KR102207352B1 - klotho knock-out porcine Models for klotho deficiency disease and the Use thereof - Google Patents

Abstract

The present invention relates to a transgenic pig for a klotho deficiency disease model and a method for manufacturing the same, and to its use, and more specifically, to using a transgenic pig that has knocked out the klotho gene as a disease animal model. .

Description

Klotho knock-out porcine models for klotho deficiency disease and the use thereof

The present invention relates to a transgenic pig for a klotho deficiency disease model and a method for producing the same, and to the use thereof, and more specifically, a transgenic pig that knocks out the klotho gene as a disease animal model. It's about things.

Rational manipulation of large DNA constructs is a major challenge for current synthetic biology and genome manipulation efforts. Recently, various techniques have been developed to address this challenge and increase the specificity and rate at which mutations can be generated.

However, it is difficult to find a model animal that expresses all human disease symptoms enough to be used as an effective disease model.

The need for diseased animal models using species closer to humans has been raised due to the distinct differences in dimension, reproduction, longevity, and behavior from humans, and primates are extremely limited due to their scarcity and cost and difficulty in breeding management. Pigs are already used in research for pathological mechanisms and treatment of various diseases as they have been recognized for disease research only in the field of disease, and have been recognized as an economical animal for a long time. It can avoid ethical problems than when using it as a disease model, and it has the advantage of being easy to maintain and manage when developing an experimental animal model because a stable breeding system is established.

Accordingly, the present inventors use pigs that are recognized for similarity with human genes, but use the CRISPR system to recognize and cut the specific sequence of human Klotho gene, and apply it as a transgenic somatic cell cloning technique, thereby knocking out the Klotho gene and effectively diseased animals. It was confirmed that the model could be produced and the present invention was completed.

It is an object of the present invention to provide a pig for a klotho deficiency disease model and a method for manufacturing the same.

Another object of the present invention is to provide a gene knockout or knockdown method using the CRISPR system, which is necessary for the method of manufacturing a pig for the disease model.

Another object of the present invention is to provide various uses of the pig for the klotho deficiency disease model.

In order to solve the above problems, the present invention

For the production of pigs for klotho deficiency disease models, artificial manipulation of the klotho gene and its use are provided. More specifically, it relates to a method for producing a pig having a klotho-deficient phenotype using artificial manipulation or modification of the klotho gene and its use.

The present invention provides a composition for manipulation or modification of klotho gene and a method of using the same for a specific purpose.

The "klotho gene" plays a crucial role in regulating aging and the development of age-related diseases in mammals. Klotho is an enzyme produced by the KL gene, which produces a type I membrane protein related to β-glucuronidase. The human klotho gene is located at 33.59-33.64 Mb of chromosome 13, and has an mRNA sequence of, for example, NM_004795. In addition, it has the protein sequence of NP_004786.

The Klotho deficiency disease may be one or more diseases selected from the group consisting of arteriosclerosis, osteoporosis, stroke and Alzheimer's.

In one embodiment of the present invention, the klotho gene is knocked out by the modification in the nucleic acid sequence of the klotho gene, klotho protein expression is inactivated, provides a Klotho deficient disease pig model.

The modification in the nucleic acid sequence may include deletion, substitution, insertion or inversion of one or more nucleic acids.

In one embodiment of the present invention, the modification of the nucleic acid may occur in the exon 3 region of the klotho gene.

In one embodiment of the present invention, modifications in the nucleic acid sequence can be artificially caused by a nuclease preparation.

For example, the nuclease agent may use one or more of the following nucleases:

(a) zinc finger nuclease (ZFN);

(b) transcription activating factor-like effector nuclease (TALEN);

(c) meganucleases; or

(d) RNA-Guided Endonuclease (RGEN)

In one embodiment, the modification in the nucleic acid sequence may be artificially manipulated by RNA-Guided Endonuclease (RGEN). The subject can be a target nucleic acid, gene, chromosome or protein.

The endonuclease is Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiop.

Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces viridochrom Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus sedomycoides, Bacillus pseudomycoides, Bacillus selenitilenduce ), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Burkholderiales bacterium , Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis ae Luginosa (Microcystis aeruginosa), Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulose syrupor bescii, Caldicelruptor bescii Candidatus Desulforudis, Clostridium bot ulinum), Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Ashidithio Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus halophilus Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Metanohalobium evestigatum, Ana Anabaena variabilis, Nodularia spumigena, Nodularia sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis , Thermosipho africanus (Thermosipho africanus) or acariochloris marina (Acaryochloris marina) can be derived from. Further examples of the Cas9 family are described in WO 2014/131833, the entire contents of which are incorporated herein by reference. It may be a Cas9 protein derived from Streptococcus pyogenes.

For example, the Klotho gene can be genetically engineered by RNA-guided endonuclease,

In the proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence constituting the Klotho gene, or in a contiguous 3bp to 25bp nucleotide sequence site located adjacent to the 5'end and/or 3'end thereof

Deletion or insertion of one or more nucleotides;

Substitution with one or more nucleotides different from the wild-type gene;

Insertion of one or more nucleotides from outside

It may include modification of one or more of the nucleic acids.

The PAM sequence may be different depending on the microorganism derived from the Cas protein. A detailed description will be described later. As a representative example, the RNA-guided endonuclease is a Cas9 protein derived from Streptococcus pyogenes, and the PAM sequence is NGG (N is A, T, C, or G).

The modification of the nucleic acid may occur at two or more sites in the genome sequence of the entire chromosome. In a specific embodiment, in at least one site, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides may be changed in the Klotho gene nucleic acid sequence. Thereby, the Klotho gene can be knocked out or knocked down.

The modification of the nucleic acid may occur in the promoter region of the gene.

The modification of the nucleic acid may occur in the exon region of the gene.

The modification of the nucleic acid may occur in the intron region of the gene.

The modification of the nucleic acid may occur in the enhancer region of the gene.

In addition, in another embodiment, the present invention provides a guide nucleic acid capable of forming a complementary bond to a target sequence among the nucleic acid sequences of a reproductive regulatory factor, for example, a Klotho gene. The target sequence may be one or more sites in the nucleic acid sequence of the Klotho gene.

The guide nucleic acid may be, but is not limited to, a nucleotide of 18 to 25 bp, 18 to 24 bp, 18 to 23 bp, 19 to 23 bp, 19 to 23 bp, or 20 to 23 bp.

For example, the following guide nucleic acids can be provided:

5'-TAGAACAAGGCTGAAGACTTCGG-3' (SEQ ID NO: 1)

In addition, in an embodiment, the present invention can provide an artificially engineered cell or embryo comprising at least one of the artificially engineered Klotho gene and products expressed therefrom.

The cell or embryo contains an inactivated Klotho gene that has undergone modification in a nucleic acid sequence, and can develop into an individual having a phenotype thereof.

Further, in an embodiment, the present invention can provide a composition for genetically engineering a Klotho gene and use thereof.

In one embodiment, a guide nucleic acid capable of forming a complementary bond to at least one portion of the nucleic acid sequence constituting the Klotho gene; And it provides a composition for Klotho genetic engineering comprising a RNA-guided endonuclease or a nucleic acid encoding the same.

The related configuration description is as described above.

In an embodiment, the present invention

In a cell or embryo containing a target genomic locus on the Klotho gene,

(a) a guide RNA capable of complementarily binding to the target genomic locus site; And

(b) RNA-guided endonuclease (RNA-Guided Endonuclease; RGEN)

It may provide a method for producing a cell or embryo in which the nucleic acid sequence of the Klotho gene has been altered, comprising the step of contacting.

The guide RNA and endonuclease may be present in one or more vectors in the form of a nucleic acid sequence, respectively, or may be present by forming a complex by binding of a guide nucleic acid and an RNA-guide endonuclease.

The contacting may be performed in vivo or ex vivo.

In one embodiment, the step of contacting may be introduced into pig cells in vitro in the form of a ribonucleoprotein (RNP) consisting of a Cas9 protein and a guide RNA.

The contacting may be performed by at least one method selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles, and PTD (Protein translocation domain) fusion protein methods.

The viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

In an embodiment, the present invention

It provides a cell line transformed with a recombinant vector for making a disease pig model that knocks out a Klotho gene, and a pig for a disease model in which the Klotho gene is knocked out using the same. In this case, the transformation may be performed by somatic cell nuclear transfer (SCNT).

The present invention includes all of the transformation vectors, cells, and nuclear transfer embryos required for the production of a Klotho deficiency disease model that can be obtained in the process of the manufacturing method of the pig for the Klotho deficiency disease model.

That is, the present invention is another specific example,

It is possible to provide a transformed cell into which a recombinant vector for constructing a Klotho deficient pig model containing a sequence encoding a guide nucleic acid and RNA-guided endonuclease having an activity of recognizing and cleaving a specific sequence of the Klotho gene was introduced, and ,

A nuclear transfer embryo of a pig formed by transplanting the nucleus of the transformed pig-derived cell into an enucleated egg may also be provided.

In embodiments, the present invention can provide a method of obtaining an individual produced by the above method and such an individual.

The individual may be one or more animals selected from the group consisting of non-human primates, cattle, horses, pigs, sheep, chickens, birds, rabbits, goats, dogs, cats and fish.

In addition, the present invention provides a variety of uses of the pig for the Klotho deficiency disease model.

As an example, there is provided a method of screening for a Klotho deficiency disease prevention and treatment, comprising:

Administering a candidate material for prevention and treatment to the pig for the Klotho deficiency disease model; And

After administration of the candidate substance, analyzing the pig tissue compared to a control group to which the candidate substance was not administered.

As such, the present invention provides an animal model of Klotho deficiency disease using a pig, which is an animal most similar to a human physiologically, and various uses thereof.

The pig for the Klotho deficiency disease model of the present invention is genetically similar to humans, is useful for genetic analysis, is useful for studying the exact cause and mechanism of the disease, as well as the optimal animal model for toxicity and safety evaluation. It will be very useful as an animal model for Klotho deficiency disease, as it can be used in various ways such as the identification of the drug, the search for therapeutic substances, and the development of diagnostic methods.

1 is a schematic representation of sgRNA specific to exon 3 of the porcine Klotho gene. The sgRNA target sequence is highlighted in red and the protospacer contiguous motif (PAM) is indicated in blue.
Figure 2 is a result of the analysis of the T7 endonuclease I (T7E1) assay for the generation of klotho gene knockout blastocysts by SCNT using porcine fibroblasts not transfected with Cas9-sgRNA RNP. [T7E1 assay is genomic in 20 replicated blastocysts. Performed using DNA (M, Marker, WT, wild type, BL, blastocyst)]
3 is a diagram showing an exon 3 editing scheme of the klotho gene of the blastocyst in FIG. 2.
4 is a photograph of a recipient's uterus (A) and fetus (B) on the 28th day of pregnancy.
FIG. 5 is a table schematically illustrating the editing scheme for exon 3 of the klotho gene of the fetus produced by SCNT using unselected porcine fibroblasts transfected with Cas9-sgRNA RNP (Fetus A, absorbed fetus; Fetus L, living) fetus).
6 is a graph showing the expression of senescence and apoptosis-related genes in klotho monallelic knockout fetal fibroblasts, (A) IGF1 signaling genes (IGF1 and IGF1R), (B) FOXO1 and antioxidant genes (MnSOD and CAT), (C) Apoptosis. It is a graph of related genes (Bax/Bcl2 ratio and Caspase3). Data are presented as mean±SEM, and groups marked with different letters within each category showed significant differences (P<0.05). The experiment was repeated at least three times (WT, wild type; Fetus L2, living fetus 2 (WT/-17bp, +12bp)).
7 is a graph comparing the expression of senescence-related genes and klotho proteins between klotho monallelic knockout and wild-type placenta, (A) IGF1 signaling genes (IGF1 and IGF1R), (B) FOXO1 and antioxidant genes (MnSOD and CAT), (C) It is a graph of Apoptosis-related genes (Bax/Bcl2 ratio and Caspase3). Data are presented as mean ± SEM, and groups marked with different letters within each category showed significant differences (P <0.05). The experiment was repeated at least three times. (D) The result of confirming the expression of klotho protein in the klotho monallelic knockout porcine placenta detected by western blot analysis (WT, wild type; Fetus L2, living fetus 2 (WT/-17bp, +12bp)).

Justice

“Cell”, “host cell”, “modified host cell” and the like refer to a particular target cell as well as progeny or potential progeny of such cells. Since certain modifications may occur laterally by mutation or environmental influences, such progeny, in fact, will not be identical to the parental cell, but are still included within the term and scope used herein.

The term "modified" or "engineered" applied to a nucleic acid is used interchangeably in the present invention, and refers to an artificial alteration in the nucleic acid sequence constituting a gene. The modification may include deletion, substitution, insertion or inversion of one or more nucleic acids. Modified or engineered nucleic acid sequences may appear in the form of altering, e.g., increasing, promoting, decreasing or disappearing the expression and/or functional activity of these nucleic acids or expression products thereof.

In particular, the terms “damage” and “destruction” as applied to a nucleic acid are used interchangeably in the present invention to mean any genetic modification that reduces or eliminates the expression and/or functional activity of a nucleic acid or its expression product. For example, disruption of a gene is included within the scope of any genetic modification that reduces or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (eg, mRNA and/or protein). Genetic modification includes complete or partial inactivation, inhibition, deletion, disruption, blockade or downregulation of a nucleic acid (eg, a gene). One embodiment of the invention includes, but is not limited to, the use of any other molecule that inhibits the activity of the fertility gene or the level or functional activity of the expression product of the fertility gene.

“Disease model animals” refer to animals with diseases very similar to human diseases. The significance of disease model animals in human disease research is based on the physiological or genetic similarity between humans and animals. In disease research, biomedical disease model animals provide materials for research on various causes, pathogenesis and diagnosis of diseases, and through the study of disease model animals, genes related to diseases can be identified, and interactions between genes can be understood. It is possible to obtain basic data to determine the possibility of practical use through the actual efficacy and toxicity test of the developed new drug candidate.

“Animal” or “experimental animal” means any mammalian animal other than humans. The animal includes animals of all ages, including embryos, fetuses, newborns and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals are laboratory animals or other animals, rabbits, rodents (e.g. mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (e.g., Chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a pig.

'Knock-out' refers to a modification on the gene sequence that results in the deterioration of the function of the target gene, preferably whereby the expression of the target gene is not detectable or meaningless. In the present invention, the knockout of the Klotho gene means that the function of the Klotho gene is substantially degraded and expression cannot be detected or exists only at an insignificant level. The knockout transformant may be a transgenic animal having a heterozygous knockout of the Klotho gene or a homozygous knockout of the Klotho gene. Knockout includes, for example, exposing the animal to a substance that promotes target gene modification, introducing an enzyme that promotes recombination at a target gene site, or other methods of directing target gene modification after birth, of a target gene. Conditional knockouts in which deformation can occur are also included.

'Target gene' refers to a nucleic acid encoding a polypeptide in a cell, unless specifically indicated otherwise. “Target sequence” means a portion of a target gene, eg, one or more exon sequences of the target gene, intron sequences or regulatory sequences of the target gene, or exon and intron sequences of the target gene, introns, unless specifically indicated otherwise. And regulatory sequences, exons and regulatory sequences, or combinations of exons, introns and regulatory sequences.

'Sequence-specific endonuclease' or'sequence-specific nuclease', unless specifically specified otherwise, recognizes and binds a polynucleotide, such as a target gene, in a specific nucleotide sequence, and the polynucleotide Refers to a protein that catalyzes single- or double-strand breaks in.

"Vector" means a nucleic acid molecule, suitably a DNA molecule, derived from, for example, a plasmid, bacteriophage, plant virus, into which a nucleic acid sequence can be inserted or replicated therein. Vectors typically contain one or more unique restriction sites and are capable of autonomously replicating in a target cell or tissue or a defined host cell, including its progenitor cell or tissue, or a sequence that has been replicated. It can be integrated into the genome of a host defined to be reproducible.

"Wild type" means a gene or gene product that has the characteristics of that gene or gene product when separated from a naturally occurring sequence. Wild-type genes are most frequently observed in the population, and thus are those that optionally represent a "normal" or "wild-type" form of the gene.

"Modified", "modified" or "mutant" means a gene or gene product that exhibits a modification in sequence and/or functional properties when compared to a wild-type gene or gene product.

“Protein,” “polypeptide,” and “peptide” include polymeric forms of amino acids of any length, including encoded and unencoded amino acids, and chemically or biochemically modified or derivatized amino acids. The term also includes modified polymers, such as polypeptides having a modified peptide backbone. These terms can be used interchangeably.

“Nucleic acid” and “polynucleotide” include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified forms thereof. These are single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, unnatural, Or a polymer comprising a derivatized nucleotide base. For convenience, nucleic acid size can be expressed in bp whether the nucleic acid is in double stranded or single stranded form.

"Complementary" of a nucleic acid means that the nucleotide sequence of one nucleic acid strand forms a hydrogen bond with another sequence on the opposite nucleic acid strand due to the orientation of its nucleic acid base group. The complementary bases of DNA are typically A and T, C and G. In RNA, these are typically C and G, U and A.

Hybridization requires that two nucleic acids contain complementary sequences, even if mismatches between bases are possible. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acid and the degree of complementarity, which are variables well known in the art.

'Nuclear transplantation' refers to a genetic engineering technology that artificially binds another cell or nucleus to an enucleated egg to have the same traits.'Nuclear transplantation' refers to an egg into which a nuclear donor cell has been introduced or fused.

A'nuclear donor cell' refers to a cell or nucleus of a cell that transfers the nucleus to a nuclear receptor, a recipient egg. "Ovum" preferably refers to a mature egg that has reached the middle of the second meiosis. In the present invention, as the nuclear donor cells, somatic cells or stem cells of pigs may be used.

A'somatic cell' is a cell other than a germ cell among cells constituting a multicellular organism, and is a differentiated cell that specializes for a certain purpose and does not become a cell other than that, and a cell that has several types of different functions. It includes cells that have the ability to differentiate.

'Stem cell' refers to a cell that can develop into any tissue. There are two basic characteristics. First, it has the ability to differentiate into cells with specific functions according to the environment, self-renewal, which creates itself through repeated division.

'Living offspring' refers to an animal that can survive outside the womb. Preferably, it refers to an animal that can survive for one second, one minute, one hour, one day, one week, one month, six months or more than one year. These animals do not require an intrauterine environment to survive.

'About' means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. , Means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "include" and "comprise" include the steps or elements presented, or groups of steps or elements, but any other step or element, or step or element It is to be understood as implying that the group of people is not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to an animal model of Klotho deficiency disease.

More specifically, it relates to a method of artificially modifying the nucleic acid sequence of Klotho and its use, to an animal model of Klotho deficiency disease.

[Klotho deficient pig model]

Pigs as a model animal have a longer survival period compared to existing rodent models, and are known to have a biomechanism similar to that of humans, so they are in the spotlight as a next-generation disease model animal species that can overcome existing limitations.

Therefore, the present inventors have tried to develop and utilize a more reliable Klotho deficiency disease animal model by confirming the induction of Klotho deficiency disease through the production and behavior of a disease model that exceeds the rodent model using a large animal pig.

The Klotho deficiency disease animal model exhibits several aging-like phenotypes similar to human early aging syndrome due to defects in klotho gene expression. It exhibits several aging-like phenotypes such as growth retardation, infertility, atherosclerosis, osteoporosis and premature death.

Klotho's monobasic polymorphism has been reported to be associated with shortened lifespan, osteoporosis, stroke and coronary artery disease (Arking et al., 2002, Kawano et al., 2002; Mullin et al., 2005, Ogata et al. ., 2002; Yamada et al., 2005). In addition, high levels of Klotho protein prolong brain cell lifespan, reduce the incidence of related diseases such as heart disease, and enhance cognitive abilities such as attention, memory, and cognition, and lack of this protein has been shown to promote the aging process. Therefore, the Klotho deficiency disease may be, for example, arteriosclerosis, osteoporosis, stroke or Alzheimer's.

In one aspect, the present invention relates to a genetically modified animal model in which Klotho gene is knocked out, preferably a pig model of Klotho deficiency disease. One embodiment of the present invention relates to the manipulation and modification of Klotho gene

In other words, it relates to a genetically modified animal, particularly a recombinant pig that knocked out the Klotho gene.

Pigs are anatomically similar in size to humans, making them not only suitable as a heterogeneous organ model, but also have many physiological and genetic similarities with humans. In addition, since it has high reproductive capacity, it is not only suitable for the development of new biological materials for production and treatment, but it is also a good animal model for toxicity and safety evaluation.

Pigs used in the present invention can be used any known type that can be appropriately selected and used by those skilled in the art.

Pigs (scientific name: SusscrofPDomestica) belong to the Suagang, Jinsuhagang, beast-fighting branch, side-abstract tree, right-headed tree (including cattle and goats), and boar family, and the number of chromosomes is 2n=38. Originally, pigs are omnivores raised as livestock for meat, but they are attracting great attention as experimental animals along with their physiological and anatomical findings similar to those of humans and high interest in animal welfare in recent years. Pigs are also attracting great attention in terms of physiological anatomy. Pigs are physiologically and anatomically similar to humans in many respects, so they are widely used throughout research.

In one embodiment, the maximum weight at maturity is 60-70 kg of mini pigs close to humans (e.g. Peteman, Moore), or small pigs that are easier to use in experiments (adult weight: 30-40 kg) Göttingen It is also possible to use mini-pigs (NIH-based), Yucatan-based mini-pigs, and micro-pigs.

[Klotho genetic manipulation or modification]

The Klotho deficiency disease pig model of the present invention may be a pig in which Klotho protein expression is inactivated.

Embodiments of the present invention include genetically engineering the Klotho gene. It can be caused by altering the Klotho gene so that it cannot be expressed, or it can occur by genetically expressing factors that will inhibit the transcription or translation of the gene.

In a specific embodiment, the concentration and/or activity of Klotho protein is at least 1%, 5%, 10%, 20%, 30 compared to control cells in which modifications to lower the level and/or activity of Klotho protein are not induced. %, 40%, 50%, 60%, 70%, 80% or 90% decrease.

Klotho is an enzyme produced by the KL gene, which produces a type I membrane protein related to β-glucuronidase. The human klotho gene is located at 33.59-33.64 Mb of chromosome 13, and has an mRNA sequence of, for example, NM_004795. In addition, it has the protein sequence of NP_004786.

In certain embodiments, it can be used by transforming the Klotho gene to knockout.

Each nucleic acid encoding Klotho may be used without limitation as long as it has a base sequence encoding Klotho known in the art.

The nucleic acid encoding Klotho can be prepared by genetic recombination methods known in the art. For example, there are PCR amplification for amplifying nucleic acids from the genome, chemical synthesis, or techniques for producing cDNA sequences.

In addition, the nucleic acid may have a nucleotide sequence encoding each functional equivalent of Klotho.

The functional equivalent is at least 70%, preferably 80%, more preferably 90% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 1,2,3 as a result of addition, substitution or deletion of amino acids. It refers to a polypeptide having a physiological activity substantially equivalent to that of the SNCA of the present invention.

At this time, the amino acid deletion or substitution is preferably located in a region not directly related to the physiological activity of the polypeptide of the present invention.

Klotho gene can be knocked out using any known method, for example, RNA-Guided Endonuclease (RGEN) can be used.

In other embodiments, manipulation of the Klotho gene can be applied to cells or embryos to inactivate.

In one embodiment, it is an in vitro cell or embryo comprising an agent that specifically binds to a target site of a chromosome of the cell or embryo, causing double-stranded DNA destruction, thereby interfering with the gene to selectively interfere with the process of formation of germ cells, the The agent may be selected from the group consisting of a targeting endonuclease and a recombinase fusion protein.

In another embodiment, the manipulation of the Klotho gene is

For example, it may include targeted changes in the Klotho gene or targeted changes in the polynucleotide of interest, including targeted changes in other desired polynucleotides.

Such targeted modifications include addition of one or more nucleotides, deletion of one or more nucleotides, substitution of one or more nucleotides, knockout of a polynucleotide of interest or a portion thereof, knockout of a polynucleotide or portion thereof of interest, a heterologous endogenous nucleic acid sequence. Including, but not limited to, substitution with a nucleic acid sequence, or a combination thereof. In a specific embodiment, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are changed to form a targeted genomic modification.

In illustrative examples, the target sequence is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 in the nucleotide sequence of the germ gene. , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology.

[Genetic manipulation method]

Klotho gene manipulation of the present invention can be performed in consideration of the gene expression control process.

In an embodiment, it may be achieved by selecting an appropriate operating means for each step in the steps of transcriptional regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translation regulation, or protein modification regulation.

In an embodiment, using RNAi (RNA interference or RNA silencing), small RNAs (sRNAs) interfere with or reduce the stability of mRNA, and in some cases, destroy the protein synthesis information to prevent transmission of the genetic information. Expression can be controlled. In an illustrative example, the expression product is an RNA molecule (e.g., siRNA, shRNA, miRNA, dsRNA, etc.) that contains a target region corresponding to the nucleotide sequence of the fertility gene and weakens or destroys the expression of the fertility gene.

In an embodiment, wild-type or variant enzymes capable of catalyzing the hydrolysis (cleavage) of bonds between DNA or RNA molecules, preferably nucleic acids in DNA molecules, can be used.

In certain embodiments, meganucleases; Zinc finger nuclease; TALE-nuclease; RNA-guided endonuclease (RNA-Guided Endonuclease; RGEN), for example, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein), etc. Gene using one or more nucleases selected from the group consisting of You can control the expression of genetic information by manipulating.

In one aspect, a composition and method for genetic engineering by targeting some or all of the non-coding or coding region of the Klotho gene can be provided.

The composition and method

In embodiments, for the desired Klotho inactivation, some of the Klotho gene nucleic acid sequences involved therein may be manipulated or modified. As a result of the manipulation, it includes all types of knock down, knock out, and knock in.

In an embodiment, a promoter region, or a transcription sequence, such as an intron or exon sequence may be targeted. Coding sequences, such as coding regions, initial coding regions, can be targeted for alteration of expression and knockout.

In an embodiment, the nucleic acid modification is one or more nucleotides, such as 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or It may be a substitution, deletion, and/or insertion of a 1 bp nucleotide.

In embodiments, it can be targeted to include deletions or mutations in one or more regions of the Klotho gene.

In embodiments, gene knockdown may target unwanted transcription sites of the Klotho gene. At this time, the exon region can be targeted.

In an embodiment, it can be used to alter the expression or activity of a Klotho gene by targeting a promoter, enhancer, intron, 3'UTR, and/or non-coding sequence of a polyadenylation signal.

The genetic modification may mean all or part of a gene (eg, one or more nucleotides) deletion, substitution, and/or regulation of the activity of a gene, such as inactivation, by insertion of one or more nucleotides. The gene inactivation refers to a modification to encode a protein that has lost its original function or suppressed or reduced expression of a gene. In addition, gene regulation includes structural modification of proteins obtained by deletion of exon sites by simultaneously targeting both intron sites surrounding one or more exons of target genes, expression of dominant negative proteins, and expression of competitive inhibitors secreted in soluble form. May mean a change in the function of a gene as a result

The genetic modification may be to inactivate the gene so that the protein encoded from the gene is not expressed in the form of a protein having an original function.

In one example, the genetic modification may be to inactivate the targeted gene by catalyzing single-stranded or double-stranded cleavage of a specific site in the targeted gene by a genome editing system including a cleavage endonuclease. .

Nucleic acid strand breaks catalyzed by cleavage endonucleases can be repaired through mechanisms such as homologous recombination or nonhomologous end joining (NHEJ). In this case, when the NHEJ mechanism occurs, a change in the DNA sequence is induced at the cleavage site, whereby the gene may be inactivated.

In this case, when the NHEJ mechanism occurs, a change in the DNA sequence is induced at the cleavage site, whereby the gene may be inactivated. Repair through NHEJ results in substitutions, insertions or deletions of short gene fragments, and can be used to induce knockouts of the corresponding gene.

The modification of the Klotho gene may be induced by one or more of the following:

1) Deletion of all or part of the gene to be modified (hereinafter referred to as'target gene'), such as 1 bp or more nucleotides of the target gene, such as 1 to 30, 1 to 27, 1 to 25, 1 to 23, Deletion of 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide,

2) 1 bp or more nucleotides of the target gene, such as 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5 , Substitution of 1 to 3, or 1 nucleotide with a nucleotide different from the original (wild type), and

3) one or more nucleotides, such as 1 to 30, 1 to 7, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to Insertion of 3 or 1 nucleotide (each independently selected from A, T, C and G) at any position of the target gene.

The modified part ('target site') of the target gene is 1bp or more, 3bp or more, 5bp or more, 7bp or more, 10bp or more, 12bp or more, 15bp or more, 17bp or more, 20bp or more, such as 1bp to 30bp, 3bp to 30bp, 5bp to 30bp, 7bp to 30bp, 10bp to 30bp, 12bp to 30bp, 15bp to 30bp, 17bp to 30bp, 20bp to 30bp, 1bp to 27bp, 3bp to 27bp, 5bp to 27bp, 7bp to 27bp, 10bp to 27bp, 12bp to 27bp, 15bp to 27bp, 17bp to 27bp, 20bp to 27bp, 1bp to 25bp, 3bp to 25bp, 5bp to 25bp, 7bp to 25bp, 10bp to 25bp, 12bp to 25bp, 15bp to 25bp, 17bp to 25bp, 20bp to 25bp, 1bp to 23bp, 3bp to 23bp, 5bp to 23bp, 7bp to 23bp, 10bp to 23bp, 12bp to 23bp, 15bp to 23bp, 17bp to 23bp, 20bp to 23bp, 1bp to 20bp, 3bp to 20bp, 5bp to 20bp, 7bp to 20bp, 10bp to 20bp, 12bp to 20bp, 15bp to 20bp, 17bp to 20bp, 21bp to 25bp, 18bp to 22bp, or 21bp to 23bp may be a contiguous nucleotide sequence site.

When the Klotho genetic modification is induced by the Cas9 protein, the genetic modification is located adjacent to the 5'end of the PAM (proto-spacer-adjacent Motif) sequence unique to the Cas9 protein according to the derived microorganism among the base sequences of each gene. Consecutive 17bp to 25bp, 20bp to 25bp, 17bp to 23bp, 20bp to 23bp, 17bp to 20bp, 21bp to 25bp, 18bp to 22bp, or 21bp to 23bp of nucleotide sequence sites, such as single-stranded or double-stranded cut, Alternatively, it may be deletion and/or insertion of one or more nucleotides in the process of repairing the cleavage.

In one example, when the Cas9 protein is derived from Streptococcus pyogenes, the PAM sequence is 5'-NGG-3' (N is A, T, G, or C).

In another example, when the Cas9 protein is derived from Streptococcus thermophiles, the PAM sequence is 5'-NNAGAAW-3' (N is each independently A, T, C or G, and W Is A or T).

In another example, when the Cas9 protein is derived from Neisseria meningitidis, the PAM sequence is 5'-NNNNGATT-3' (N is each independently A, T, C, or G). .

In another example, when the Cas9 protein is derived from Streptocuccus aureus, the PAM sequence is 5'-NNGRR(T)-3' (N is each independently A, T, C, or G And R is A or G, and (T) means an optionally inclusive sequence).

In another example, when a Cpf1 protein is used, the PAM sequence is 5'-TTN-3' (N is A, T, C or G).

Therefore, in a representative embodiment of the present invention,

The function of the Klotho gene is reduced or eliminated by genetically modifying or manipulating at least one site of the nucleic acid sequence constituting the Klotho gene, that is, a site where nucleic acid modification can occur.

The target sequence may simultaneously target two or more sites among the nucleic acid sequences constituting the Klotho gene.

In one embodiment, one or more of the nucleic acid sequences constituting the Klotho gene may be targeted using one kind of guide RNA. Thereby, the Klotho gene can be knocked out or knocked down.

In one embodiment, two or more kinds of guide RNAs may be used to target at least one portion of the nucleic acid sequence constituting the Klotho gene. Thereby, the Klotho gene can be knocked out or knocked down.

In one embodiment, the decrease in the level and/or activity of the Klotho gene may be due to a modification of the nucleic acid sequence constituting the Klotho gene, for example, a modification in a non-coding region, a coding region, and/or an intron.

Such Klotho gene nucleic acid sequence modifications include, but are not limited to, addition, deletion or substitution of nucleotides in the genome.

Insertion of one or more nucleotides into the Klotho gene nucleic acid sequence,

Deletion of one or more nucleotides from the Klotho gene nucleic acid sequence,

Substitution of one or more nucleotides in the Klotho gene nucleic acid sequence,

Knockout of a Klotho gene nucleic acid sequence or a portion thereof,

Melting of the Klotho gene nucleic acid sequence or a portion thereof, replacing the endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or

It may include a Klotho gene nucleic acid sequence change including the combination.

Accordingly, in a specific embodiment, it may be degraded or eliminated by destroying a gene encoding a protein or polypeptide having the activity of the Klotho gene. In a specific embodiment, in at least one site, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are changed in the Klotho gene nucleic acid sequence. Using a variety of methods, additional targeted modifications can be made.

In one embodiment, the decrease in the level and/or activity of Klotho protein may be due to genetic modification of the Klotho gene (ie, genetic modification in regulatory regions, coding regions, and/or introns, etc.).

Such genetic modifications include, but are not limited to, addition, deletion or substitution of nucleotides to the genome.

These genetic modifications, for example,

Insertion of one or more nucleotides into the Klotho gene,

Deletion of one or more nucleotides from the Klotho gene,

Substitution of one or more nucleotides in the Klotho gene,

Knockout of the Klotho gene or part thereof,

Klotho gene changes, including melting of the Klotho gene or a portion thereof, substitution of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or a combination thereof.

Thus, in specific embodiments, the activity of the Klotho polypeptide can be reduced or eliminated by disrupting the gene encoding the Klotho polypeptide. In a specific embodiment, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are changed in the Klotho gene. Using a variety of methods, further targeted genetic modifications can be made.

[Nuclease system]

In an exemplary embodiment of the present invention, the cleavage nuclease system or components of such a system can be used to modify the genome of the Klotho gene. It is collectively referred to as a nuclease agent. In a preferred example, an RNA-Guided Endonuclease (RGEN) system can be used.

CRISPR / Cas system

An exemplary embodiment of the present invention is to induce sequence-induced double strand cleavage by introducing components of the CRISPR system, including CRISPR-related nuclease Cas9 and sequence-specific guide RNA (gRNA), into cells. Using the capabilities of the CRISPR system to induce the cleavage.

Components of the CRISPR system, including CRISPR-related nuclease Cas9 and sequence specific guide RNA (gRNA), can be introduced into cells as encoded on one or more vectors, such as plasmids.

The CRISPR/Cas system contains transcripts and other elements involved in inducing the expression or activity of the Cas gene. The CRISPR/Cas system can be a Type I, Type II or Type III system. The methods and compositions disclosed herein use a CRISPR/Cas system by using a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of nucleic acids.

RNA guide Endonuclease

Cas proteins generally contain at least one RNA recognition or binding domain. These domains can interact with guide RNA (gRNA, described in more detail below).

Nuclease domains have catalytic activity for nucleic acid cleavage. Cleavage involves breaking the covalent bond of a nucleic acid molecule. Cleavage can produce blunt ends or staggered ends, and can be single-stranded or double-stranded.

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl0d, CasF , CasG, CasH, Csy1, Csy2, Csy3, Cse1(CasA), Cse2(CasB), Cse3(CasE), Cse4(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf1966, Cpf1 and their homologues or modifications thereof A modified version.

The Cas protein can be derived from the type II CRISPR/Cas system. For example, the Cas protein may be a Cas9 protein or may be derived from a Cas9 protein.

Cas9 protein is, for example, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiop.

Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces viridochrom Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus sedomycoides, Bacillus pseudomycoides, Bacillus selenitilenduce ), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Micros

Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii ), Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degencii (Ammonifex degensii), Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Pinegoldia magna Finegoldia magna), Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferroooxidans feridanxthiobacillus ), Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoaltero Monas haloplanktis (Pseudoalteromonas haloplanktis), Ktedonobacter racemifer, Metanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nodularia sp., Arthrospira maxima, Arthrospira platensis , Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis (Petrotoga mobilis) ), Thermosipho africanus (Thermosipho africanus) or Acaryochloris marina (Acaryochloris marina). Further examples of the Cas9 family are described in WO 2014/131833, the entire contents of which are incorporated herein by reference. The Cas9 protein or derivative thereof from Streptococcus pyogenes is a preferred enzyme.

Cpf1 protein is Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, Eubacterium eligens. . (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (Uplasmo112), Candida term derived from Methanoplasma, or Methanoplasma However, it is not limited thereto.

The Cas protein may be a wild-type protein (ie, a naturally occurring one), a modified Cas protein (ie, a Cas protein variant), or a fragment of a wild-type or modified Cas protein. Cas proteins may also be active variants or fragments of wild-type or modified Cas proteins. Active variants or fragments are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% relative to the wild-type or modified Cas protein or portion thereof. , 99% or more sequence identity, wherein the active variant retains the ability to cleave at the desired cleavage site, thus retaining nick-inducing or double-stranding inducing activity. Methods for measuring nick-inducing or double-strand break-inducing activity are known.

Cas proteins can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity and/or enzymatic activity. Cas proteins can also be modified to change other activities or properties of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or the Cas protein can be cleaved to remove domains that are not essential to the function of the protein or optimize the activity of the Cas protein.

One or two nuclease domains may be deleted or mutated, which may no longer be functional or result in decreased nuclease activity.

When one nuclease domain is deleted or mutated, the resulting Cas protein (e.g., Cas9) may be referred to as a nickase, and single-stranded cleavage in the CRISPR RNA recognition sequence in the double-stranded DNA. Can be generated.

If the two nuclease domains are deleted or mutated, the resulting Cas protein (eg, Cas9) will have a reduced ability to cleave both strands of double-stranded DNA. Examples of mutations that convert Cas9 to nickase may be D10A or H939A or H840A in the RuvC domain of sp.Cas9.

Cas protein can also be a fusion protein. For example, the Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcription activation domain, or a transcription repressor domain. The fused domain or heterologous polypeptide may be located within the N-terminal, C-terminal, or Cas protein.

In addition, the Cas protein can be fused to a heterologous polypeptide. Heterologous peptides include, for example, nuclear localization signals (NLS) such as SV40 NLS for targeting the nucleus, mitochondrial localization signals for targeting mitochondria, ER retention signals, and the like. These signals can be located at the N terminus, C terminus or anywhere within the Cas protein.

Cas proteins can also be linked to the cell permeable domain. For example, the cell permeable domain is derived from an HIV-1 TAT protein, a TLM cell permeable motif derived from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetrating peptide derived from herpes simplex virus, or a polyarginine peptide sequence. Can be.

Cas proteins may also include heterologous polypeptides, such as fluorescent proteins, purification tags, or epitope tags to facilitate tracking or purification.

Cas protein can be provided in any form. For example, the Cas protein may be provided in the form of a protein such as a Cas protein complexed with gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (eg, messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon optimized for efficient translation into the protein in a particular cell or organism.

The nucleic acid encoding the Cas protein can be stably integrated into the genome of a cell and can be operably linked to a promoter active in the cell. Alternatively, the nucleic acid encoding the Cas protein can be operably linked to the promoter of the expression construct.

Guide RNA ( gRNA )

"Guide RNA" or "gRNA" includes an RNA molecule that binds to a Cas protein and targets the Cas protein at a specific location in the target DNA. The guide RNA may comprise two segments, a “DNA target segment” and a “protein binding segment”. “Segment” includes a segment, section or region of a molecule, such as a continuous stretch of nucleotides in RNA. Some gRNAs contain two separate RNA molecules, a "activator-RNA", a crRNA and a "targeter-RNA", a tracrRNA. Another gRNA is a single RNA molecule (single RNA polynucleotide), also referred to as “single molecule gRNA”, “single guide RNA” or “sgRNA”.

The crRNA and the corresponding tracrRNA hybridize to form a gRNA. The crRNA provides a single-stranded DNA targeting segment that hybridizes to a CRISPR RNA recognition sequence. If used for modification in a cell, the complete sequence of a given crRNA or tracrRNA molecule can be designed to be specific to the kind in which the RNA molecule will be used.

The DNA targeting segment (crRNA) of a given gRNA contains a nucleotide sequence that is complementary to the sequence of the target DNA.

The DNA targeting segment of the gRNA interacts sequence-specifically with the target DNA through hybridization (ie, base pairing). As such, the nucleotide sequence of the DNA targeting segment may vary, and the gRNA and target DNA determine the location within the target DNA to interact with. The DNA targeting segment of the gRNA of interest can be modified to hybridize to the desired sequence within the target DNA.

The DNA targeting segment can have a length of about 12 nucleotides to about 100 nucleotides. For example, the DNA targeting segment can be from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt. , From about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.

The tracrRNA can be of any form (eg, full length tracrRNA or active partial tracrRNA) and of various lengths.

The complementarity ratio between the DNA targeting sequence and the CRISPR RNA recognition sequence in the target DNA is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, at least 97%, at least 98%, at least 99%, or 100%).

The guide RNA has additional desirable characteristics, such as modified or regulated stability; Intracellular targeting; Tracking using fluorescent labels; It may include a modification or sequence that provides a binding site for the protein or protein complex.

Examples of such modifications include, for example, 5'caps (eg 7-methylguanylate caps (m7G)); 3'polyadenylated tail (ie, 3'poly(A) tail); Riboswitch sequences (eg, taking into account regulated stability and/or regulated accessibility by proteins and/or protein complexes); Stability control sequence; It may include a sequence that forms a dsRNA double helix structure (ie, a hairpin).

The guide RNA can be provided in any form. For example, the gRNA can be provided in the form of two molecules (separated crRNA and tracrRNA) or RNA as one molecule (sgRNA) and optionally in the form of a complex with a Cas protein. The gRNA can also be provided in the form of DNA encoding the RNA. DNA encoding gRNA may encode a single RNA molecule (sgRNA) or an isolated RNA molecule (eg, isolated crRNA and tracrRNA).

The DNA encoding the gRNA can be stably integrated into the genome of the cell and operably linked to a promoter active in the cell. Alternatively, the DNA encoding the gRNA can be operably linked to the promoter of the expression construct. For example, the human U6 promoter can be used.

Cleavage of nucleic acids

The Cas protein can cleave a nucleic acid at a site inside or outside the nucleic acid sequence present in the target DNA to which the DNA targeting segment of the gRNA will bind.

A “cleavage site” includes a nucleic acid site at which a Cas protein causes a single or double strand break. For example, CRISPR complex formation may be performed at or near a nucleic acid sequence present in the target DNA to which the DNA targeting segment of the gRNA is bound (e.g., 1, 2, 3, 4, 5, 6, 7, from the nucleic acid sequence. Within 8, 9, 10, 20, 50 or more base pairs) of one or two strands.

The cleavage site may be on only one or both strands of the nucleic acid. The cleavage site can be at the same location on both strands of the nucleic acid (which produces a blunt end), or can be at a different site on each strand (which produces a staggered end).

Site-specific cleavage of the target DNA by Cas9 can occur in the target DNA at a position determined by (i) base pairing complementarity between the gRNA and the target DNA and (ii) a short motif referred to as PAM.

A Protospacer adjacent motif (PAM) can be flanked by a CRISPR RNA recognition sequence. Optionally, the CRISPR RNA recognition sequence may be flanked by PAM. For example, the cleavage site of Cas9 can be about 1 to about 10, or about 2 to about 5 base pairs (eg, 3 base pairs) upstream or downstream of the PAM sequence.

Klotho Guide RNA for genetic manipulation

In one embodiment of the present invention, a guide RNA sequence for genetic manipulation of the Klotho gene is provided. For example, a guide RNA sequence corresponding to the Klotho gene target sequence is provided. For example, the guide RNA sequence shown in FIG. 1 can be provided.

In one embodiment of the present invention, there is provided a CRISPR-related nuclease that interacts with a guide RNA corresponding to a target sequence for genetic manipulation of a Klotho gene, for example, forming a complex.

In one embodiment of the present invention, each nucleic acid modification product in which artificial genetic manipulation has occurred in the target sequence site of the Klotho gene and an expression product thereof are provided.

In addition, in one embodiment, the present invention is a nucleic acid in the target site of the Klotho gene It provides a complex of'guide RNA-CRISPR related nuclease' used for modification.

In particular, a gRNA molecule comprising a domain capable of complementary binding to a target site from a gene, for example, an isolated or non-naturally derived gRNA molecule, and a DNA encoding the same can be provided.

In addition, when two or more gRNAs are used to locate two or more cleavage events, such as double stranded or single stranded breaks, in a target nucleic acid, two or more cleavage events may be generated by the same or different Cas9 proteins.

The gRNA is, for example,

Can target more than 2 sites in the Klotho gene,

Two or more sites can be targeted for each Klotho gene,

Can independently induce double-stranded and/or single-stranded cleavage of the Klotho gene,

It is also possible to induce the insertion of an external nucleotide at the cleavage site of the Klotho gene.

In addition, as another specific embodiment of the present invention, the nucleic acid constituting the complex of'guide RNA-CRISPR-related nuclease' is

(a) a sequence encoding a gRNA molecule complementary to a target site sequence in the Klotho gene as disclosed herein; And

(b) a sequence encoding a CRISPR-related nuclease may be included.

In this case, (a) may be present in two or more depending on the target site, and (b) may use the same or two or more nucleases.

In an embodiment, the nucleic acid is an enzymatically inactive editor protein or a fusion protein thereof (e.g., transcriptional repressor domain fusion protein) sufficiently close to the knockdown target site to reduce, reduce or inhibit the function or expression of the Klotho gene. ) Can be configured to target.

TALEN system

TALEN refers to a protein comprising a transcriptional activator-like (TAL) effector binding domain and a nuclease domain, and includes not only the functional monomer TALEN itself, but also others that require dimerization with other monomeric TALENs. .

TALEN is used to mean a TALEN or a pair of TALENs engineered to work together to remove DNA at the same site. TALENs working together can be referred to as left-TALEN and right-TALEN, and refer to the handedness of DNA or TALEN-pairs.

TALEN induces the genetic engineering of immortal human cells by two major eukaryotic DNA repair pathways, non-homologous end binding (NHEJ) and homologous repair.

In some embodiments, a TAL effector can be used to target other protein domains (eg, non-nuclease protein domains) for a particular nucleotide sequence. For example, the TAL effector is, without limitation, a DNA 20 interacting enzyme (e.g., methylase, topoisomerase, integrase, transferase or ligase), a transcriptional activator or inhibitor, or a histone. It can be bound to a protein domain from a protein that interacts with or modifies other proteins. Examples of applications of the TAL effector fusion include, for example, producing or modifying epigenetic regulatory elements, site-specific insertion, deletion, or repair in DNA, regulating gene expression, and chromatin structure. Includes changing.

zinc Finger Nuclease system

Zinc-finger nuclease (ZFN) is an artificial restriction enzyme produced by fusing zinc finger DNA-binding domains. The zinc finger domain can be engineered to target the desired DNA sequences, which allows the zinc-finger nuclease to target specific sequences within the complex genome. Taking advantage of the endogenous DNA repair machine, these reagents can be used to modify the genome of higher organisms. ZFN can be used in a method of inactivating genes.

The zinc finger DNA-binding domain has about 30 amino acids and folds into a stable structure. Each finger binds primarily to a triplet within the DNA matrix. Amino acid residues at critical positions contribute most of the sequence-specific interactions with the DNA site. These amino acids can be changed while retaining the remaining amino acids to preserve the essential structure.

Binding to longer DNA sequences is achieved by binding several domains simultaneously. Other functional groups such as non-specific FokI cleavage domain (N), transcription activator domain (A), transcription repressor domain (R) and methylase (M) are fused to ZFP, respectively, and ZFN, zinc finger transcriptional activity Factor (ZFA), zinc finger transcription inhibitory factor (ZFR), and zinc finger methylase (ZFM).

Materials and methods for using zinc fingers and zinc finger nucleases to produce genetically engineered animals are described, for example, in US Pat. No. 8,106,255, US 2012/0192298, US 2011/0023159, and US patents. It is disclosed in 2011/0281306.

Composition for genetic manipulation

In one embodiment of the present invention, a composition for genetic manipulation or modification of the Klotho gene and a method of using the same are provided.

In one embodiment, the composition for genetic manipulation or modification of the Klotho gene may include components of the RNA-Guided Endonuclease (RGEN) system.

For example, it provides a composition for genome manipulation comprising a guide RNA or a nucleic acid molecule encoding the same and an RNA-guided endonuclease (RNA-Guided Endonuclease; RGEN) or a nucleic acid molecule encoding the same.

For example, (i) a guide RNA or a DNA molecule encoding the guide RNA, or (ii) a guide RNA or a DNA molecule encoding the guide RNA and an RNA-guide endonuclease or the RNA-guide endonuclease It provides a Klotho gene inactivation composition comprising a gene encoding a clease.

For example, (i) guide RNA or a DNA molecule encoding the guide RNA,

Or (ii) the guide RNA or a DNA molecule encoding the guide RNA, and an RNA-guide endonuclease or a gene encoding the RNA-guide endonuclease.

The composition may be provided in the form of an expression cassette.

[Introduction into cells]

One embodiment of the present invention relates to a method for introducing a composition for genetic manipulation or modification of the Klotho gene into a pig cell.

For example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection ( Transgenic animals by DEAEDextran-mediated transfection), polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells It can be introduced into cells for fabrication.

In one embodiment of the present invention, for the genetic manipulation or modification, for example, knockout, gene inactivation, or to obtain the expression of a specific gene, or for other purposes, various nucleic acids are introduced into the cell. I can.

Nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids, such as synthetic bases or alternating backbones. . Nucleic acid molecules can be double-stranded or single-stranded.

The nucleic acid may be included in a vector.

A vector may refer to an expression vector or vector system and is a set of components necessary to effect DNA insertion into a genome or other targeted DNA sequence, such as an episome, a plasmid, or even a viral/phage DNA segment.

Viral vectors used for gene transfer in animals can be used. In addition, non-viral vectors can be used.

Many different types of vectors are known. For example, plasmid and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids are typically origins of replication, appropriate promoters, and optional enhancers, and any essential ribosome binding sites, polyadenylation sites, splice donors and receptor sites, transcription termination sequences, and 5'flanking non-transcriptional Have a sequence. Examples of vectors are: plasmid (which may be another type of carrier), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus ( For example, ASV, ALV or MoMLV), and transposons (eg, Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

In one embodiment, the target nucleic acid sequence may be operably linked to a regulatory region such as a promoter.

In one embodiment, additional regulatory regions can be introduced together. Polyacetylation sequences, translational regulatory sequences (eg, internal ribosome entry segment, IRES), enhancers, inducible elements or introns, but are not limited thereto.

In one embodiment, nucleic acid constructs encoding signal peptides or selectable markers may be used.

In one embodiment, the exogenous nucleic acid encodes a polypeptide. It may include a tag sequence encoding "tag".

The nucleic acid can be delivered by a non-viral vector delivery method.,

For example, Cas9 and sgRNA in the form of mRNA can be packaged and delivered in lipid nanoparticles. DNA or nucleic acids that make up CRISPR/Cas9 with negative ions can form nanoparticles by electrostatically complexing with substances with positive ions, which can lead to receptor-mediated endocytosis or phagocytosis. Can penetrate. Naturally-occurring polymers, synthetic polymers, and lipids are the most widely known cationic materials for delivering nucleic acids.

For example, it can be delivered in the form of a Cas9/sgRNA complex.

Cas9 protein and sgRNA are made into RNP (ribonucleoprotein) form before administration, and then delivered to desired cells using a delivery material such as a liposome. The RNP form is expressed at the same time as it is delivered into cells, and when delivered in the aforementioned mRNA form, there is no delay until it is translated into a protein, and its expression is temporary. Since sgRNA and Cas9 proteins are introduced into cells in the form of RNP complex, they are introduced in the Active Cas9 protein state, so there is an advantage that editing can be performed faster and with minimal off-target effects. In one embodiment of the present invention, sgRNA and Cas9 protein were introduced into pig embryonic cells in the form of a RNP (Ribonucleoprotein) complex.

In one embodiment, the present invention can provide a porcine cell having an inactivated or removed Klotho gene.

For example, it relates to a method for introducing a recombinant vector containing a composition for Klotho knockout into a nuclear donor cell, and to a cell into which the recombinant vector has been introduced.

For example, it relates to a method for introducing an RNP complex comprising a composition for Klotho knockout into a nuclear donor cell, and to a cell into which the RNP complex has been introduced.

In one embodiment, the cells may be porcine embryonic cells, somatic cells, or stem cells.

In certain embodiments, for example, but not limited to, cumulus cells, epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, embryo-derived cells, placental cells, and embryonic cells. In addition, adult stem cells derived from various tissues of origin, for example, stem cells derived from tissues such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, or skin (epithelial) may be used. The non-human host embryo may generally be an embryo comprising a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, a 32-cell stage, a 64-cell stage, a morula, or a blastocyst. .

More preferably, it may be fetal-derived cells, adult fibroblasts, and cumulus cells. Most preferably, fibroblasts isolated from fetuses and adults of pigs are used. The characteristics of these cells are that a large number of cells can be obtained during initial separation, cell culture is relatively easy, and culture and manipulation in vitro are easy.

The embryonic cells, somatic cells, or stem cells provided as nuclear donor cells can be obtained from a method of preparing a surgical specimen or a biopsy specimen using a conventional method known in the art.

Meanwhile, the nuclear donor cells transformed with a Klotho gene knockout vector for making a Klotho gene deficiency disease pig model according to the present invention may be proliferated and cultured according to a method known in the art.

A suitable medium is any use that can be developed in the laboratory for the cultivation of animal cells and, in particular, mammalian cells, or prepared in the laboratory with the appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and/or micronutrients Any possible medium can be used.

The medium is any basic medium suitable for animal cell growth, as a non-limiting example, and the basic medium generally used for culture includes MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute). Medium), K-SFM (Keratinocyte Serum Free Medium), and in addition, any medium used in the industry can be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM/F12 medium, PCM medium, M199/F12 (mixture) (GIBCO), and MSC expansion medium (Chemicon) may be selected from the group consisting of.

To this basal medium, anabolic sources of carbon, nitrogen and micronutrients, such as, but not limited to, serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources can be added.

It will be apparent that one of ordinary skill in the art can select or combine a suitable medium and properly culture it by a known method. In addition, it is obvious that the culture can be cultured while controlling conditions such as suitable culture environment, time, temperature, etc. based on common knowledge in this field.

[Transgenic animals]

Chimeric animal

In one embodiment of the present invention, in order to produce a pig in which klotho protein expression is inactivated, embryos may be conceived under suitable conditions.

For example, the somatic cell nucleus transplantation method (SCNT) described above can be used using somatic cells of a transformed pig.

In one embodiment, the present invention relates to a method for producing a transgenic pig for a Klotho deficiency disease model in which Klotho gene is knocked out by producing litter by somatic cell nucleus transplantation (SCNT), and a pig for a Klotho deficiency disease model prepared thereby will be.

The somatic cell nucleus transplantation method (SCNT) can use a known method.

In one embodiment, the present invention is a method for preparing a nuclear transfer egg of a pig formed by transplanting the nucleus of a pig-derived somatic cell, embryonic cell, or stem cell transformed with a composition that knocks out the Klotho gene into an enucleated egg, and prepared thereby Nuclear transfer embryos can be provided.

In another embodiment, the method of preparing a transgenic pig for a Klotho deficiency disease model by knocking out a Klotho gene comprising the step of producing a litter by transplanting the nuclear transfer egg into the fallopian tube of a surrogate mother, and the Klotho gene produced by it It provides a pig for Klotho deficiency disease model, characterized in that the knock-out.

In certain embodiments, the present invention includes a method for producing a genetically engineered animal,

The method includes exposing the embryo or cell to an mRNA encoding a targeting nuclease (e.g., meganuclease, zinc finger, TALEN, intraocular RNA, recombinase fusion molecules),

Cloning cells within the surrogate mother or transplanting embryos within the surrogate mother, wherein the targeting nuclease specifically binds to an embryo or an intracellular target chromosome site to cause a change in the cellular chromosome, and the surrogate mother is at the target chromosome site. Genetically engineered animals can be conceived.

[Usage]

One embodiment of the present invention provides a use of a pig in which the klotho gene is knocked out by modification in the nucleic acid sequence of the klotho gene, and the expression of the klotho protein is inactivated.

Pigs for klotho deficiency disease model knocked out of the klotho gene according to the present invention can produce litter through mating, and transfer of the foreign gene is possible in the future.

Therefore, the pig characterized in that the klotho gene of the present invention is knocked out has the advantage of easy reproducibility, and is very useful as an animal model of klotho deficiency disease.

That is, the klotho deficiency disease model pig of the present invention may be used in various ways, such as the identification of the klotho deficiency disease mechanism, the search for therapeutic substances, and the development of diagnostic methods.

Therefore, in another aspect, the present invention includes various uses of a pig for a klotho deficiency disease model produced by the above method.

For example, the animal model according to the present invention can be used in studies necessary to treat or prevent klotho deficiency disease, for example, as a method of screening a test drug.

In one embodiment, the screening method of the present invention

1) administering a prophylactic and therapeutic candidate substance to the klotho deficiency disease model pig;

2) After administration of the candidate substance, the pig tissue may be compared with a control group to which the candidate substance was not administered and analyzed.

The candidate substance is preferably any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, but is not limited thereto. The compound may be a novel compound or a widely known compound. These candidate substances may form salts.

As a method of administering such a candidate substance, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration can be appropriately selected according to the symptoms of the target animal and the properties of the candidate substance. In addition, the dosage of the candidate substance can be appropriately selected according to the administration method or the properties of the candidate substance.

[Example]

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples.

One Animals and reagents

The animals used in this study were maintained by R&F farm (Boryeong, Korea) and Gyeonggi livestock veterinary research farm (Osan, Korea). All animal experiments were performed with the approval of the user manual (SNU-160613-16) of the animal laboratory of Seoul National University Hospital.

2. CRISPR / Cas9 System Target doing Klotho's Design and design

The sgRNA capable of recognizing the porcine klotho gene was designed using the online CRISPR design tool (http:/zifit.partners.org/ZiFiT/Disclaimer.aspx). The DNA sequence information for the designed sgRNA is 5'-TAGAACAAGGCTGAAGACTTCGG-3' (SEQ ID NO: 1).

The protospacer contiguous motif (PAM) and sgRNA target sequence can be identified by blue and red fonts, respectively (Fig. 1). The specificity of the designed sgRNA was confirmed by searching for similar pig sequences in GenBank. The sgRNA was designed for double-stranded cleavage in exon 3 of klotho.

3. CRISPR / Cas9 of ribonucleoproteins relay

To induce double-strand breaks (DSBs) in wild-type porcine fetal fibroblasts using ribonucleotroteins (RNPs), 9x10 ⁵ cells were 1400 V, 30 ms pulse width and nucleofection set to pulse number 1 (Neon , Invitrogen) transfected in vitro with sgRNA (7.2 μg) and Cas9 protein (28.8 μg) mixed in advance. Transfected cells were performed in 4-well cell culture dishes. One to two days after transfection, non-selected cell populations were used directly for somatic cell nuclear transplantation. Cas9 protein in storage solution (20mM HEPES pH 7.5, 150mM KCl, 1mM DTT and 10% glycerol) was mixed with sgRNA dissolved in nuclease-free water and incubated for 10 minutes at room temperature before use.

4. Somatic cells Nuclear Transplantation Department Bae Ai Sik

After 40 hours of in vitro maturation culture, the oocyte complex (COC) was removed by gently pipetting with 0.1% hyaluronidase. The molten oocytes were incubated for 10 minutes in Tyrode's albumin lactate pyruvate (TALP) medium containing 5 μg/mL of Hoechst 33342 and observed under an inverted microscope equipped with epifluorescence.

The oocytes were fixed with a micropipette for a post, and the translucency was partially dissected with a fine glass needle to make a slit near the first pole body.

Denucleation was performed by aspirating the cytoplasm adjacent to the first polar body containing the metaphase II chromosome with a suction pipette in TALP medium containing 7.5 μg/mL cytochalasin B. Then, using a fine pipette, the trypsin-treated fetal fibroblasts having a smooth cell surface were transferred to the upper cavity of the enucleated oocytes.

They were balanced with a fusion solution (0.26M mannitol solution containing 0.5mM HEPES and MgSO4) for 4 minutes, and then fused with 20 µl of the fusion solution with a single DC pulse of 1.2 kV/cm for 30 μs using an electric pulse machine (LF101). ; NepaGene, Chiba, Japan). After 30 minutes, the fused couplet was balanced with an activation solution (0.28 M mannitol solution containing 0.5 mM HEPES, 0.1 mM CaCl ₂ and MgSO ₄ ) for 4 minutes, and then transferred to a chamber with two electrodes containing the activation solution, BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA, USA) was used to activate a single DC pulse of 1.5 kV/cm for 30 μs.

Electrically activated embryos were cultured with Porcine Zygote Medium-5 (PZM-5, Funakoshi Corporation, Tokyo, Japan) at 39°C in 5% O2, 5% CO2 and 90% N2 for 7 days.

5. Bae Ai Sik And fetal recovery

After activation, on the 1st to 2nd day, postoperative estrus was observed, and on the 2nd day, 1-4 cell stage SCNT embryos were transferred to naturally circulating recipient pigs. Midventral laparotomy was performed under general anesthesia with Isoflurane (Isoflulane).

The reproductive organs were exposed and SCNT fetuses (150-250 embryos) were transferred to the fallopian tubes of recipient pigs. The fetus was diagnosed through ultrasound on the 25th day of pregnancy (the day of SCNT is considered as day 0), and the fetus recovered on the 28th day after transfer.

6. Primary culture of pig fetal fibroblasts

Replicated fetal fibroblasts were isolated from 7 cloned pig embryos and cultured.

The euthanized fetus was dissected into three parts: head, body, and tail.

Only the body part of the fetus was washed 3 times with phosphate-buffered saline (PBS; Gibco, Carlsbad, CA) containing 1% Penicillin/Streptomycin (P/S; Gibco) and 60 containing Dulbecco's Modified Eagle's Medium (DMEM; Gibco) Cut into small pieces in mm dishes.

The well-dissociated tissue was centrifuged for 2 minutes at 1,500 rpm. The supernatant was discarded, the pellet was resuspended in DMEM, and then centrifuged at 1,500 rpm for 2 minutes. These processes were repeated twice. Finally, discard the supernatant and turn the pellet over the tube several times to prepare 20% fetal bovine serum (FBS, Gibco), 1% P/S, 1% non-essential amino acids (NEAA, Gibco) and 100 mM β-mercaptoethanol (β-ME). It was resuspended in DMEM containing this. The suspension was transferred to a cell culture dish by changing the culture medium every 2-3 days for ~10 days. These primary cells were cultured, stretched, and frozen at -196°C for later use. Cell culture was maintained in DMEM containing 20% FBS, 1% P/S, 1% NEAA, and 100 mM β-ME.

7. T7E1 analysis

Genomic DNA was extracted using ExgeneTM cell SV (GeneAll Biotech, Seoul, Korea) according to the manufacturer's instructions. Using the primers shown in Table 1, a PCR amplification product containing the CRISPR/Cas9 target site was generated. The T7E1 assay was analyzed as previously described (Kim et al., 2009). In summary, PCR amplification products were denatured at 95℃ and analyzed to form heteroduplex DNA, and then T7 endonuclease 1 (ToolGen Inc., Seoul) was treated with 5 units at 37℃ for 20 minutes, followed by 2% agarose gel electrolysis. It was analyzed by Yeongdong.

8. Deep sequencing

The on-target region was amplified with genomic DNA and used for library construction.

Bidirectional read sequencing was performed on the same amount of PCR amplification products using Illumina MiSeq (v2, 300 cycles). Rare sequence reads that occur only once were excluded to eliminate errors related to sequencing reactions and amplification. Insertions or deletions around the CRISPR/Cas9 cleavage site (3bp upstream of PAM) were considered to be CRISPR/Cas9-induced mutations.

[Table 1]

Primers used for T7E1 assay and deep sequencing

The primer sequences in Table 1 are sequentially named as SEQ ID NOs: 2 to 5.

9. Quantitative real-time PCR

All samples were stored at -80°C until analysis. Total RNA was extracted according to the manufacturer's protocol using the Easy-Spin Total RNA Extraction Kit (iNtRON), and the total RNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). CDNA was synthesized using Maxime RT Premix (iNtRON) according to the manufacturer's protocol. PCR plate with 1 μl of cDNA, 0.4 μl of forward primer (10 pmol/μl), 0.4 μl of reverse primer (10 pmol/μl), 10 μl of SYBR Premix Ex Taq (Takara) and 8.2 μl of Nuclease-free water (Ambion) (MicroAmp optical 96-well reaction plate) was made. The reaction was carried out for 40 cycles, and the cycling parameters were as follows: denaturation at 95° C. for 15 seconds, annealing at 60° C. for 1 minute and extension at 72° C. for 1 minute. All oligonucleotide primer sequences are shown in Table 2. For each gene, in the order of Forward and Reverse, the primer sequences in Table 2 are sequentially named as SEQ ID NOs: 6 to 23. Expression of each target gene was quantified by comparison with the expression of the internal control gene (GAPDH) using the equation R = 2- ^{[ΔCt sample -ΔCt control]} .

[Table 2]

Real-time PCR primer list

10. Western blot analysis

The tissue was finely crushed by a homogenizer, washed with phosphate buffer (PBS, pH 7.4) and ice-cold lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 0.1% afro). Tinine, 0.1% SDS, 1mM PMSF). The lysate was centrifuged (13,000 rpm, 4° C. for 10 minutes). 10 μg of protein from the supernatant was fractionated by 10% SDS-PAGE and electrophoresed onto a PVDF membrane. Blots were blocked overnight with 5% skim milk at 4% C. The next day, PVDF membranes were prepared with rabbit anti-klotho polyclonal antibody (1:1000) and rabbit anti-β-actin (1:5000) diluted in 5% skim milk Tris-buffered saline (TBST) containing 0.1% Tween 20. Incubated for 2 hours. Then, the membrane was washed three times with TBST for 10 minutes, and incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1:5000) for 1 hour. The blot was confirmed using Pierce SuperSignal West Pico Chemiluminescent System (Thermo Fisher Scientific). The density of the Immunoblot was scanned with an image acquisition system (VilberLourmat, Fusion SL3500).

11. Statistical Analysis

Statistical analysis was performed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA).

Percent data (eg, fusion, cleavage and blastocyst production rates) were compared with the experimental group by Man-Whitney's test. Data are expressed as mean±SEM. <0.05 was judged to be statistically significant.

Example: 1 CRISPR - sgRNA RNPs Targeted by klotho Transfected Unselected Using selection cells After SCNT Embryonic development And Genome editing Efficiency evaluation

The pre-transplant development of embryos after SCNT was compared with Cas9-sgRNA RNPs (klotho gene targeting) or untransformed fibroblasts (Table 3).

[Table 3]

Post-SCNT development of pig embryos before transplantation and comparison of transfected or non-transfected fibroblasts with Cas9-sgRNA RNPs (targeting the klotho gene)

A total of 253 SCNT embryos were made using untransformed donor cells (n=58) and transformed donor cells (n=195). Some of them developed cysts (n=6 and 20), and there was no significant difference in the rate of cyst formation.

However, there was a significant difference in the fusion rate between the non-transfected group and the transfected group.

In the T7E1 analysis, 20 (65%) of cysts derived from Cas9-sgRNA RNPs transfected cells showed klotho genetic modification (FIG. 2A ). To confirm fertilization, we performed an in-depth sequencing analysis and 8 cysts showed 40.0% monolithic deformation and 5 cysts showed 25.0% double antagonism (Table 4 and FIG.

[Table 4]

DNA editing ratio for the klotho gene of blastocysts produced by SCNT using porcine fibroblasts not transfected with Cas9-sgRNA RNP

Example 2 : Bae Ai Sik And klotho Knockout Fetal cell line Evaluation of genome editing efficiency after establishment

Based on the high fertilization rate in embryos before transplantation, non-selected donor cells transfected with Klotho targeting CRISPR-sgRNA RNP were embryonicated after SCNT.

A total of 936 SCNT embryos were transferred to 5 recipients, and 1 recipient became pregnant (Table 5).

[Table 5]

Results of somatic cell nuclear transplantation for embryo transfer to recipient

After 28 days, 16 cloned embryos were recovered.

Nine of them were absorbed and 7 of them survived (Figure 3). We made a klotho knockout fetal cell line by primary culture using 7 live fetus body parts, and deep sequencing was performed using the remaining tissues of alive and surviving fetus.

In in-depth sequencing analysis, 4 out of 9 absorbed fetuses (44.4%) and 3 out of 7 surviving fetal tissues (42.9) showed single-order fertilization (Table 6 and FIG. 4).

[Table 6]

Example 3: Effect of klotho monallelic knockout on gene expression of fetal fibroblasts and preimplantation development of cloned embryos from cell lines

The gene expression of Klotho monallelic knockout fetal fibroblasts (Fetus L2) and wild-type fetal fibroblasts was compared. In this experiment, the expression of genes related to senescence (IGF1 signaling gene, FOXO1 and antioxidant gene) and cell death was evaluated. As shown in Figure 6, the expression of IGF1 and IGF1R was significantly reduced in the fetal (Fetus) L2 fibroblasts. In addition, Fetus L2 fibroblasts significantly reduced the expression of their downstream target genes (MnSOD and CAT) with FOXO1 and antioxidant functions. Regarding apoptosis-related genes, the Bax/Bcl2 ratio and Caspase3 expression were significantly increased in Fetus L2 fibroblasts. Although there were significant changes in the expression of senescence-related genes in klotho monallelic knockout fetal fibroblasts, there were no significant differences in cleavage rate and blastocyst generation rate between wild type and cloned embryos derived from Fetus L2 fibroblasts (82.8% and 15.2, respectively). % vs. 85.5% and 14.3%) (Table 7).

[Table 7]

Comparison of pre-transplantation development of SCNT embryos derived from wild-type and klotho monallelic knockout porcine fibroblasts

Example 4: Result of comparison of expression of klotho monallelic knockout fetal fibroblast cloned SCNT embryos and aging-related genes and klotho protein in fetal placenta

As shown in Table 8, a total of 2088 SCNT embryos cloned from the klotho monallelic knockout pigcine fibroblast (Fetus L2 and L3) were delivered to 11 synchronized recipients. Seven of the 11 recipient subjects (63.6%) were pregnant. But all of them stopped. To find out why the klotho monallelic knockout fetus was aborted, placenta was recovered from the klotho monallelic fetus and wild-type (artificially fertilized) fetus at 4 to 5 weeks, and the expression of aging-related genes and klotho protein was investigated (Fig. 7). . The expression of IGF1, FOXO1 and downstream target genes (MnSOD and CAT) was significantly reduced in the Fetus L2 placenta. In the case of apoptosis-related genes, the Bax/Bcl2 ratio and Caspase3 expression were significantly increased in Fetus L2 placenta. As a result of confirming the expression of klotho protein in the placenta of fetuses cloned from Fetus L2 fibroblasts using western blotting, the expression of klotho was relatively low (Fig. 7).

[Table 8]

Delivery outcome of cloned SCNT embryos from klotho monallelic knockout porcine fetal fibroblasts

<110> seoul national university <120> klotho knock-out porcine Models for klotho deficiency disease and the use thereof <130> CP18-186 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding guide RNA <400> 1 tagaacaagg ctgaagactt cgg 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Klotho(T7E1)F <400> 2 cctcaagtag taaaaccctc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Klotho(T7E1)R <400> 3 ggttttgtca gctgacttac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Klotho (Deep sequencing) F <400> 4 cttgctcttg tcctctttcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Klotho (Deep sequencing) R <400> 5 caacaattcc ccaagcaaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F <400> 6 gtcggttgtg gatctgacct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R <400> 7 ttgacgaagt ggtcgttgag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IGF1 F <400> 8 aggaggctgg agatgtactg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IGF1 R <400> 9 tggcatgtca ttcttcactc 20 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IGF1R F <400> 10 attcgcacca atgcttca 18 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> IGF1R R <400> 11 agggcgggtt ccacttc 17 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXO1 F <400> 12 cattgagcgc ttagactgtg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXO1 R <400> 13 tctcagttcc tgctgtcaga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MNSOD F <400> 14 gcttacagat tgctgcttgt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MNSOD R <400> 15 aaggtaataa gcatgctccc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT F <400> 16 ttaatccatt cgatctcacc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT R <400> 17 ggcggtgagt gtcaggatag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX F <400> 18 tgcctcagga tgcatctacc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX R <400> 19 aagtagaaaa gcgcgaccac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCL2 F <400> 20 agggcattca gtgacctgac 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCL2 R <400> 21 cgatccgact caccaatacc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CASPASE3 F <400> 22 cgtgcttcta agccatggtg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CASPASE3 R <400> 23 gtcccactgt ccgtctcaat 20

Claims (16)

As a pig model for age-related diseases,
Contains an artificial modification in the nucleic acid sequence of the klotho gene in the genomic DNA sequence of the pig,
The modification is a deletion, substitution, insertion or inversion of one or more nucleic acids,
The modification is present only in one of the homologous chromosomes,
At this time, the pig, when compared to the wild type, pig model, characterized in that it comprises cells having reduced expression of one or more genes selected from the group consisting of IGF1, IGFR1, FOXO1, MnSOD, and CAT genes.
delete The method of claim 1,
Pig model, characterized in that the modification of the nucleic acid occurs in the exon 3 region of the klotho gene
delete The method of claim 1,
The klotho gene is genetically engineered by RNA-guided endonuclease,
In the proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence constituting the klotho gene, or in a contiguous 3bp to 25bp nucleotide sequence site located adjacent to the 5'end and/or 3'end thereof
Deletion or insertion of one or more nucleotides;
Substitution with one or more nucleotides different from the wild-type gene;
Insertion of one or more nucleotides from outside
Modification of one or more nucleic acids of
Pig model comprising a.
delete The method of claim 1,
Klotho deficiency disease is a pig model, characterized in that at least one disease selected from the group consisting of arteriosclerosis, osteoporosis, stroke and Alzheimer's.









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