KR20190038449A - 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-deficient disease model, a method for producing the same, and uses thereof. More particularly, the present invention relates to the use of a transgenic pig having klotho gene knocked out as an animal disease model.

Description

Klotho gene knockout disease model animal and its use {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, a method for producing the same, and a use thereof, and more particularly, to a transgenic pig using a klotho gene knock-out transformed pig as an animal model of disease .

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

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

Due to the marked differences in dimenomening, breeding, lifespan and behavior with humans, the need for animal models using more closely related species has been raised, and in the case of primates, extremely limited due to the scarcity and cost and difficulties of breeding management Pigs have been recognized as similar to human anatomy physiologically in disease studies, and have already been used for the pathological mechanism and treatment of various diseases. In particular, It is possible to avoid ethical problems as compared with the case of using as a disease model, and a stable breeding system is established, so that it is easy to maintain and manage when developing an animal model.

Accordingly, the present inventors have found that, by using a pig recognized to have similarity with a human gene, the Klotho gene is knocked out by applying the transformed somatic cell cloning technique using the CRISPR system which recognizes and cleaves a specific sequence of the human Klotho gene, And the present invention has been completed.

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

It is another object of the present invention to provide various uses of pigs for the klotho deficiency disease model.

In order to solve the above problems,

provides an artificial manipulation of the klotho gene and its use for the production of pigs for the klotho-deficient disease model. More particularly, the present invention relates to a method of producing pigs having a klotho deficient phenotype and their use by artificial manipulation or modification of the klotho gene.

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

The " klotho gene " plays a crucial role in regulating the development and aging of aging-related diseases in mammals. Klotho is an enzyme produced by the KL gene, which produces I-type membrane proteins associated with [beta] -glucuronidase. The human klotho gene is located at 33.59-33.64 Mb of chromosome 13, for example, having the mRNA sequence of NM_004795. It also has the protein sequence of NP_004786

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

In one embodiment of the present invention, a Klotho-deficient disease pig model is provided in which the expression of klotho protein is knocked out by deformation in the nucleic acid sequence of the klotho gene.

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

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

In one embodiment of the invention, a modification in a nucleic acid sequence can be artificially caused by a nuclease agent.

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

(a) zinc finger nuclease (ZFN);

(b) transcription activator-like effector nuclease (TALEN);

(c) meganuclease; or

(d) RNA-Guided Endonuclease (RGEN)

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

The endonuclease may be selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus,

Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces spp. Streptosporangium roseum, Streptosporangium roseum, Alicyclobaclus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Bacillus spp. ), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, and the like. , Polaromonas na For example, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, , Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus de sulphorus (Candida albicans), Candida albicans Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Peltomaculorum thermoprofus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas spp., Pseudoalteromonas spp. haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microorganisms such as < RTI ID = 0.0 & Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus or Acari sp. It can be derived from keulroriseu Marina (Acaryochloris marina). Additional 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 an RNA-guided endonuclease,

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

Deletion or insertion of one or more nucleotides;

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

Insert one or more nucleotides from outside

Lt; RTI ID = 0.0 > of < / RTI >

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

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

Modifications of the nucleic acid can occur in the promoter region of the gene.

Modifications of the nucleic acid can occur in the exon region of the gene.

Modifications of the nucleic acid can occur in the intron region of the gene.

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

Further, in another embodiment, the present invention provides a guide nucleic acid capable of forming a complementary bond, respectively, to a target sequence of a reprogramming factor, for example, a nucleic acid sequence of Klotho gene. The target sequence may be at least one of the nucleotide sequences of the Klotho gene.

The guide nucleic acid may be, without limitation, a nucleotide of 18-25 bp, 18-24 bp, 18-23 bp, 19-23 bp, 19-23 bp, or 20-23 bp.

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

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

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

The cell or embryo may contain an inactivated Klotho gene that has undergone transformation in the nucleic acid sequence and may develop into an individual having a phenotype of the Klotho gene.

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

In one embodiment, a guide nucleic acid capable of forming a complementary bond to at least one site of the nucleic acid sequence constituting the Klotho gene; And an RNA-guide endonuclease or a nucleic acid encoding the same.

The description of the relevant configuration is as described above.

In an embodiment,

In cells or embryos containing the target genomic locus on the Klotho gene,

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

(b) RNA-Guided Endonuclease (RGEN)

Contacting the transformed cells or embryos with a nucleic acid sequence of the Klotho gene.

The guide RNA and the endonuclease may be present in one or more vectors each in the form of a nucleic acid sequence or may be present by complexing with a guide nucleic acid and the binding of an RNA-guide endonuclease.

The contacting step may be carried out in vivo or ex vivo.

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

The step of contacting may be carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) 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 provides a cell line transformed with a recombinant vector for producing a disease pig model in which the Klotho gene is knocked out, and a pig for a disease model in which the Klotho gene is knocked out. At this time, the transformation can be performed by somatic cell nuclear transfer (SCNT).

The present invention includes all the transformation vectors, cells, and nuclear transfer embryos necessary for the production of a Klotho-deficient disease model, which can be obtained in the process of the method for producing pigs for the Klotho-deficient disease model.

That is, according to another embodiment of the present invention,

A transformed cell into which a recombinant vector for constructing a Klotho-deficient disease pig model containing a sequence encoding a guide nucleic acid having an activity of cleaving and cleaving a Klotho gene-specific sequence and an RNA-guide endonuclease is introduced can be provided ,

 The nucleus of the transfected porcine-derived cells can also be provided with a porcine nuclear transfer embryo formed by implantation into a enucleated oocyte.

In an embodiment, the invention can provide a method and such an entity for obtaining an individual produced by said method.

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

The present invention also provides various uses of pigs for the Klotho deficiency disease model.

As an example, there is provided a method of screening for a preventive and therapeutic agent for Klotho deficiency disease, comprising:

Administering a prophylactic and therapeutic agent candidate to the pig for the Klotho deficiency disease model; And

After administration of the candidate substance, the tissue of the pig is analyzed in comparison with a control group to which the candidate substance is not administered.

Thus, the present invention provides animal models of Klotho deficiency disease using pigs that are the most physiologically similar to humans, and their various uses.

The Klotho-deficient model pig of the present invention is an animal model that is genetically similar to humans, useful for genetic analysis and useful for studying precise cause and mechanism of disease, and also suitable for toxicity and safety assessment. Klotho deficiency disease mechanism , It is very useful as an animal model of Klotho deficiency disease since it can be used variously for identification of therapeutic substances, development of diagnostic methods, and the like.

Figure 1 is a schematic representation of sgRNA specific for exon 3 of the porcine Klotho gene. The sgRNA target sequence is highlighted in red and the protospacer proximal motif (PAM) in blue.
Figure 2 shows the results of assay of T7 endonuclease I (T7E1) assay for generation of klotho gene knockout blastocyst by SCNT using porcine fibroblasts not transfected with Cas9-sgRNA RNP [T7E1 assay was performed in 20 replicate blastocysts with genomic DNA (M, Marker, WT, wild type, BL, blastocyst)]
Fig. 3 is a diagram showing the exon 3 editing plan of the klotho gene of the blastocyst in Fig. 2; Fig.
Fig. 4 is a photograph of the recipient's uterus (A) and fetus (B) on the 28th day of pregnancy.
FIG. 5 is a graphical representation of the editing scheme for exon 3 of the fetal klotho gene produced by SCNT using non-selected porcine fibroblasts transfected with Cas9-sgRNA RNP (Fetus A, absorbed fetus; Fetus L, living fetus).
(A) IGF1 signaling genes (IGF1 and IGF1R), (B) FOXO1 and antioxidant genes (MnSOD and CAT), (C) Apoptosis Related genes (Bax / Bcl2 ratio and Caspase3). Data were presented as mean ± SEM, with significant differences between the groups indicated by different letters within each category (P <0.05). The experiment was repeated at least three times (WT, wild type; Fetus L2, living fetus 2 (WT / -17 bp, + 12 bp)).
(A) IGF1 signaling genes (IGF1 and IGF1R), (B) FOXO1 and antioxidant genes (MnSOD and CAT), and (C) Apoptosis-related genes (Bax / Bcl2 ratio and Caspase3). Data were presented as mean ± SEM, with significant differences between the groups indicated by different letters within each category (P <0.05). The experiment was repeated at least three times. (WT, wild type; Fetus L2, living fetus 2 (WT / -17bp, + 12bp)) was detected in the clotho monallelic knockout pig placenta detected by Western blot analysis.

Justice

&Quot; Cell ", " host cell ", " transformed host cell " and the like refer to a progeny or potential progeny of such a cell as well as a specific target cell. Such a offspring, in fact, would not be identical to the parent cell, but is still included within the scope and range of terms used in the present invention, since certain modifications may occur later in time by mutation or environmental effects.

The term " modified " or " engineered ", as applied to nucleic acids, is used interchangeably herein to refer to the artificial alteration of a nucleic acid sequence constituting a gene. Such alterations may include deletion, substitution, insertion or inversion of one or more nucleic acids, and the like. A modified or engineered nucleic acid sequence may appear as an alteration, for example, an increase, enhancement, decrease or loss of expression and / or functional activity of these nucleic acids or an expression product thereof.

In particular, the terms "damage" and "destruction" as applied to nucleic acids are used interchangeably herein to refer to any genetic modification that reduces or eliminates the expression and / or functional activity of a nucleic acid or an expression product thereof. For example, destruction 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 (e.g., mRNA and / or protein). Genetic modification includes complete or partial inactivation, repression, deletion, disruption, containment or downregulation of a nucleic acid (e.g., a gene). One embodiment of the invention includes, but is not limited to, the use of reproductive gene activity or any other molecule that inhibits the level or functional activity of the expression product of the reproductive gene.

"Disease model animal" refers to an animal with a disease that closely resembles a human disease. Diseases in the study of human disease The significance of model animals is due to their physiological or genetic similarities between humans and animals. Biomedical disease models in disease studies Animals provide research materials for various causes of diseases, pathogenesis and diagnosis, and research on disease-related animals to identify disease-related genes and understand the interactions between genes And the basic efficacy and toxicity test of the developed new drug candidates will provide basic data for judging the possibility of practical use.

An "animal" or "laboratory animal" means any mammal other than a human. The animal includes animals of all ages, including embryos, fetuses, neonates and adults. Animals for use in the present invention can be used, for example, from commercial sources. Such animals include, but are not limited to, laboratory animals or other animals, rabbits, rodents (such as mice, rats, hamsters, gerbils and guinea pigs), cows, sheep, pigs, goats, horses, (Eg, chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a pig.

&Quot; Knock-out &quot; refers to a modification of a gene sequence that results in a degradation of a target gene, preferably by which target gene expression is not detectable or meaningless. In the present invention, knockout of the Klotho gene means that the function of the Klotho gene is substantially lowered so that the expression can not be detected or exists only at a meaningless 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. In knock-outs, for example, in the case of exposing the animal to a substance that promotes target gene degeneration, introducing an enzyme that promotes recombination at the target gene site, or other methods of directing target gene modification after birth, Conditional knock-outs in which deformation can occur are also included.

A &quot; target gene &quot; refers to a nucleic acid encoding an intracellular polypeptide unless specifically stated otherwise. The term " target sequence ", as used herein, refers to a portion of a target gene, for example, one or more exon sequences of a target gene, an intron sequence or a control sequence of a target gene or an exon and intron sequence of a target gene, And control sequences, exons and control sequences, or combinations of exons, introns, and control sequences.

"Sequence-specific endonuclease" or "sequence-specific nuclease" refers to a polynucleotide that recognizes and binds a polynucleotide, eg, a target gene, in a particular nucleotide sequence, unless specifically stated otherwise, &Lt; / RTI &gt; refers to a protein that catalyzes single- or double-strand breaks in E. coli.

&Quot; Vector " means a nucleic acid molecule, preferably a DNA molecule, derived from, for example, a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence can be inserted or replicated. The vector typically contains one or more unique restriction sites and may autonomously replicate in a defined host cell comprising a target cell or tissue or a progenitor cell or tissue, Can be integrated into the genome of the host defined to be regenerable.

&Quot; Wild type " means a gene or gene product having the characteristics of the gene or gene product when separated from a naturally occurring sequence. Wild-type genes are most frequently observed in populations, and thus are arbitrarily representative of the "normal" or "wild-type" form of the gene.

&Quot; Modified ", " modified " or " mutant " refers to a gene or gene product that exhibits a change in sequence and / or functional properties as compared to a wild-type gene or gene product.

&Quot; Protein ", " polypeptide ", and " peptide " include polymeric forms of amino acids of any length, including encoded and unencrypted 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 may be used interchangeably.

&Quot; Nucleic acid " and " polynucleotide " include polymer forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified forms thereof. These include 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, Or a polymer comprising a derivatized nucleotide base. For convenience, the nucleic acid size may be expressed in bp, whether the nucleic acid is double stranded or single stranded.

By " complementarity " of a nucleic acid is meant 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 nucleotide base group. The complementary bases of DNA are typically A, T, C, and G. In RNA, these are typically C and G, U and A.

Hybridization requires that both nucleic acids contain complementary sequences, although mismatches between bases are possible. Conditions suitable for hybridization between two nucleic acids depend on the nucleic acid length and degree of complementarity, which are well known in the art.

'Nuclear transplantation' refers to a genetic manipulation technique that allows an enucleated oocyte to artificially bind other cells or nuclei to the same trait. "Nuclear oocyte" refers to an oocyte into which nuclear donor cells are introduced or fused.

'Nuclear donor cells' refers to nuclei of cells or cells that deliver nuclei to recipient oocytes, which are nuclear receptors. The term &quot; oocyte &quot; preferably refers to a mature oocyte that has reached the middle stage of the second meiosis. In the present invention, porcine somatic cells or stem cells can be used as the nuclear donor cells.

The term "somatic cell" refers to a cell other than a germ cell that constitutes a multicellular organism, a cell that is specialized for a specific purpose and does not become any other cell, and a cell having several different functions Cells that have the ability to differentiate.

"Stem cell" refers to a cell that can develop into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.

"Living offspring" refers to an animal that can survive outside the uterus. Preferably, it refers to an animal that can survive for 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months, or 1 year. The animal does not require an intrauterine environment for survival.

By "about" is meant a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprises " and " comprising ", when not explicitly required in the context, include a stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

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

More particularly, the present invention relates to a method for artificially transforming a nucleic acid sequence of Klotho and an animal model of Klotho deficiency disease using the same.

[Klotho-deficient pig model]

As a model animal, pigs have a longer survival period than conventional rodent models and are known to have a similar biological mechanism to humans, and are thus attracting attention as a next-generation disease model animal species that can overcome the limitations of the prior art.

Therefore, the present inventors have developed a more reliable animal model of Klotho deficiency disease by utilizing the large animal pig, confirming the induction of Klotho deficiency disease through the production and behavior of a disease model which goes beyond the rodent model, and to utilize it.

Klotho deficient disease animal models exhibit phenotypes such as multiple aging similar to human premature aging syndrome due to defects in klotho gene expression. Growth retardation, infertility, arteriosclerosis, osteoporosis, and premature death.

It has been reported that Klotho's single nucleotide polymorphisms are 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, higher levels of Klotho protein may prolong the life span of brain cells, reduce the incidence of related diseases such as heart disease, strengthen cognition such as attention, memory, cognition, and accelerate the aging process. Therefore, Klotho deficiency diseases can be, for example, arteriosclerosis, osteoporosis, stroke or Alzheimer's.

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

That is, it relates to a genetically modified animal, in particular a recombinant pig knockout Klotho gene.

Pigs are anatomically similar in size to organs and are suitable as heterogeneous organs, as well as physiologically and genetically similar to humans. In addition, it has high breeding capacity, which makes it suitable for the development of biological materials for production and treatment as well as an animal model that is good for toxicity and safety evaluation.

The pigs to be used in the present invention can be any known kind which can be appropriately selected and used by a person skilled in the art.

Pigs (Scientific name: SusscrofPDomestica) belong to the subspecies, descent descent, emerald-side subspecies, right titles (including cattle, chlorine) and wild boar, and the number of chromosomes is 2n = 38. Originally, pigs have attracted much attention as experimental animals as well as ovarian animals raised with cattle, with a similar interest in physiological anatomical findings and a high interest in animal welfare in recent years. The pigs are attracting much attention as physiological anatomy. Pigs are widely used throughout the study because they are similar to humans in many physiological and anatomical points.

In one embodiment, a small pig (typical example: Pittman, Moore) of 60-70 kg of maximum body weight at maturity is used, or a small money (adult: 30-40 kg) (NIH system), a Yucatan mini-pig, a micro-pig, or the like may be used.

[Klotho genetic modification or modification]

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

Embodiments of the invention include genetically engineering Klotho genes. By altering the Klotho gene such that it can not be expressed, or by genetically expressing factors that would inhibit transcription or translation of the gene.

In a specific embodiment, the concentration and / or activity of the Klotho protein is at least 1%, 5%, 10%, 20%, 30%, 30%, 30% %, 40%, 50%, 60%, 70%, 80% or 90%.

Klotho is an enzyme produced by the KL gene, which produces I-type membrane proteins associated with [beta] -glucuronidase. The human klotho gene is located at 33.59-33.64 Mb of chromosome 13, for example, having the mRNA sequence of NM_004795. It also has the protein sequence of NP_004786

In certain embodiments, the Klotho gene can be used to transform into knockout.

Each of the nucleic acids encoding Klotho can be used without limitation as long as it has a nucleotide sequence encoding Klotho known in the art.

The nucleic acid encoding Klotho can be prepared by a recombinant method known in the art. For example, PCR amplification for amplifying a nucleic acid from a genome, chemical synthesis, or a technique for producing a cDNA sequence.

The nucleic acid may also have a nucleotide sequence encoding each functional equivalent of Klotho.

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

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

Any of the known methods can be used to knock out the Klotho gene and use, for example, RNA-Guided Endonuclease (RGEN).

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

In one embodiment, the cell or embryo is an in vitro cell or embryo that specifically binds to a chromosomal target site of a cell or an embryo to cause double stranded DNA breakage to interfere with the gene to selectively inhibit the germ cell formation process, The agent may be selected from the group consisting of a targeting endonuclease and a recombinant enzyme fusion protein.

In other embodiments, manipulation of the Klotho gene

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

Such targeted modifications include the addition of one or more nucleotides, deletion of one or more nucleotides, substitution of one or more nucleotides, knockout of the polynucleotide of interest or a portion thereof, dissolution of the polynucleotide of interest or a portion thereof, Substitution with a nucleic acid sequence, or combinations thereof. In a specific embodiment, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are altered to form a targeted genomic modification

In an illustrative example, 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 reproductive gene. , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%.

[Genetic manipulation method]

The Klotho gene manipulation of the present invention can be performed considering the gene expression regulatory process.

In embodiments, selection may be made of suitable manipulation means for each step in transcription regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translational regulation or protein modification regulation.

In embodiments, RNAi (RNA interference or RNA silencing) can be used to prevent small RNAs (sRNAs) from interfering with mRNA, degrading stability and, in some cases, destroying protein synthesis 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 reproductive gene and weakens or destroys the expression of the reproductive gene.

In embodiments, wild-type or variant enzymes that can catalyze the hydrolysis (cleavage) of DNA or RNA molecules, preferably bonds between nucleic acids in DNA molecules, can be used.

In certain embodiments, a meganuclease; Zinc finger nuclease; TALE-nuclease; RNA-Guided Endonuclease (RGEN), for example, one or more nucleases selected from the group consisting of CRISPR / Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) The expression of the genetic information can be controlled.

In one aspect, compositions and methods for targeting and genetically manipulating some or all of the non-coding or coding regions 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 may be engineered or modified. As a result of the operation, it includes knock down, knock out, and knock in forms.

In embodiments, a promoter region, or a transcription sequence, such as an intron or exon sequence, may be targeted. A cipher sequence, for example a cipher region, an initial cipher region, can be targeted for alteration of expression and knockout.

In an embodiment, the nucleic acid modification is carried out in the presence of 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, Deletion, and / or insertion of 1 bp nucleotides.

In an embodiment, it can be targeted to include deletions or mutations in one or more of the Klotho genes.

In an embodiment, the gene knockdown can target the transcription site of the unwanted 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 the Klotho gene by targeting a non-coding sequence of a promoter, enhancer, intron, 3'UTR, and / or polyadenylation signal.

The genetic modification may be a regulation, eg, inactivation, of a gene by deletion, substitution, and / or insertion of one or more nucleotides, all or a portion of the gene (eg, one or more nucleotides). The gene inactivation means that the gene is inhibited from expression or downregulation of the gene or modified to encode a protein whose original function is lost. In addition, gene regulation can be achieved by simultaneously targeting both intron regions surrounding one or more exons of the target gene, resulting in structural modification of the protein resulting from deletion of the exon region, expression of dominant negative forms of protein, and expression of competitive inhibitors secreted in soluble form This may be a change in function of the gene due to the result

The gene 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 the original function

In one example, the transgenesis may be by inactivating the targeted gene by catalyzing a single stranded or double stranded cleavage of a specific site in a gene targeted by a genetic correction system comprising a truncation endonuclease .

Nucleic acid strand breaks catalyzed by truncated 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 is made in the DNA sequence at the cleavage site, whereby the gene can be inactivated.

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

Modifications of the Klotho gene may be derived from one or more of the following:

(1) deletion of all or part of the target gene (hereinafter referred to as a "target gene"), such as 1 to 30 nucleotides, 1 to 27 nucleotides, 1 to 25 nucleotides, 1 to 23 nucleotides, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide,

2) a nucleotide of 1 bp or more, 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 , One to three, or one nucleotide to the original (wild type) and to a different nucleotide, and

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

A portion of the target gene that is modified (the 'target site') may be at least 1 bp, 3 bp, 5 bp, 7 bp, 10 bp, 12 bp, 15 bp, 17 bp, 20 bp, 3bp to 30bp, 5bp 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, 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, 20 bp to 23 bp, 3 bp to 23 bp, 5 bp to 23 bp, 7 bp to 23 bp, 10 bp to 23 bp, 12 bp to 23 bp, 15 bp to 23 bp, 17 bp to 23 bp, 20 bp to 23 bp, 1 bp to 20 bp, 3 bp to 20 bp, 20bp, 7bp to 20bp, 10bp to 20bp, 12bp to 20bp, 15bp to 20bp, 17bp to 20bp, 21bp 25 bp, 18 bp to 22 bp, or 21 bp to 23 bp.

When the Klotho gene modification is induced by the Cas9 protein, the gene modification is located adjacent to the 5 'end of the PAM (proto-spacer-adjacent motif) sequence unique to Cas9 protein according to the microorganism derived from the nucleotide sequence of each gene Such as single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, single stranded or double stranded, Or deletion and / or insertion of one or more nucleotides in the course of repairing said cleavage.

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

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

In another example, when the Cas9 protein is from Streptococcus aureus, the PAM sequence is 5'-NNGRR (T) -3 ', where N is each independently A, T, C or G , R is A or G, and (T) means an optionally included 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 an exemplary embodiment of the present invention,

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

The target sequence may target two or more sites of the nucleic acid sequence constituting the Klotho gene at the same time.

In one embodiment, one or more of the nucleotide sequences constituting the Klotho gene may be targeted using one kind of guide RNA. This allows the Klotho gene to knock out or knock down.

In one embodiment, two or more kinds of guide RNAs can be used to target one or more of the nucleotide sequences constituting the Klotho gene. This allows the Klotho gene to knock out or knock down.

In one embodiment, the level and / or degradation of the Klotho gene may be due to variations in the nucleic acid sequence that make up the Klotho gene, such as a noncoding region, a coding region, and / or a modification in an intron or the like.

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

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

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

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

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

Substitution of the endogenous nucleic acid sequence with a heterologous nucleic acid sequence which is the dissolution of a Klotho gene nucleic acid sequence or a portion thereof,

Lt; RTI ID = 0.0 &gt; Klotho &lt; / RTI &gt;

Thus, in a specific embodiment, it can be reduced or eliminated by destroying a gene or the like that encodes a protein or polypeptide having the activity of a Klotho gene. In a specific embodiment, at least one or more sites, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are changed in the Klotho gene nucleic acid sequence. Using various methods, it is possible to cause additional targeted deformations.

In one embodiment, the level and / or degradation of the Klotho protein may be due to genetic modification (i. E., Regulatory region, coding region, and / or genetic modification in the intron, etc.) of the Klotho gene.

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

Such genetic modification can be used, 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 a portion thereof,

Substitution of the endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or a combination thereof, which is the dissolution of the Klotho gene or a portion thereof.

Thus, in a specific embodiment, 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, additional targeted genetic modifications can be made.

[NUCLEASE SYSTEM]

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

CRISPR / Cas  system

Exemplary embodiments of the present invention include methods of inducing sequence-directed double-strand breaks by introducing components of a CRISPR system, including CRISPR-related nuclease Cas9 and sequence-specific guide RNA (gRNA) And utilizing the capabilities of the CRISPR system to induce said cleavage.

The components of the CRISPR system, including the CRISPR-related nuclease Cas9 and the sequence-specific guide RNA (gRNA), can be introduced into cells in one or more vectors, e.g., coded on a plasmid.

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

RNA guide Endonuclease

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

The nuclease domain has catalytic activity for nucleic acid cleavage. Cleavage involves the destruction of the covalent bond of a nucleic acid molecule. Cleavage can produce a smooth or staggered end 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) (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 (CasA), Casse, CasH, Csy1, Csy2, Csy3, Cse1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1 and Cu1966, A modified version.

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

The Cas9 protein can be, for example, a protein selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus,

Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces spp. Streptosporangium roseum, Streptosporangium roseum, Alicyclobaclus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Bacillus spp. ), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Mucrose,

Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, and the like. ), Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonipex degeneration Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Pinegoldia magna ( Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, For example, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anna spp. For example, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Such as Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatori sp. a sp.), Petrotoga mobilis, Thermosipho africanus or Acaryochloris marina. Additional 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.

The Cpf1 protein may be selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. For example, Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp (GW2011_GWA_33_10), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus methanoplasma termitum and Eubacterium eligens. . (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens But is not limited thereto.

The Cas protein can be a wild-type protein (i.e., one that occurs in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. The Cas protein may also be an active variant or fragment of the wild type or modified Cas protein. The active variant or fragment is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 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-strand break inducing activity. Methods for measuring nick induction or double-strand break induction activity are known.

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

One or two nucleases domains may be deleted or mutated, resulting in either no longer functional or a decrease in nuclease activity.

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

When two nuclease domains are deleted or mutated, the resulting Cas protein (e. G. Cas9) will degrade the ability to cleave two strands of double stranded DNA. An example of a mutation that converts Cas9 to nick carase is D10A or H939A or H840A in the RuvC domain of sp. Cas9.

The Cas protein may also be a fusion protein. For example, the Cas protein can be fused to a cleavage domain, a reproductive deformation domain, a transcription activation domain, or a transcription repression factor domain. The fused domain or heterologous polypeptide may be located at the N-terminus, the C-terminus, or within the Cas protein.

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

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

The Cas protein may also comprise a heterologous polypeptide, e. G. A fluorescent protein, a purified tag or an epitope tag, to facilitate tracking or purification.

The Cas protein may 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 a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid encoding a Cas protein, such as RNA (e. G., Messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon-optimized for efficient translation into a protein from a particular cell or organism.

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

The guide RNA ( gRNA )

A " guide RNA " or " gRNA " includes RNA molecules that bind Cas proteins and target Cas proteins at specific locations within the target DNA. The guide RNA may comprise two segments, a " DNA target segment " and a " protein binding segment. &Quot; A " segment " includes a segment, section or region of a molecule, such as a continuous stretch of nucleotides of RNA. Some gRNAs include two separate RNA molecules: the "activator-RNA" crRNA and the "targeter-RNA" tracrRNA. Another gRNA is a single RNA molecule (a single RNA polynucleotide), also referred to as a "single molecule gRNA", "single guide RNA" or "sgRNA".

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

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

The DNA targeting segment of the gRNA interacts with the target DNA in a sequence-specific manner through hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA targeting segment can be different and determines the location in the target DNA with which the gRNA and the target DNA will interact. The DNA targeting segment of the subject gRNA may be modified to hybridize to the desired sequence in the target DNA.

The DNA targeting segment may have a length of from about 12 nucleotides to about 100 nucleotides. For example, the DNA targeting segment may comprise about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, 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 in any form (e. g., full-length tracrRNA or active tracrRNA) and in various lengths.

The complementarity ratio between the DNA targeting sequence in the target DNA and the CRISPR RNA recognition sequence 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 97%, at least 98%, at least 99%, or 100%).

The guide RNA may have additional desirable characteristics, for example, modified or controlled stability; Intracellular targeting; Tracking with fluorescent labels; A binding site for a protein or protein complex, and the like.

Examples of such modifications include, for example, 5 'caps (e.g., 7-methyl guanylate cap (m7G)); 3 'polyadenylated tail (i.e., 3' poly (A) tail); Riboswitch sequences (e.g., considering controlled stability and / or controlled access by protein and / or protein complexes); Stability control sequence; dsRNA &lt; / RTI &gt; duplex structure (i. e., a hairpin), and the like.

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

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

Cleavage of nucleic acid

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

&Quot; Cleavage site " includes nucleic acid sites where the Cas protein causes single strand breaks or double strand breaks. For example, CRISPR complex formation may be performed at or near the nucleic acid sequence present in the target DNA to which the DNA targeting segment of the gRNA will bind (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs).

The cleavage site may be on only one or two strands of the nucleic acid. The cleavage site may be at the same position (producing a smooth end) of the two strands of the nucleic acid, or at different sites of each strand (creating a staggered terminus).

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

PAM (Protospacer adjacent motif) can be flanked by CRISPR RNA recognition sequences. Optionally, CRISPR RNA recognition sequences can be flanked by PAM. For example, the cleavage site of Cas9 can be from about 1 to about 10, or from about 2 to about 5 base pairs (e.g., three base pairs) upstream or downstream of the PAM sequence.

Klotho  Guide RNA for gene manipulation

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

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

In addition, in one embodiment, the present invention provides a nucleic acid Provides a complex of 'guide RNA-CRISPR-related nuclease' used for transformation.

In particular, it is possible to provide a gRNA molecule including a domain capable of complementary binding with a target site from the gene, for example, an isolated or non-native gRNA molecule and a DNA encoding the same.

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

The gRNA may be, for example,

Two or more sites in the Klotho gene can be targeted,

Two or more sites may be targeted for each of the Klotho genes,

It is possible to independently induce double stranded and / or single strand breaks of the Klotho gene

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

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

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

(b) a sequence encoding a CRISPR-related nuclease.

At this time, the above (a) may exist in two or more depending on the target site, and (b) may be homologous or two or more kinds of nuclease.

In an embodiment, the nucleic acid is an enzymatically inactive enzyme protein or its fusion protein close enough to the knockdown target position to reduce, reduce or suppress the function or expression of the Klotho gene (e. G., Transcriptional repressor domain fusion ). &Lt; / RTI &gt;

TALEN  system

TALEN refers to a protein comprising a transcriptional activator-like (TAL) effector binding domain and a nuclease domain and includes, as such, a functional monomer TALEN as well as others requiring dimerization with other monomer TALEN .

TALEN is used to mean one TALEN or a pair of TALENs that have been engineered to work together to remove DNA at the same site. TALEN working together can be referred to as left -TALEN and right -TALEN and refers to the handedness of DNA or TALEN-pair.

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

In some embodiments, the TAL effector can be used to target other protein domains (e. G., Non-nuclease protein domains) for a particular nucleotide sequence. For example, the TAL effector may be, without limitation, a DNA 20 interacting enzyme (e.g., methylase, topoisomerase, integrase, transposase or ligase), a transcriptional or repressor, Can be coupled to protein domains from proteins that interact with or modify other proteins. Examples of such TAL effector fusion applications include, for example, producing or transforming welfare regulatory elements, site-specific insertion, deletion, or repair in DNA, regulating gene expression, and chromatin structure .

zinc Finger  Nuclease system

Zinc-Finger Nuclease (ZFN) is an artificial restriction enzyme created by fusion of 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 the advantage of an endogenous DNA repair machine, the reagents can be used to transform the genome of higher organisms. ZFN can be used to inactivate genes.

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

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

Materials and methods for using zinc finger and zinc finger nuclease to produce genetically engineered animals are described, for example, in U.S. Patent No. 8,106,255, U.S. Patent Application No. 2012/0192298, U.S. Patent No. 2011/0023159, 0.0 &gt; 2011/0281306. &Lt; / RTI &gt;

Genetic engineering composition

In one embodiment of the invention, compositions for genetic manipulation or modification of the Klotho gene and methods for their use are provided.

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

For example, there is provided a genomic engineering composition comprising a guide RNA or a nucleic acid molecule encoding it and an 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 DNA molecule encoding the guide RNA or the guide RNA and an RNA-guide endonuclease or the RNA- There is provided a Klotho gene inactivating composition comprising a gene encoding a cysteine protease.

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

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

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

[Introduction to Cells]

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

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

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

Nucleic acid means 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 . The nucleic acid molecule may be double-stranded or single-stranded.

The nucleic acid may be included in a vector.

A vector can refer to an expression vector or vector system and is a set of components necessary to cause DNA insertion into a genomic or other targeted DNA sequence, such as an episome, a plasmid, or even a virus / phage DNA segment.

Viral vectors used for gene transfer in animals can be used. Non-viral vectors may also be used.

Many different kinds of vectors are known. For example, plasmids and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids are typically derived from a recombinant plasmid containing a replication origin, a suitable promoter, and an optional enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 ' Sequence. Examples of vectors are: plasmids (which may be carriers of other types of vectors), adenoviruses, adeno-associated viruses (AAV), lentiviruses (e.g., modified HIV-1, SIV or FIV), retroviruses (E.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

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

In one embodiment, additional regulatory regions may be introduced together. But are not limited to, polyacetylation sequences, detoxification control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements or introns.

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. and may include a tag sequence encoding " tag ".

The nucleic acid may be delivered in a non-viral vector delivery mode.

For example, Cas9 and sgRNA in the form of mRNA can be packaged and delivered to lipid nanoparticles. DNA or nucleic acids that make up the anion CRISPR / Cas9 can be electrostatically complexed with cationic materials to form nanoparticles, which can be expressed in cells as receptor-mediated endocytosis or phagocytosis It can penetrate. The most widely known cationic materials for delivering nucleic acids are naturally-occurring polymers, synthetic polymers and lipids

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

Cas9 protein and sgRNA are made into RNP (ribonucleoprotein) form before administration and then delivered to desired cells using a carrier such as liposome. RNP is expressed at the same time as it is transduced into the cell and there is no delay in translocation to the protein when it is transferred into the aforementioned mRNA form, and there is a temporary advantage of the expression. Since sgRNA and Cas9 protein are introduced into the cells in the form of RNP complex, they are introduced into the active Cas9 protein state, so that it is possible to perform the editing with a quicker off-target effect. In one embodiment of the present invention, sgRNA and Cas9 protein were introduced into porcine germ cells in the form of RNP (Ribonucleoprotein) complex

In one embodiment, the present invention can provide porcine cells with an inactivated or removed Klotho gene.

For example, a recombinant vector comprising a composition for Klotho knockout is introduced into a nuclear donor cell, and a cell into which the recombinant vector is introduced.

For example, a method for introducing an RNP complex containing a composition for Klotho knockout into a nuclear donor cell, and a cell into which the RNP complex is introduced.

In one embodiment, the cell may be an embryonic cell, somatic cell, or stem cell of a porcine.

In certain embodiments, for example, but not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal derived cells, embryonic cells and embryonic cells. Also, adult stem cells derived from various origins can be used, such as adult stem cells such as fat, uterus, bone marrow, muscle, placenta, cord blood or skin (epithelium). Non-human host embryos can generally be embryos 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, .

More preferably, it may be a fetal-derived cell, an adult fibroblast, or a cumulus cell. Most preferably, fibroblasts isolated from fetal and adult pigs are used. The characteristics of these cells are that they can obtain a large number of cells at the initial separation, have relatively easy cell culture, and are easy to cultivate and manipulate in vitro.

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

Meanwhile, the nuclear donor cell transformed with the Klotho gene knockout vector for producing a Klotho gene deficient disease pig model according to the present invention can be proliferated and cultured according to a method known in the art.

Suitable media are those which have been developed for the cultivation of animal cells and in particular mammalian cells, or for any use which may be produced in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients Possible media can be used.

The medium may be any basic medium suitable for animal cell growth, including but not limited to MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) and K-SFM (Keratinocyte Serum Free Medium). In addition, there is no limit to the use of any medium used in the industry. Preferably, the cells are cultured in a culture medium such as? -MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / (GIBCO), and MSC expansion medium (Chemicon).

To this base medium may be added an assimilative source of carbon, nitrogen and micronutrients, as a non-limiting example, a serum source, a growth factor, an amino acid, an antibiotic, a vitamin, a reducing agent, and / or a sugar source.

It will be apparent to those skilled in the art that suitable media may be selected or combined and cultured appropriately in a known manner. In addition, it is apparent that culturing can be carried out while adjusting conditions such as a suitable culture environment, time, temperature and the like on the basis of ordinary knowledge in this field.

[Transgenic Animal]

Chimeric  animal

In one embodiment of the invention, the embryo can be conceived under suitable conditions to produce pigs in which klotho protein expression is inactivated.

For example, the somatic cell nuclear transfer method (SCNT) described above can be used using somatic cells transformed into pigs.

In one embodiment, the present invention relates to a method for producing a transformed pig for a Klotho-deficient disease model in which a Klotho gene has been knocked out by producing a live body by somatic cell nuclear transfer (SCNT), and a pig for the Klotho- will be.

For somatic cell nuclear transfer (SCNT), known methods can be used.

In one embodiment, the present invention provides a method for preparing a porcine-derived somatic cell transformed with a composition knocking Klotho gene, a nucleus of an embryo cell or a stem cell into a enucleated oocyte, Nuclear transfer embryos can be provided.

In another embodiment of the present invention, there is provided a method for producing a transformed pig for a Klotho-deficient disease model in which a Klotho gene is knocked out, comprising the step of transplanting the nuclear transfer embryo into a tubulus of a surrogate mother to produce a live egg, and a Klotho gene Wherein the knockout animal is a knockout animal.

In certain embodiments, the invention encompasses a method of producing a GM animal,

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

Cloning a cell in a surrogate mother, or transplanting embryos in a surrogate mother, wherein the targeting nuclease specifically binds to an embryonic or intracellular target chromosomal region to cause a change in a cell chromosome, Genetically engineered animals can be conceived.

[Usage]

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

The klotho gene knockout pig model for the klotho deficiency disease model according to the present invention is capable of production of live plants through crosses and is capable of transferring the foreign gene later.

Therefore, pigs characterized by knockout of the klotho gene of the present invention are very useful as an animal model of klotho deficiency disease, because they have an advantage of reproducibility.

That is, the present invention can be applied to the identification of the mechanism of klotho deficiency disease, the search for therapeutic substances, and the development of diagnostic methods using pig model of klotho deficiency disease.

Therefore, the present invention, in a different aspect, encompasses various uses of pigs for the model of klotho deficiency diseases produced by the method.

For example, an animal model according to the present invention can be used in a research required to treat or prevent klotho-deficient disease, for example, as a method of screening a test drug.

In one embodiment, the screening method of the present invention comprises

1) administering a prophylactic and therapeutic agent candidate to a pig model of klotho deficiency disease;

2) After administration of the candidate substance, the tissue of the pig may be analyzed in comparison with a control group to which the candidate substance is not administered.

Preferably, the candidate substance is 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. The compound may be a novel compound or a well-known compound. These candidate substances may form salts.

The method of administering the candidate substance may be suitably selected in accordance with the symptoms of the subject animal, the nature of the candidate substance, etc., for example, by oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. The dose of the candidate substance can be appropriately selected in accordance with the method of administration, the nature of the candidate substance, and the like.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments.

One Animals and reagents

The animals used in this study were maintained by the R & F farm (Boryeong, Korea) and the Livestock Farm (Osan, Korea). All animal studies were conducted with the approval of SNU-160613-16, SNU-160613-16.

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

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

The protospacer proximal motif (PAM) and sgRNA target sequences can be identified by blue and red fonts, respectively (Figure 1). The specificity of the designed sgRNA was verified by searching for similar pig sequences in GenBank. sgRNA was designed for double strand cleavage in exon 3 of klotho.

3. CRISPR / Cas9 ribonucleoproteins  relay

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

4. Somatic cells Nuclear transfer Embryo implantation

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

The oocytes were fixed with a micropipette for holding and a transparent rod was partially dissected with a fine glass needle to form a slit near the first polar body.

Enucleation was performed by aspirating the cytoplasm adjacent to the first polar body containing Medium II chromosome with a suction pipette in a TALP medium containing 7.5 μg / mL cytochalasin B. Next, using a fine pipette, trypsinized fetal fibroblasts with a smooth cell surface were transferred to the Uranium river of enucleated oocytes.

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

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

5. Embryo implantation  And fetal recovery

On the second day after ovarian hornings were observed on days 1 and 2 after activation, the 1-4 cell stage SCNT fetuses were transferred to recipient pigs that were spontaneously circulating. We performed midventral laparotomy under general anesthesia with isoflurane (isoflurane).

The reproductive organs were exposed and SCNT embryos (150-250 embryos) were transferred to the oviducts of the receiving pigs. On the 25th day of pregnancy (SCNT day was regarded as 0 day), the fetus was diagnosed by ultrasound. The fetus was recovered on the 28th day after the transfer.

6. Primary culture of porcine fetal fibroblast

Cloned fetal fibroblasts were isolated and cultured in 7 replicated pig embryos.

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

The fetal body was washed three times with phosphate-buffered saline (PBS; Gibco, Carlsbad, Calif.) Containing 1% Penicillin / Streptomycin (P / S; Gibco) and incubated with Dulbecco's Modified Eagle's Medium (DMEM; Gibco) I cut into small pieces in a mm dish.

The well dissociated tissue was centrifuged at 1,500 rpm for 2 minutes. The supernatant was discarded and the pellet was resuspended in DMEM and centrifuged at 1,500 rpm for 2 minutes. These procedures were repeated twice. Finally, the supernatant was discarded and the pellet was inverted several times with tubes containing 20% fetal bovine serum (FBS, Gibco), 1% P / S, 1% non essential amino acid (NEAA, Gibco) and 100 mM β- mercaptoethanol &Lt; / RTI &gt; was resuspended in DMEM. 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, expanded, 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 Exgene ™ cell SV (GeneAll Biotech, Seoul, Korea) according to the manufacturer's instructions. The primers set forth in Table 1 were used to generate PCR amplifications containing CRISPR / Cas9 target sites. The T7E1 assay was analyzed as previously described (Kim et al., 2009). In summary, the PCR amplification was denatured at 95 ° C and subjected to agarization to form heteroduplex DNA. T7 endonuclease 1 (ToolGen Inc., Seoul) was then treated at 5 ° C for 20 minutes at 37 ° C, followed by 2% agarose gel electrophoresis Respectively.

8. Deep sequencing

The target region was amplified by genomic DNA and used for library construction.

Bi-directional read sequencing was performed using the same amount of PCR amplification using Illumina MiSeq (v2, 300 cycles). Except for rare sequence readings that occur only once for sequencing reactions and for errors associated with amplification. Insertions or deletions around the CRISPR / Cas9 cleavage site (3 bp upstream of PAM) were considered CRISPR / Cas9-induced mutations.

[Table 1]

Primers used for T7E1 assay and dip sequencing

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. 1 μl of the cDNA, 0.4 μl (10 pmol / μl) of the forward primer, 0.4 μl (10 pmol / μl) of the reverse primer, 10 μl of the SYBR Premix Ex Taq (Takara) and 8.2 μl of the Nuclease- (MicroAmp optical 96-well reaction plate). The reaction was carried out for 40 cycles and the cycling parameters were: denaturation at 95 ° C for 15 sec, annealing at 60 ° C for 1 min and extension at 72 ° C for 1 min. All oligonucleotide primer sequences are shown in Table 2. Expression of each of the target genes was quantified relative to the expression of the internal control gene (GAPDH) using the equation R = 2- ^{[? Ct sample-? CT control]} .

[Table 2]

Real-time PCR primer list

10. Western blot analysis

The tissues were finely pulverized by a homogenizer and washed with phosphate buffer (PBS, pH 7.4) and resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1% Triton X- Thymine, 0.1% SDS, 1 mM PMSF). The lysates were centrifuged (13,000 rpm, 4 ° C for 10 min). Ten micrograms of protein from the supernatant was fractionated by 10% SDS-PAGE and electroporated onto a PVDF membrane. The blot was blocked with 4% C to 5% skim milk overnight. The next day, a PVDF membrane was incubated with rabbit anti-klotho polyclonal antibody (1: 1000) and rabbit anti-β-actin (1: 5000) diluted in 5% skim milk Tris-buffered saline containing 0.1% Tween 20 And incubated for 2 hours. Membranes were then washed three times for 10 minutes each with TBST and incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1: 5000) for 1 hour. Blots were identified using Pierce SuperSignal West Pico Chemiluminescent System (Thermo Fisher Scientific). Immunoblot density 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).

Percentage data (for example, fusion, cleavage and blastocyst production rates) were compared with the experimental group by Man-Whitney's test. Data were expressed as mean ± SEM. <0.05 was considered statistically significant.

Examples: 1 CRISPR - sgRNA RNPs  Targeted by klotho  Transfected Non-selected  Using selective cells After SCNT Embryo development  And Genome editing  Efficiency evaluation

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

[Table 3]

Comparison of pre-transplantation development of fetal embryos after SCNT and transfected or bitransfected fibroblasts with Cas9-sgRNA RNPs (targeting the klotho gene)

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

However, there was a significant difference in the fusion rate between the transfected and transfected groups.

In the T7E1 assay, 20 (65%) of the cysts derived from Cas9-sgRNA RNPs transfected cells showed klotho gene modification (Fig. 2A). To confirm the correction, we performed in-depth sequencing analysis. Eight cysts showed 40.0% unilateral deformity and five cysts showed 25.0% double confrontation (Table 4 and Figure 2B).

[Table 4]

The DNA editing ratio of the blot analysis of the blot analysis of the blot analysis of the clot gene of blastocysts produced by SCNT using porcine fibroblasts not transfected with Cas9-sgRNA RNP

Example  2 : Embryo implantation  And klotho Knockout Fetal cell line  Evaluation of genome editing efficiency after establishment

Based on the high fertilization rate in the pre-implantation embryo, Klotho-transfected non-selected donor cells targeting CRISPR-sgRNA RNP were embryo-implanted after SCNT.

A total of 936 SCNT embryos were transferred to 5 beneficiaries, and one beneficiary conceived (Table 5).

[Table 5]

Result of somatic cell nuclear transfer for embryo transfer to beneficiary

Sixteen embryos cloned after 28 days were recovered.

Nine of them were absorbed and 7 of them survived (Figure 3). We performed a primary culture using 7 live fetal body parts to make a klotho knockout fetal cell line and dip sequencing using the surviving remaining fetal tissue.

In deep-seated sequencing, four of the nine absorbed fetuses (44.4%) and three of the seven viable fetal tissues (42.9) showed single-phase modifications (Table 6 and Figure 4).

[Table 6]

Example 3: Gene expression of fetal fibroblasts and the effect of klotho monallelic knockout on the developmental development of embryos cloned in cell lines

Klotho monallelic knockout Fetal fibroblasts (Fetus L2) and wild-type fetal fibroblasts were compared for gene expression. In this experiment we evaluated the expression of genes related to senescence (IGF1 signaling gene, FOXO1 and antioxidant genes) and apoptosis. As shown in Figure 6, the expression of IGF1 and IGF1R was significantly reduced in Fetus L2 fibroblasts. In addition, Fetus L2 fibroblasts significantly reduced the expression of downstream target genes (MnSOD and CAT) by FOXO1 and antioxidant function. Regarding the apoptosis related gene, the expression of Bax / Bcl2 and Caspase3 was significantly increased in Fetus L2 fibroblasts. There was a significant change in the expression of aging-related genes in the klotho monallelic knockout fetal fibroblast, but there was no significant difference in the rate of cleavage and the rate of blastocyst formation 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-implantation development of SCNT embryos derived from wild-type and klotho monallelic knockout pig fibroblasts

Example 4: Comparison of expression of klotho protein and senescence-related genes of fetal-derived placenta and SCNT embryo transferred from klotho monallelic knockout fetal fibroblast

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

[Table 8]

klotho monallelic knockout Delivery of SCNT embryos cloned from fetal fibroblasts

Claims (16)

The knockout of the klotho gene by a modification of the nucleotide sequence of the klotho gene,
Klotho deficient disease pig model with inactivated klotho protein expression.
The method according to claim 1,
Wherein the strain in the nucleic acid sequence comprises deletion, substitution, insertion or inversion of one or more nucleic acids.
The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the exon 3 region of the klotho gene.
The method according to claim 1,
Wherein the strain in the nucleic acid sequence is artificially caused by a nuclease agent.
The method according to claim 1,
The klotho gene is genetically engineered by RNA-guided endonuclease,
(3bp to 25bp) nucleotide sequence located adjacent to the 5 'end and / or the 3' end of the proto-spacer-adjacent motif (PAM) sequence in the nucleic acid sequence constituting the klotho gene
Deletion or insertion of one or more nucleotides;
Substitution with one or more nucleotides that is different from the wild type gene;
Insert one or more nucleotides from outside
Lt; RTI ID = 0.0 &gt;
And a pig model.
6. The method of claim 5,
Wherein 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 method according to claim 1,
Wherein the Klotho deficiency disease is one or more diseases selected from the group consisting of atherosclerosis, osteoporosis, stroke, and Alzheimer's disease.
a guide nucleic acid capable of forming a complementary bond to at least one site of the nucleic acid sequence constituting the klotho gene, or a nucleic acid encoding the guide nucleic acid; And
RNA-guided endonuclease or nucleic acid encoding it
Composition for klotho gene knockout
9. The method of claim 8,
The RNA-guided endonuclease is
Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus (Streptococcus aureus) ), A Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein.
9. The method of claim 8,
Wherein the guide nucleic acid has the nucleotide sequence of SEQ ID NO: 1.
9. The method of claim 8,
Wherein the composition is in the form of a ribonucleoprotein (RNP) composed of Cas9 protein and sgRNA.
In pig cells or embryos containing the target genomic locus on the klotho gene,
(a) a guide RNA capable of complementarily binding to the target genomic locus region; And
(b) RNA-Guided Endonuclease (RGEN)
A method for producing a cell or an embryo in which the nucleic acid sequence of the klotho gene has been changed,
13. The method of claim 12,
Wherein the step of contacting is carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods.
13. The method of claim 12,
Characterized in that said step of contacting is introduced into pig cells in vitro in the form of RNP (ribonucleoprotein) consisting of Cas9 protein and guide RNA
14. The method of claim 13,
Wherein the viral vector is at least one selected from the group consisting of retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV).
13. The method of claim 12,
Wherein the method is performed by somatic cell nuclear transfer (SCNT).

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