertilization, and in vitro culture of embryos

[0308] Motile sperm were purified using the Percoll gradient method. Sperm derived from semen thawed at 35°C were filtered by centrifugation at 1500 rpm on a Percoll discontinuous gradient (45%-90%) for 15 minutes. To prepare a 45% Percoll solution, 1 mL of TALP was added to 1 mL of 90% Percoll. The sperm pellet was centrifugated at 1500 rpm for 5 minutes and washed twice by adding 3 mL of TALP. Motile sperm purified by the Percoll gradient method were used for fertilization. Along with mature oocytes, 1-2X sperm were fertilized in 45 μl of IVF-TALP medium coated with mineral oil (Nidacon, Cat. No. NO-100) in a humid atmosphere of 5% CO2. 6 Sperm with motile sperm counts of 100 / mL were cultured. Eighteen hours after in vitro fertilization, cumulus cells were removed from the zygotes. These zygotes were cultured in a medium protected by two levels of chemically defined mineral oil at a temperature of 38.5°C in a 5% O2, 5% CO2, and 90% N2 atmosphere. The zygotes were cultured into embryos.

[0309] [Example 4] Microinjection

[0310] When microinjection was performed, Cas9 mRNA and sgRNA were divided into four groups to find the most suitable concentrations. (CB; TE only microinjection; RNA1X; Cas9 mRNA: 100 ng / μL, sgRNA: 50 ng / μL; RNA2X; Cas9 mRNA: 200 ng / μL, sgRNA: 100 ng / μL; RNA4X; Cas9 mRNA: 400 ng / μL, sgRNA: 200 ng / μL). 18 hours after in vitro fertilization, Cas9 mRNA (sigma-Aldrich, Cat. No. CAS9MRNA) and sgRNA were synthesized using the GeneArt Precision gRNA Synthesis Kit (Thermofisher, Cat. No. A29377) and injected into zygotes using a microinjector (Eppendorf, Femtojet®). Seven days after microinjection, preimplantation embryos were retrieved and myostatin deletion was observed, or they were implanted in the surrogate mother's uterus.

[0311] The microinjection method is illustrated in Figure 2.

[0312] Figure 5 schematically illustrates the results of experiments in which Cas9 mRNA and sgRNA were divided into four groups.

[0313] From the results above, the proportion of blastocysts in both RNA1X and RNA2X was similar to that of the wild type. Looking at the modification rates, the RNA2X group showed significantly higher modification rates than the other RNA1X and RNA4X groups. Therefore, the RNA2X concentration was determined to be the most suitable, and experiments were then conducted using the concentrations of Cas9 mRNA: 200 ng / μl and sgRNA: 100 ng / μl used in the RNA2X group.

[0314] Figures 3 and 4 show the myostatin modification of embryos after microinjection.

[0315] Figure 3 shows that when myostatin in embryos is modified by performing the T7E1 assay, unlike the wild type, another band below 530 bp is observed, similar to the positive control. As a result, it can be confirmed that the myostatin gene is modified in the embryo.

[0316] Figure 4 shows the results of myostatin-modified morphology of embryos obtained by performing Sanger sequencing.

[0317] As can be seen from the results above, in the embryo, base pairs 1, 2, 3, 10, and 12 of the second exon of the myostatin gene were deleted, and one base pair of the second exon of the myostatin gene was inserted.

[0318] Thus, unlike animals, we confirmed that embryos can have various modified forms as a result of indels, as well as forms in which only 12 base pairs of the myostatin gene in exon 2 are deleted. However, the manipulated cells of this application refer only to manipulated cells that have the myostatin gene in which 12 base pairs of exon 2 are deleted.

[0319] [Example 5] Embryo transfer and pregnancy diagnosis

[0320] Blastocysts were stored in PBS supplemented with 20% FBS. Using a non-surgical method, the blastocysts were transferred into the uterus of each surrogate mother around day 7 via the cervical approach (estrus = day 0 = fusion day). Fifty days after estrus, the surrogate mothers were examined by rectal examination and ultrasound to observe embryo survival and pregnancy. Subsequently, pregnant cows were regularly confirmed by rectal examination and ultrasound.

[0321] To observe the changes in appearance during the growth process of cow No. 17 (Figure 6) after giving birth, photographs were taken at one-month intervals from one month postpartum to four months of age.

[0322] As can be seen in the photo above, cow No. 17 is a cow in which 12 base pairs are deleted in the second exon of the myostatin gene. Muscle development is visible to the naked eye, and the appearance of muscle development is even more pronounced after 3 months.

[0323] [Example 6] T7E1 assay

[0324] Genomic DNA was extracted from transgenic primary cells using a DNA extraction kit (DNeasy Blood & Tissue Kit, Qiagen, Cat. No. 69504). MSTN primers were designed using PRIMER3 software. PCR conditions were 94°C for 5 minutes, 94°C for 20 seconds, 57°C for 30 seconds, 72°C for 35 seconds, and 72°C for 5 minutes for 35–40 cycles.

[0325] In Figure 7, the results of 12 base pairs of the myostatin gene in cow No. 17, one of the cows born in Example 5, were confirmed by the T7E1 assay.

[0326] Based on the above results, the T7E1 assay confirmed that, unlike the wild type, cattle No. 17 had a differently located cleavage band, and consequently, cattle No. 17 possessed a modified myostatin gene.

[0327] [Example 7] Gene expression by real-time PCR

[0328] Total RNA was extracted from primary cultured cells using the RNeasy® Mini Kit (Qiagen, Cat. No. 74106), and complementary DNA was synthesized from 1 μg of RNA to cDNA EcoDry® Premix (OligodT) using RNA (Takara, Cat. 639543). Gene expression analysis was performed using the SYBR Green method in QuantStudio 3 (Applied Biosystems, Model No. A28132), and the relative cycle threshold (CT) values ​​were normalized by GAPDH. The primers used in the above example are listed under sequence identification numbers 87 to 90.

[0329] [Table 6]

[0330] [Example 8] Analysis of MSTN off-target effects

[0331] Using Cas-OFFinder software and a fast and versatile algorithm for searching for potential off-target sites of Cas9 RNA-derived endonucleases, we investigated potential off-target effects attributable to CRISPR / Cas9 in three MSTN mutant calves. We set the number of mismatches to 3 at the MSTN target site used in the experiment, and identified five nucleotide sequences that target the entire bovine gene. Primers targeting each of the five sequences were named sequence identification numbers 89 to 100, and off-target effects based on primer location were confirmed by T7E1 analysis.

[0332] In Figure 8, the T7E1 assay was performed using the following primer sequences to determine the location of potential off-target effects.

[0333] As the results show, unlike the positive control, truncated bands for confirming off-target effects were not identified at all five locations. Therefore, as a result, it was confirmed that there are no off-target effects of CRISPR / Cas9 at potential off-target sites, and that the guide RNA and Cas9 mRNA used in one embodiment of the present invention act in a target-specific manner.

[0334] [Table 7]

[0335] [Example 9] Targeted deep sequencing

[0336] First, target sites were amplified to approximately 500 bp in size from extracted genomic DNA using KAPA HiFi HotStart DNA polymerase (Roche, Cat. No. #KK2502) according to the manufacturer's protocol. The amplicons were then re-amplified to a maximum size of 230 bp, and subsequently amplified using TruSeq HT double-index primers to add adapter and index sequences for the Illumina sequencing platform to each sample. The primers used in this study are listed under sequence identification numbers 101 and 102. Pooled PCR ampoules were purified using a PCR Refining Kit (MGmed) and sequenced using a paired-end sequencing system (2 x 150 bp) on a MiniSeq (Illumina). Indel rates in the deep sequencing data were quantified using Cas-Analyzer.

[0337] The results of targeted deep sequencing can be seen in Figures 9-13.

[0338] Figure 9 shows the results of targeted deep sequencing of 17 cattle and wild-type cattle born by transferring engineered embryos containing the modified myostatin gene of this application into surrogate mothers. As can be seen from these results, unlike wild-type cattle, the indel rates of cattle No. 6, No. 14, and No. 17 were 10.45%, 45.4%, and 99.98%, respectively.

[0339] A more detailed list of the results in Figure 9 can be seen in Figures 10-13.

[0340] Referring to Figure 10, it was confirmed that the base pair of the second exon of the myostatin gene is not altered in wild-type cattle. The gray box represents the PAM sequence. The underlined sequence is the protospacer sequence. The gray box and underline are used in exactly the same way in Figures 10-13.

[0341] In Figure 11, it was confirmed that 12 base pairs in the second exon of the bovine myostatin gene No. 6 were deleted. Upon checking the indel rate, the 12 base pairs were found to be deleted at 10.45%, which confirmed the reading result.

[0342] In Figure 12, it can be seen that in cattle No. 14, 12 base pairs within the base pairs of the second exon of the myostatin gene are determined in the same manner as in Figure 11. The indel rate in cattle No. 14 was 45.4%.

[0343] Figure 13 shows that 12 base pairs in the second exon of bovine myostatin gene 17 are deleted. The indel rate of bovine gene No. 17 was confirmed to be 99.98%.

[0344] Based on the results above, in this application, targeted deep sequencing of cattle born after the induction of modification of myostatin's second exon confirmed that the bases of myostatin's second exon were not modified. However, in all three cattle in which modification was confirmed, it was found that only 12 base pairs of the second exon were deleted.

[0345] [Table 8]

[0346] [Example 10] Culture of primary cells

[0347] Primary cells derived from bovine ear skin were obtained using a biopsy punch. The bovine auricles were cut into small pieces using a sterile surgical scalpel, washed multiple times, and cultured for 4-18 hours at 38°C in HANK's equilibrium salt solution (Gibco®, Cat. No. 14175095) supplemented with collagenase (Collagenase type I, Gibco®, Cat. No. 17-017). After one night, the dispersed cells were washed multiple times in DMEM (Gibco®, Cat. No. 21068028) medium and supplemented with 10% fetal serum (Gibco®, Cat. No. GIB-11150-059) and 1% penicillin / streptomycin (Gibco®, Cat. No. GIB-11150-059). Cat. No. 15140148), 1% non-essential amino acids (Gibco®, Cat. No. 11140050), and 100 ml β-mercaptoethanol (Sigma-aldrich, Cat. No. M3418).

[0348] In Figure 7, where the T7E1 assay was performed during the process of the above example, it was performed after culturing primary cells.

[0349] In Figure 14, the levels of myostatin mRNA expression in wild-type cattle and cattle No. 14 and No. 17 were compared and plotted after culturing primary cells of cattle No. 14 and No. 17 born in this application.

[0350] As can be seen from the results above, the amount of myostatin mRNA expression in primary cells of bovine No. 14 and No. 17 was reduced by more than 60% in the case of bovine No. 14 and by 80% in the case of bovine No. 17.

[0351] Therefore, it was confirmed that myostatin mRNA expression was reduced in cattle in which 12 base pairs of the second exon of the myostatin gene described in this application were deleted, compared to wild-type cattle.

[0352] [Example 11] Germline transmission in MSTN knockout cattle

[0353] Experimental method

[0354] 1) Donor management and egg collection (Ovum Pick Up; OPU)

[0355] Random MSTN mutant donor cows with different estrous cycles, implanted using vaginal progesterone devices (Repro360, Cue-mate), were intramuscularly injected with 2.0 mg of estradiol benzoate. On days 4 and 5, 200 mg of FSH (Kawasaki Pharm, Antonin R-10) was administered to the donors every 12 hours in four divided doses (57, 57, 43, and 43 mg). Prior to OPU, the P4 instrument was immediately removed on the 7th day.

[0356] For OPU (Ovarian Pulse Purification), donor cows were restrained from bovine crushing. Epidural anesthesia was administered using 5 mL of 2% lidocaine (Daihan, DAIHAN Lidocaine, Korea). The ovaries were fixed transrectally and kept on the probe of an ultrasound device. A trained OPU technician performed the OPU procedure using an ultrasound device (Esaote, MyLab One) connected to a 7.5 MHz transrectal transducer probe with a follicular aspiration guide (WTA, catalog no. 10283). Follicular puncture was performed using an 18G OPU threaded needle (WTA, catalog no. 17927), and follicular fluid was collected in a 50 mL tube. Oocytes from the follicular fluid were collected under a stereomicroscope and used for in vitro fertilization. The remaining small amount of follicular fragments were used for primary culture.

[0357] 2) Semen collection

[0358] Semen was collected from MSTN mutant bulls using electroejaculation. (Three times per bull). Before semen collection, the foreskin was cut, the opening was washed with clean water, and then dried with a clean paper towel to minimize contamination. Electroejaculation was performed using a manually controlled electroejaculator, ElectroJac6 (Ideal® Instruments Neogen Corporation, Michigan, USA, Lansing), which had a 6.5 cm diameter rectal probe attached with three ventral electrodes spaced approximately 1 cm apart, with the electrodes fully inserted into the rectum toward the abdominal cavity. The number of electrical stimulations was increased until the bull ejaculated. Each stimulation was lasted 8–10 seconds, with a pause of approximately 2.0 seconds before the next stimulation was applied. When semen secretion became opaque, the collection tube was placed on the penis to collect the semen. The ejaculated semen was transferred to the laboratory at 25°C within 30 minutes.

[0359] 3) Freezing and thawing of semen

[0360] Semen samples were used for cryopreservation if they showed general motility of 60% or higher. The semen samples were bulked up at 37°C using Optixcell® (IMV Technologies). The bulked semen was equilibrated at 4°C for 3 hours and then placed in 0.5 mL straws. The filled straws were placed in a dedicated rack 5 cm above liquid nitrogen and exposed to liquid nitrogen vapor for 15 minutes, and then placed in a cryogenic bath filled with liquid nitrogen (-196°C). The cryopreserved sperm were thawed in a 37°C water bath for 45 seconds.

[0361] 4) Sperm motility assay

[0362] To analyze and quantify sperm motility, the IVOS-II CASA (Computer Assisted Sperm Analysis Program) system was used according to the manufacturer's instructions. Briefly, frozen semen was thawed, incubated, and purified using the same protocol as used for IVF. Three μl of sperm were then packed into a sperm analysis chamber (Leja slide) and analyzed by CASA. Frozen straws from three different bovines were used. To eliminate technical error, each semen sample was analyzed three times, and the mean value of the CASA results was used for statistical evaluation.

[0363] 5) In vitro fertilization and culture

[0364] Motile sperm were selected using the Percoll gradient method as previously described. Briefly, semen from F0 bovine follicular 6 100 sperm / mL were cultured. After 18 hours of co-incubation in CO2 at 38.5°C, cumulus cells were removed from the putative zygotes. The zygotes were cultured in a two-step chemically defined medium coated with mineral oil at 38.5°C in an atmosphere of 5% O2, 5% CO2, and 90% N2.

[0365] Experimental results

[0366] 1) Germline transmission in MSTN mutant cows

[0367] After performing OPU, all 45 oocytes were collected (n=3). Following in vitro fertilization with wild-type frozen-thawed semen, the 45 oocytes were cultured, resulting in the formation of 5 blastocysts (12.5±10.9%) (Figure 15a). Selected blastocysts were transferred to 5 recipients. Pregnancy was confirmed in one recipient by rectal examination and ultrasound (Figure 15b). Furthermore, the MSTN mutation was confirmed by culturing the follicular fluid obtained during OPU (Figure 16). T7E1 assay and Sanger sequencing confirmed the same mutation as in the F0 females in both the remaining embryos and follicular fluid-derived cells (Figures 17 and 18). These results demonstrate the success of germline transmission in MSTN mutant cattle using the OPU technique.

[0368] Figure 15 shows the results of germline transmission in MSTN mutant females, including the production of MSTN mutant blastocysts derived from MSTN mutant bovine oocytes (a) and representative images of pregnancy diagnosis using an ultrasound device at day 30 (b).

[0369] Figure 16 shows images of somatic cells derived from follicular fluid obtained by the OPU process ((a): MSTN mutant female, (b): wild type).

[0370] Figure 17 shows the results of the T7E1 assay (a) and sequencing data (b) of MSTN mutant female blastocysts (M: marker; WT: wild type; 1: MSTN mutant female; N: negative control group; P: T7E1 positive control group).

[0371] Figure 18 shows the results of the T7E1 assay (a) and sequencing data (b) from somatic cells of follicular fluid (1: MSTN mutant female (not wild-type); 2: MSTN mutant female (not wild-type)).

[0372] 2) Germline transmission in MSTN mutant males

[0373] Semen was collected from male bovines (F0) with a 10.5% mutation by electroejaculation. The samples were frozen for in vitro fertilization and thawed for sperm motility. CASA showed significant differences between F0 and wild-type samples in forward cells (%), VCL, ALH, and BCF. However, LIN and STR did not show significant differences between F0 and wild-type samples (Figure 19). Furthermore, no adverse effects on embryonic development and competence were observed when semen samples were used for in vitro fertilization. Oocytes collected from slaughterhouses were fertilized with frozen and thawed semen and cultured to develop into blastocysts. A total of 335 oocytes (replica number = 3) were used. Of these, 261 oocytes (78.9 ± 10.8%) cleaved, forming 166 blastocysts (50.5 ± 6.8%) (Figure 20). The total cell count was 81.3 ± 20.6 (n=20). Mutations were analyzed in 117 blastocysts, and 15 blastocysts showed MSTN mutations (12.7 ± 3.1%) (Figure 21).

[0374] Figure 19 shows an overview of semen from the MSTN male founder, analyzed by Computer-Assisted Semen Analysis.

[0375] Figure 20 shows a photograph of a representative blastocyst (a blastocyst produced in vitro using semen from an MSTN mutant bovine) as a result of verified germline transfer from an MSTN mutant bovine.

[0376] Figure 21 shows the mutation rates of the MSTN gene in blastocysts derived from semen of MSTN mutant bovine fertilized in vitro (1-6: randomly selected blastocysts). The upper panel (a) shows the results for T7E1, and the lower panel (b) shows the sequencing results for the MSTN target site.

Claims

1. A transgenic animal possessing a myostatin gene that has been artificially modified in the genome, The artificial modification is located in the second exon of the myostatin gene. The aforementioned artificial modification involves the deletion of 12 base pairs in the second exon that correspond to the region encoding the amino acid sequence of leucine, tryptophan, isoleucine, and tyrosine, compared to the amino acid sequence of myostatin in wild-type animals. The transgenic animal expresses less myostatin gene mRNA than the wild-type animal and expresses mature myostatin protein with the same sequence as the wild-type animal. Transgenic animals.

2. The aforementioned transgenic animal is a mammal other than a human. The transgenic animal according to claim 1.

3. The aforementioned transgenic animal is a member of the Bovidae subfamily. The transgenic animal according to claim 1.

4. The promyostatin protein expressed in the body of the aforementioned bovine subfamily animal has an amino acid sequence represented by sequence identification number:

30. The transgenic animal according to claim 3.

5. A manipulated cell having an artificially modified myostatin gene in its genome, The artificial modification is located in the second exon of the myostatin gene. The aforementioned artificial modification involves the deletion of 12 base pairs in the second exon corresponding to the region encoding the amino acid sequence of leucine, tryptophan, isoleucine, and tyrosine, compared to the amino acid sequence of myostatin in wild-type cells. The manipulated cells express less myostatin gene mRNA than wild-type cells and express mature myostatin protein with the same sequence as wild-type cells. Manipulated cells.

6. The aforementioned cells are at least one selected from embryonic cells, stem cells, and somatic cells. The manipulated cells according to claim 5.

7. The aforementioned cells were obtained from mammals. The manipulated cells according to claim 5 or 6.

8. The aforementioned cells were obtained from animals of the Bovidae subfamily. The manipulated cells according to claim 5 or 6.

9. The promyostatin protein expressed in the body of the manipulated cells has an amino acid sequence represented by sequence identification number 30. The manipulated cell according to claim 8.

10. A guide RNA having a guide sequence capable of forming a complementary bond with a target sequence, or DNA encoding such a guide RNA; and Cas protein or nucleic acid sequence encoding it; Equipped with, The aforementioned target sequence includes one or more sequences selected from sequence identification numbers 38 to 60. The aforementioned guide sequence includes one or more sequences selected from sequence identification numbers 62 to 84. A composition for modifying the myostatin gene.

11. The Cas protein is selected from Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Staphylococcus aureus, and Cas12a protein (conventional CPF1: Prevotella and Francisella 1). The composition according to claim 10.

12. The composition comprises a plasmid vector containing DNA encoding the guide RNA and DNA encoding the Cas protein. The composition according to claim 10 or 11.

13. The composition comprises a viral vector containing DNA encoding the guide RNA and DNA encoding the Cas protein. The composition according to claim 10 or 11.

14. The aforementioned viral vector is at least one selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, poxvirus vectors, and herpes simplex virus vectors. The composition according to claim 13.

15. The above composition is formed in a ribonucleoprotein (RNP), which is a complex of the guide RNA and the Cas protein. The composition according to any one of claims 10 to 14.

16. The step of contacting cells or embryos with the composition according to any one of claims 10 to 15. Equipped with, A method for producing engineered cells or embryos having an artificially modified myostatin gene.

17. The contact step is carried out by one or more methods selected from microinjection, electroporation, liposome use, plasmid use, viral vector use, nanoparticle use, and PTD (protein translocation domain) fusion protein method. The method according to claim 16.

18. A method for producing transgenic animals having an artificially modified myostatin gene, A step of preparing an engineered embryo having an artificially modified myostatin gene by contacting the embryo with the composition described in claim 10; and The step of transferring the manipulated embryo to the surrogate mother; Equipped with, The transgenic animals produced express less of the artificially modified myostatin mRNA than the myostatin mRNA expressed in wild-type animals. method.

19. The transgenic animal is a mammal other than a human. The method according to claim 18.

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