Transgenic animals and transgenic embryos producing modified nucleases

Abstract

To provide transgenic animals and transgenic embryos that produce modified nuclease components.SOLUTION: The transgenic animal (or embryo) of the invention that produces a component of a modified nuclease comprises: a first cell having a genome including a first toolbox; and a second cell having a genome including a second toolbox, where the first toolbox and the second toolbox each comprises at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of binding specifically to a target site, where the first toolbox is located at a first locus of the genome of the first cell, the second toolbox is located at a second locus of the genome of the second cell, and the first locus and the second locus are different from each other.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The details disclosed herein relate to transgenic animals and transgenic embryos that produce components of the engineered nucleases.

[0002] In particular, the details disclosed herein relate to chimeric transgenic animals and chimeric transgenic embryos whose genomes include polynucleotides that encode components of modified nucleases.

[0003] More specifically, the details disclosed herein relate to transgenic animals and transgenic embryos comprising a first cell having a genome in which a polynucleotide encoding a component of a modified nuclease is located at a first locus, and a second cell having a genome in which a polynucleotide encoding a component of a modified nuclease is located at a second locus different from the first locus.

[0004] Additionally, details disclosed herein relate to transgenic animals and transgenic embryos comprising a first cell having a genome in which a first polynucleotide encoding a component of a modified nuclease is located at a first locus, and a second cell having a genome in which a second polynucleotide encoding a component of a modified nuclease is located at the first locus, where the sequence of the first polynucleotide differs from the second polynucleotide. [Background technology]

[0005] In a current trend, genetic manipulation using gene editing tools, including CRISPR / Cas9, is being applied to various species of animals and plants.

[0006] Conventional genetic engineering has relied on the method of providing gene editing tools to cells or individuals from outside. However, this method has the problem that when gene editing tools are injected from outside into cells or individuals, the efficiency of genetic engineering is low.

[0007] When transgenic animals are used that have a genome into which a gene encoding a gene editing tool has been inserted, the gene editing tool can be expressed in the animal or cell, resulting in greater efficiency of genetic manipulation.

[0008] Additionally, when a transgenic animal having a genome in which a gene encoding a gene editing tool is inserted is used, multiple animals in which various genes are knocked in or knocked out can be prepared. In the above, a transgenic animal having a genome in which a gene encoding a gene editing tool is inserted can be used as a platform technology.

[0009] Furthermore, when the transgenic animal is a large animal, the utility may be better than in a small animal.

[0010] Therefore, there is a need to develop larger animals whose genomes are inserted with genes encoding gene editing tools. However, genetic engineering in larger animals has progressed slowly compared to smaller animals due to technical limitations.

[0011] [Technical issue] In one embodiment, the disclosure relates to a transgenic animal comprising a polynucleotide encoding a component of an engineered nuclease.

[0012] In another embodiment, the disclosure relates to a transgenic embryo comprising a polynucleotide encoding a component of an engineered nuclease.

[0013] It is an object of the present disclosure to enable more efficient genetic manipulation using transgenic animals or embryos capable of expressing components of engineered nucleases.

[0014] Another object of the present disclosure is to prepare a plurality of cells, embryos and / or animals in which various genes have been knocked in or knocked out using transgenic animals or transgenic embryos capable of expressing components of the engineered nucleases.

[0015] [Technical solution] According to one aspect of the disclosure, the present invention provides a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, the target site being an endopolynucleotide of the animal or an exopolynucleotide located between a first ITR sequence and a second ITR sequence comprised in the genome of the animal, wherein the first toolbox is present at a first locus in the genome of the first cell and the second toolbox is present at a second locus in the genome of the second cell, the first locus being different from the second locus.

[0016] According to another aspect of the present disclosure, the present invention provides a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, the target site being an endopolynucleotide of the animal or an exopolynucleotide located between a first ITR sequence and a second ITR sequence contained in the genome of the animal, wherein the sequence of the first toolbox is different from the sequence of the second toolbox, the first toolbox is present at a first locus in the genome of the first cell and the second toolbox is present at a second locus in the genome of the second cell, and the first locus is identical to the second locus.

[0017] According to one aspect of the disclosure, the present invention provides a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, the target site being an endopolynucleotide of the embryo or an exopolynucleotide located between a first ITR sequence and a second ITR sequence comprised in the genome of the embryo, wherein the first toolbox is present at a first locus in the genome of the first cell and the second toolbox is present at a second locus in the genome of the second cell, the first locus being different from the second locus.

[0018] According to another aspect of the present disclosure, the present invention provides a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, the target site being an endopolynucleotide of the embryo or an exopolynucleotide located between a first ITR sequence and a second ITR sequence contained in the genome of the embryo, the exopolynucleotide being located between the first ITR sequence and the second ITR sequence in the genome of the transgenic embryo, the sequence of the first toolbox differs from the sequence of the second toolbox, the first toolbox is present at a first locus in the genome of the first cell and the second toolbox is present at a second locus in the genome of the second cell, and the first locus is identical to the second locus.

[0019] According to one aspect of the disclosure, the present invention provides a transgenic animal comprising a first cell having a genome comprising a first toolbox and a target site, and a second cell having a genome comprising a second toolbox and a modification site, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to the target site, the target site comprises a first region, a second region, and a third region, the first region is located at a 5' end of the second region, the third region is located at a 3' end of the second region, the modification site comprises a fourth region, a fifth region, and a sixth region, the fourth region is located at a 5' end of the fifth region, and the sixth region is located at a 3' end of the fifth region, wherein the sequence of the first region is identical to the sequence of the fourth region, the sequence of the third region is identical to the sequence of the sixth region, and the sequence of the second region differs from the sequence of the fifth region.

[0020] According to another aspect of the disclosure, the present invention provides a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a target site, and a second cell having a genome comprising a second toolbox and a modification site, wherein the first toolbox and the second toolbox each comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to the target site, the target site comprises a first region, a second region, and a third region, wherein the first region is located at a 5' end of the second region, the third region is located at a 3' end of the second region, the modification site comprises a fourth region, a fifth region, and a sixth region, wherein the fourth region is located at a 5' end of the fifth region, and the sixth region is located at a 3' end of the fifth region, wherein the sequence of the first region is identical to the sequence of the fourth region, the sequence of the third region is identical to the sequence of the sixth region, and the sequence of the second region differs from the sequence of the fifth region.

[0021] According to one aspect of the disclosure, the present invention provides a method for preparing a transgenic embryo in which a component of a modified nuclease is expressed, the method comprising microinjecting a vector into a fertilized egg or embryo, the vector comprising a transposon gene and a polynucleotide encoding a component of the modified nuclease, the vector being a plasmid vector or a viral vector.

[0022] According to another aspect of the present disclosure, the present invention provides a method for preparing a transgenic embryo in which a component of a modified nuclease is expressed, the method comprising the steps of i) preparing a transgenic donor cell in which a component of a modified nuclease is expressed, and ii) transferring a nucleus of the transgenic donor cell into an enucleated egg.

[0023] According to a further aspect of the present disclosure, the present invention provides a method for preparing a transgenic animal in which a component of a modified nuclease is expressed, the method comprising the steps of i) preparing a transgenic embryo in which a component of the modified nuclease is expressed, and ii) implanting the transgenic embryo into the uterus of a surrogate mother.

[0024] According to one aspect of the disclosure, the invention provides a method for preparing an embryo having a genome comprising a gene edit that has occurred at a target site present in the genome, the method comprising providing a guide nucleic acid capable of binding to a fertilized egg or embryo, the fertilized egg or embryo having a genome comprising a polynucleotide encoding an RNA-guided endonuclease, the guide nucleic acid being in the form of RNA or incorporated into a plasmid or viral vector.

[0025] According to another aspect of the disclosure, the present invention provides a method for preparing an embryo having a genome comprising a gene edit that occurs at a target site present in the genome, the method comprising the steps of i) preparing a transgenic donor cell having a genome comprising a polynucleotide encoding an RNA-guided endonuclease and the gene edit that occurs at the target site present in the genome, and ii) transferring a nucleus of the transgenic donor cell into an enucleated egg.

[0026] According to a further aspect of the present disclosure, the present invention provides a method for preparing an animal having a genome comprising a gene edit that has occurred at a target site present in the genome, the method comprising the steps of i) preparing an embryo having a genome comprising a polynucleotide encoding an RNA-guided endonuclease and a gene edit that has occurred at a target site present in the genome, and ii) implanting the embryo into the uterus of a surrogate mother.

[0027] According to yet a further aspect of the present disclosure, the present invention provides a method for preparing an embryo having a genome comprising a gene edit that occurred at a target site present in the genome, the method comprising providing at least one of materials and conditions capable of affecting an expression regulator to a fertilized egg or embryo, the fertilized egg or embryo having a genome comprising: i) a polynucleotide encoding an RNA-guided endonuclease; ii) a polynucleotide encoding a guide nucleic acid capable of binding to the target site; and iii) an expression regulator located at least one of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid.

[0028] According to a further aspect of the present disclosure, the present invention provides a method for preparing an embryo having a genome comprising a gene edit that has occurred at a target site present in the genome, the method comprising the steps of i) preparing a transgenic donor cell having a genome, the genome comprising a polynucleotide encoding an RNA-guided endonuclease, a polynucleotide encoding a guide nucleic acid capable of binding to the target site, and an expression regulator located at at least one of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid, the genome comprising a gene edit that has occurred at the target site present in the genome; and ii) transferring a nucleus of the transgenic donor cell into an enucleated oocyte.

[0029] According to yet a further aspect of the present disclosure, the present invention provides a method for preparing an animal having a genome comprising a gene edit that has occurred at a target site present in the genome, the method comprising the steps of i) preparing an embryo having a genome, the genome comprising a polynucleotide encoding an RNA-guided endonuclease, a polynucleotide encoding a guide nucleic acid capable of binding to the target site, and an expression regulator located at at least one of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid, the genome comprising the gene edit that has occurred at the target site present in the genome; and ii) implanting the embryo into the uterus of a surrogate mother. [Effects of the invention] The technology disclosed in this specification provides the following advantages.

[0030] According to one embodiment disclosed herein, a transgenic animal can be provided that includes a polynucleotide encoding a component of a modified nuclease. Additionally, a transgenic animal that can perform genetic manipulation more effectively can be provided. Furthermore, a platform transgenic animal can be provided for preparing cells, embryos and / or animals with various genes knocked in or knocked out.

[0031] According to another embodiment disclosed herein, a transgenic embryo may be provided that includes a polynucleotide encoding a component of a modified nuclease. Additionally, a transgenic embryo may be provided that allows more efficient genetic manipulation. Furthermore, a platform transgenic embryo may be provided for preparing cells, embryos and / or animals in which various genes are knocked in or knocked out. [Brief description of the drawings]

[0032] [Figure 1] 1 shows a toolbox containing polynucleotides encoding components of engineered nucleases. [Diagram 2]1 shows several embodiments of polynucleotides encoding components of engineered nucleases.

[0033] [Diagram 3] Shows the chromosomes formed from the genome present in a cell. [Figure 4] Several forms of toolboxes that can be inserted into chromosomes are shown.

[0034] [Diagram 5] Shown are somatic and germline cells, each with a genome with a toolbox inserted.

[0035] [Figure 6] Shown are a 1-cell fertilized egg having a genome with a toolbox inserted therein, as well as 2-cell, 4-cell, 8-cell, and 16-cell embryos separated from the 1-cell fertilized egg.

[0036] [Figure 7] Shown are 2-cell, 4-cell, 8-cell and 16-cell embryos in which the toolbox is inserted into the genome of several cells.

[0037] [Figure 8] The figure shows a toolbox that contains a polynucleotide encoding an RNA-guided endonuclease that does not have an expression regulatory element, and a polynucleotide encoding a guide nucleic acid, and shows a form in which a specific target site is cleaved in the genome into which the toolbox is inserted. [Figure 9] 1 shows several embodiments of a toolbox containing expression regulators. [Figure 10] Several embodiments of expression regulators are shown.

[0038] [Figure 11] 1 shows several embodiments of a toolbox that includes a polynucleotide having a PAM sequence.

[0039] [Figure 12] 1 illustrates the process of gene editing in a toolbox containing a polynucleotide with a PAM sequence.

[0040] [Figure 13] 1 shows another process of gene editing in the toolbox containing a polynucleotide with a PAM sequence.

[0041] [Figure 14] 13 shows yet another process of gene editing in a toolbox that includes a polynucleotide having a PAM sequence.

[0042] [Figure 15] The present invention shows a process for knocking in a polynucleotide encoding a target protein and a polynucleotide encoding a linker into a donor polynucleotide, as well as a process for producing the target protein from a cell into which the donor polynucleotide has been knocked in or from a transgenic animal containing the cell.

[0043] [Figure 16] We show how the Surface Toolbox selects cells with inserted genomes.

[0044] [Figure 17] 1 shows how to select cells in which gene editing has occurred in the Surface Toolbox.

[0045] [Figure 18] 1 shows a genome with a suicide toolbox inserted, a cell with a genome with a suicide toolbox inserted, a form in which gene editing has occurred in the suicide toolbox, and a cell in which gene editing has occurred in the suicide toolbox.

[0046] [Figure 19]The morphology of a genome into which the SRY knockout toolbox has been inserted, and a genome in which the SRY gene has been knocked out in a genome into which the SRY knockout toolbox has been inserted, are shown.

[0047] [Figure 20] 1 shows a genome into which the XY chromosome sorting toolbox has been inserted, and the process by which embryos with XX chromosomes and embryos with XY chromosomes are sorted by the XY chromosome sorting toolbox.

[0048] [Figure 21] An embodiment of the excision toolbox in which expression of the transposase can be regulated by inducible and tissue-specific promoters is shown.

[0049] [Figure 22] FIG. 1 shows another embodiment of the excision toolbox in which expression of the transposase can be regulated by inducible and tissue-specific promoters.

[0050] [Diagram 23] 1 shows an embodiment of the excision toolbox in which expression of the recombinase can be regulated by inducible and tissue-specific promoters.

[0051] [Figure 24] A portion of a vector containing a gene encoding green fluorescent protein (green fluorescent protein gene) and a portion of a vector containing a gene encoding red fluorescent protein (red fluorescent protein gene) are shown.

[0052] [Diagram 25] 1 shows an image showing the expression of a fluorescent protein in a donor cell having a genome into which a toolbox containing a gene encoding a fluorescent protein (fluorescent protein gene) has been inserted.

[0053] [Figure 26]1 shows images showing the results of RT-PCR indicating the presence or absence of mRNA expression of the green fluorescent protein gene or the red fluorescent protein gene in the cloned embryo, and the results of DNA PCR confirming the insertion of the green fluorescent protein gene or the red fluorescent protein gene in the genome of the cloned embryo.

[0054] [Figure 27] 13 shows an image showing the expression of a fluorescent protein in a cloned embryo having a genome into which a toolbox containing a fluorescent protein gene has been inserted.

[0055] [Figure 28] The partial configuration of some vectors is shown, with each vector containing a fluorescent protein gene.

[0056] [Figure 29] 1 shows a transgenic cow having a genome into which a toolbox containing a gene encoding a yellow fluorescent protein (yellow fluorescent protein gene) has been inserted, and an image showing the results confirming the expression of yellow fluorescent protein in the transgenic cow.

[0057] [Diagram 30] 1 shows an image illustrating the results confirming the expression of green fluorescent protein in a transgenic cow having a genome into which a toolbox containing a gene encoding green fluorescent protein has been inserted.

[0058] [Diagram 31] 1 shows a transgenic cow having a genome into which a toolbox containing an expression regulator, a green fluorescent protein gene and a red fluorescent protein gene has been inserted, as well as images showing results confirming expression of red fluorescent protein when the transgenic cow is treated with a substance that affects the expression regulator.

[0059] [Diagram 32]1 shows images showing DNA PCR and RT-PCR results confirming the insertion and transcription of the green fluorescent protein gene in a transgenic cow having a genome into which a toolbox including an expression regulator, a green fluorescent protein gene and a red fluorescent protein gene has been inserted, as well as DNA PCR results confirming that the green fluorescent protein gene has been removed and only the red fluorescent protein gene is present following treatment with a substance that affects the expression regulator in the transgenic cow.

[0060] [Diagram 33] 1 shows a calf having a genome into which a toolbox containing a fluorescent protein gene has been inserted, and an image showing expression of the fluorescent protein in primary cells of the calf.

[0061] [Diagram 34] 13 shows an image illustrating the results of a DNA PCR analysis confirming the presence of a fluorescent protein gene in the genome of a calf having a genome into which a toolbox containing a gene encoding a fluorescent protein has been inserted.

[0062] [Diagram 35] 1 shows images showing the difference in the expression level of fluorescent proteins according to the number of toolboxes containing the green fluorescent protein gene inserted into the genome.

[0063] [Diagram 36] 1 shows images showing visual and ELISA results confirming expression of green fluorescent protein in the milk of cows whose genomes have a toolbox inserted containing the green fluorescent protein gene.

[0064] [Figure 37] FIG. 1 shows a schematic diagram showing the sgRNA expression vector and Cas9 expression vector for knocking out the green fluorescent protein gene inserted into the genome of a cell.

[0065] [Figure 38]1 shows images showing the visual result of no expression of green fluorescent protein and the result confirming indels in the green fluorescent protein gene after transfection of sgRNA expression vector and Cas9 expression vector into bovine fibroblast cells having a genome with a toolbox inserted that contains a fluorescent protein gene.

[0066] [Figure 39] 1 shows an image showing the result of donor DNA being knocked in when primary cultured cells having a genome with a toolbox inserted containing the green fluorescent protein gene are transfected with gRNA capable of targeting the gene encoding green fluorescent protein, Cas9, and donor DNA (puromycin resistance gene).

[0067] [Diagram 40] 1 shows images showing that green fluorescent protein is not expressed in primary cells transfected with an sgRNA expression vector targeting green fluorescent protein, a CRISPR / Cas9 expression vector, and a donor DNA (puromycin resistance gene) expression vector.

[0068] [Diagram 41] FIG. 1 shows a schematic diagram illustrating the process by which a donor polynucleotide targeting the green fluorescent protein gene present in the genome of bovine primary cells is knocked in.

[0069] [Diagram 42] An image is shown showing the red fluorescent protein gene targeting the green fluorescent protein gene in bovine primary cells with the genome into which the green fluorescent protein gene is inserted, knocked in, and then expressed.

[0070] [Diagram 43]1 shows a schematic diagram of a vector containing a toolbox containing the spCas9 gene, and images of a transgenic embryo and a transgenic cow, each of which has a genome into which the toolbox containing the spCas9 gene has been inserted.

[0071] [Diagram 44] 1 shows visual results showing the expression of red fluorescent protein in primary cultured cells of transgenic cattle having a genome into which a toolbox containing the spCas9 gene has been inserted, DNA PCR results showing that the spCas9 gene is inserted into the genome of the transgenic cattle, and RT-PCR results showing the expression of the spCas9 gene inserted into the genome of the transgenic cattle.

[0072] [Diagram 45] FIG. 1 shows a schematic illustrating the process of knocking out a targeted gene using a cow with a genome inserted with the Cas9 gene.

[0073] [Figure 46] 1 shows images showing the results of knocking out the PRNP gene, beta-lactoglobulin (BLG), retinoblastoma 1 (Rb1), Nanog, p53, and beta-casein (BCN) genes using cows with genomes inserted with spCas9 genes.

[0074] [Figure 47] 1 shows images showing the results of electrophoresis and sequence analysis confirming knockout of the PRNP gene after injection of a guide nucleic acid targeting the PRNP gene into blastocysts prepared using bovine gametes having a genome with an spCas9 gene inserted therein.

[0075] [Figure 48]This shows images demonstrating that red fluorescent protein is expressed in primary cultured cells of calves obtained by natural mating of cattle having a genome with the spCas9 gene inserted therein, and that the spCas9 gene and the Fat1 gene were inserted into the calf genome by DNA PCR.

[0076] [Figure 49] This shows an image showing the results of electrophoresis confirming indels in the PRNP gene after transfection of sgRNA targeting the PRNP gene into primary cultured cells of a transgenic cow having an spCas9 gene inserted into its genome.

[0077] [Figure 50] Schematic diagram showing a portion of a donor polynucleotide vector for HDR and a portion of a donor polynucleotide vector for HITI. [Figure 51] This shows an image illustrating the results of confirming knock-in of the mCherry gene by HITI. [Figure 52] 1 shows images illustrating the results of confirming knock-in of the mCherry gene by HDR.

[0078] [Diagram 53] 1 shows an mCherry gene knock-in embryo prepared via somatic cell nuclear transfer, and an image showing mCherry expression in the mCherry gene knock-in embryo.

[0079] [Figure 54] A schematic showing parts of the final expression vector to allow expression of spCas9 and sgRNA is shown.

[0080] [Figure 55]1 shows images showing the expression of red fluorescent protein in primary cells of transgenic bovines with genomes inserted with a toolbox comprising a polynucleotide encoding spCas9 gene and sgRNA, and fPCR results confirming indels in the beta-lactoglobulin gene in fibroblasts in transgenic bovines.

[0081] [Figure 56] FIG. 1 shows a schematic diagram illustrating a portion of a vector capable of regulating expression of an RNA-guided endonuclease.

[0082] [Figure 57] This shows images illustrating the results of confirming the presence or absence of indels in target genes by DNA PCR, depending on whether or not cells capable of regulating the expression of an RNA-guided endonuclease were treated with Cre recombinase.

[0083] [Figure 58] An image showing the sequence of the HDR donor vector is shown, where the shaded areas of the entire sequence represent the first and second homology arms, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] According to one embodiment provided herein, a transgenic embryo may be provided having a genome into which a polynucleotide encoding a component of a modified nuclease has been inserted.

[0085] For example, a transgenic fertilized egg or a transgenic embryo having a genome including a polynucleotide encoding an RNA-guided endonuclease included between the first and second ITR sequences can be provided. Specifically, the embryo can be an artiodactyl embryo. Furthermore, a transgenic fertilized egg or a transgenic embryo can be provided that further includes a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the genome of the fertilized egg or the embryo between the first and second ITR sequences. In this case, the expression regulator can be included in at least one of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid.

[0086] In another example, a transgenic embryo may be provided comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox, the first toolbox being present at a first locus and the second toolbox being present at a second locus, the first locus and the second locus being different. The first toolbox and the second toolbox may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the embryo or an exopolynucleotide contained in the genome of the embryo, and may be included between the first ITR sequence and the second ITR sequence. In this case, the sequence of the first toolbox may be the same as or different from the sequence of the second toolbox.

[0087] In another example, a transgenic embryo may be provided comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox, the first toolbox being present at a first locus and the second toolbox being present at a second locus, the first locus and the second locus being identical to each other. The first toolbox and the second toolbox may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the embryo or an exopolynucleotide contained in the genome of the embryo, and may be included between the first ITR sequence and the second ITR sequence. In this case, the sequence of the first toolbox is different from the sequence of the second toolbox. The genome of the first cell may further include a third toolbox having an identical sequence to the first toolbox. The genome of the second cell may further include a fourth toolbox having an identical sequence to the second toolbox. The transgenic embryo may further comprise a third cell having a genome that does not include a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid.

[0088] In another example, a transgenic embryo may be provided, comprising a first cell having a genome including a first toolbox and a target site, and a second cell having a genome including a target site. The first toolbox may include at least one of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to the target site, and the genome of the second cell may not include a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to the target site. Additionally, ITR sequences may be further included at the 5' end and 3' end of the first toolbox. In this case, the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be the same or different from each other, and the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be the same or different from each other. The target site may be an endopolynucleotide. Specifically, the target site may be a 18-25 bp base sequence present in the genome of the transgenic embryo. The target site may be an exopolynucleotide. The target site may be a sequence adjacent to the 5'-end or 3'-end of the PAM sequence. The target site and the PAM sequence may be included between the first ITR sequence and the second ITR sequence.

[0089] According to another embodiment provided herein, there may be provided a method for preparing a transgenic embryo having a genome inserted with a polynucleotide encoding a component of a modified nuclease.

[0090] One method for preparing a transgenic embryo may include microinjecting a vector containing a polynucleotide encoding a transposon gene and a component of a modified nuclease into a fertilized egg or embryo. Additionally, the method may include microinjecting a transposase capable of interacting with the transposon gene into a fertilized egg or embryo. The transposase may be in the form of a protein, a polypeptide, or a polynucleotide encoding the transposase. The polynucleotide may be contained in a plasmid vector or a viral vector. Furthermore, the polynucleotide encoding the transposase may be incorporated into a single vector together with the polynucleotide encoding the transposon gene and the component of the modified nuclease, and then microinjected into a fertilized egg or embryo. One type of transgenic embryo that may be prepared by the above method may comprise a first cell having a genome in which a polynucleotide encoding a first modified nuclease component is contained in a first locus, and a second cell having a genome in which a polynucleotide encoding a second modified nuclease component is contained in a second locus different from the first locus. In this case, the sequence of the polynucleotide encoding the first modified nuclease component and the sequence of the polynucleotide encoding the second modified nuclease component may be the same or different from each other. Another type of transgenic embryo that can be prepared by the above method may comprise a first cell having a genome in which a polynucleotide encoding a first modified nuclease component is contained in a first locus, and a second cell having a genome in which a polynucleotide encoding a second modified nuclease component is contained in the first locus. In this case, the sequence of the polynucleotide encoding the first modified nuclease component and the sequence of the polynucleotide encoding the second modified nuclease component may be the same or different from each other.

[0091] Another method for preparing a transgenic embryo may include preparing a transgenic donor cell in which a component of a modified nuclease is expressed, and transferring the nucleus of the transgenic donor cell into an enucleated egg. The preparation of the transgenic donor cell may include transforming the cell with a vector containing a polynucleotide encoding a transposon gene and a component of the modified nuclease. Additionally, the preparation of the transgenic donor cell may further include transforming the cell with a transposase capable of interacting with the transposon gene. The transposase may be in the form of a protein, polypeptide, or a polynucleotide encoding the transposase. The polynucleotide may be contained in a plasmid vector or a viral vector. Furthermore, the polynucleotide encoding the transposase may be incorporated into a single vector together with the polynucleotide encoding the transposon gene and the component of the modified nuclease, and then microinjected into a fertilized egg or embryo.

[0092] According to yet another embodiment provided herein, a transgenic animal may be provided having a genome into which a polynucleotide encoding a component of a modified nuclease has been inserted.

[0093] For example, a transgenic animal can be provided having a genome comprising a polynucleotide encoding an RNA-guided endonuclease contained between the first and second ITR sequences. Specifically, the animal can be an artiodactyl. In addition, a transgenic animal can be provided in which a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the animal is further comprised between the first and second ITR sequences. In this case, the expression regulator can be comprised in one or more of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid.

[0094] In another example, a chimeric transgenic animal may be provided, comprising a first cell having a genome including a toolbox and a second cell having a genome not including a toolbox. Specifically, a transgenic animal may be provided, comprising a first cell having a genome including a first toolbox and a target site, and a second cell having a genome including a target site. The first toolbox may include at least one polynucleotide of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to the target site, and the genome of the second cell may not include a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to the target site. Additionally, ITR sequences may be further included at the 5' and 3' ends of the first toolbox. In this case, the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be the same or different from each other, and the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be the same or different from each other. The target site may be an endopolynucleotide. Specifically, the target site may be a base sequence of 18 bp to 25 bp present in the genome of the transgenic animal. The target site may be an exopolynucleotide. The target site may be a sequence adjacent to the 5' end or 3' end of the PAM sequence. The target site and the PAM sequence may be included between the first ITR sequence and the second ITR sequence.

[0095] In another example, a transgenic animal may be provided that includes a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox, where the first toolbox is present at a first locus and the second toolbox is present at a second locus, and the first and second loci are different. The first and second toolboxes may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the animal or an exopolynucleotide that may be included in the genome of the animal and between the first and second ITR sequences. In this case, the sequence of the first toolbox and the sequence of the second toolbox may be the same or different from each other.

[0096] In another example, a transgenic animal may be provided comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox, the first toolbox being present at a first locus and the second toolbox being present at a second locus, the first locus and the second locus being identical to each other. The first toolbox and the second toolbox may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the animal or an exopolynucleotide that may be included in the genome of the animal and may be included between the first ITR sequence and the second ITR sequence. In this case, the sequence of the first toolbox and the sequence of the second toolbox are different from each other. The genome of the first cell may further include a third toolbox having an identical sequence to the first toolbox. The genome of the second cell may further include a fourth toolbox having an identical sequence to the second toolbox. The transgenic animal may further comprise a third cell having a polynucleotide encoding an RNA-guided endonuclease and a genome that does not include a polynucleotide encoding a guide nucleic acid.

[0097] According to another exemplary embodiment provided herein, a method can be provided for preparing a transgenic animal having a genome inserted with a polynucleotide encoding a component of a modified nuclease.

[0098] A method for preparing a transgenic animal may include microinjecting a vector containing a polynucleotide encoding a transposon gene and a component of a modified nuclease into a fertilized egg or embryo. Additionally, preparing a transgenic embryo may further include microinjecting a transposase capable of interacting with the transposon gene into the fertilized egg or embryo.

[0099] Another method for preparing a transgenic animal may include preparing a transgenic donor cell in which the components of the modified nuclease are expressed, and transferring the nucleus of the transgenic donor cell into an enucleated egg of the animal. The step of preparing the transgenic donor cell may include transforming the cell with a vector comprising a polynucleotide encoding a transposon gene and a component of the modified nuclease. Additionally, the preparation of the transgenic donor cell may further comprise transforming the cell with a transposase capable of interacting with the transposon gene. The transposase may be in the form of a protein, polypeptide, or polynucleotide encoding the transposase. The polynucleotide may be contained in a plasmid vector or a viral vector. Furthermore, the polynucleotide encoding the transposase may be in the form in which the polynucleotide is contained in a single vector together with the transposon gene and the polynucleotide encoding the component of the modified nuclease.

[0100] According to one embodiment provided herein, a transgenic embryo may be provided having a gene edited genome.

[0101] For example, a transgenic embryo can be provided having a genome that includes a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid between the first ITR sequence and the second ITR sequence, and the endo polynucleotide is knocked out. At this time, the guide nucleic acid can specifically bind to the endo polynucleotide. The form in which the endo polynucleotide is knocked out can be i) a form in which at least one nucleotide is not included in the sequence of the endo polynucleotide, ii) a form in which at least one nucleotide is further included in the sequence of the endo polynucleotide, and iii) a form in which at least one nucleotide is deleted from the sequence of the endo polynucleotide and at least one nucleotide is further included.

[0102] In another example, a transgenic embryo may be provided that includes a first cell having a genome including a first toolbox and a target site, and a second cell having a genome including a second toolbox and a modification site. The sequence of the first toolbox may be the same as or different from the sequence of the second toolbox. The target site may be an endopolynucleotide. The target site may be an exopolynucleotide. The modification site may be a target site sequence that has been changed by gene editing. Specifically, the target site may include a first region, a second region, and a third region, and the modification sequence may include a fourth region, a fifth region, and a sixth region. In this case, the sequence of the first region is identical to the sequence of the fourth region, the sequence of the third region is identical to the sequence of the sixth region, and the sequence of the second region is different from the sequence of the fifth region. The second region and the fifth region may each include a PAM sequence. The third region and the sixth region may each include a PAM sequence. The sequence of the fifth region may be in the form of i) at least one nucleotide not included in the sequence of the second region, ii) at least one nucleotide additionally included in the sequence of the second region, or iii) at least one nucleotide deleted from the sequence of the second region and at least one additional nucleotide included. In the cases of ii) and iii), the at least one additional nucleotide may include one or more of an editing-compatible component, a polynucleotide encoding a protein or RNA, a polynucleotide encoding a non-functional polypeptide, a polynucleotide encoding an untranslated RNA, a non-transcribed polynucleotide, an artificial intron, and an expression regulator.

[0103] In yet another example, a transgenic embryo may be provided that includes a first cell having a genome including a first toolbox and a target site, and a second cell having a genome that does not include the toolbox but includes a modified gene. In this case, the first toolbox may include one or more of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid. The modified site may be a target site sequence that has been changed by gene editing. Specifically, the target sequence may include a first region, a second region, and a third region, and the modified sequence may include a fourth region, a fifth region, and a sixth region. In this case, the sequence of the first region is identical to the sequence of the fourth region, the sequence of the third region is identical to the sequence of the sixth region, and the sequence of the second region is different from the sequence of the fifth region. The sequence of the fifth region may be i) in a form in which at least one nucleotide is not included in the sequence of the second region, ii) in a form in which at least one nucleotide is further included in the sequence of the second region, and iii) in a form in which at least one nucleotide is deleted from the sequence of the second region and at least one nucleotide is further included.

[0104] According to another embodiment provided herein, a transgenic animal may be provided having a gene edited genome.

[0105] For example, a transgenic animal can be provided that has a genome that includes a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid between the first ITR sequence and the second ITR sequence, and that has an endo polynucleotide knocked out. In this case, the guide nucleic acid can specifically bind to the endo polynucleotide. The form in which the endo polynucleotide is knocked out can be any one of the following: i) a form in which at least one nucleotide is not included in the sequence of the endo polynucleotide; ii) a form in which at least one nucleotide is further included in the sequence of the endo polynucleotide; and iii) a form in which at least one nucleotide is deleted from the sequence of the endo polynucleotide and at least one nucleotide is further included.

[0106] In another example, a transgenic animal may be provided that includes a first cell having a genome including a first toolbox and a target site, and a second cell having a genome including a second toolbox and a modification site. The sequence of the first toolbox may be the same as or different from the sequence of the second toolbox. In this case, the first toolbox and / or the second toolbox may include one or more of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid. The target site may be an endopolynucleotide. The target site may be an exopolynucleotide. The modification site may be a target site whose sequence has been changed by gene editing. Specifically, the target sequence may include a first region, a second region, and a third region, and the modification sequence may include a fourth region, a fifth region, and a sixth region. In this case, the sequence of the first region is identical to the sequence of the fourth region, the sequence of the third region is identical to the sequence of the sixth region, and the sequence of the second region is different from the sequence of the fifth region. The second region and the fifth region may include a PAM sequence. The third and sixth regions may include a PAM sequence. The sequence of the fifth region may be: i) at least one nucleotide not included in the sequence of the second region; ii) at least one nucleotide further included in the sequence of the second region; and iii) at least one nucleotide deleted from the sequence of the second region and at least one additional nucleotide included. In the case of ii) and iii), the at least one additional nucleotide may include one or more of an editing-compatible component, a polynucleotide encoding a protein or RNA, a polynucleotide encoding a non-functional polypeptide, a polynucleotide encoding an untranslated RNA, a non-transcribed polynucleotide, an artificial intron, and an expression regulator. The target site and the modification site may be adjacent to the PAM sequence. The target site and the modification site may each include a first ITR sequence at the 5' end and a second ITR sequence at the 3' end.

[0107] [Terminology definition] [Transformation (genetic modification)] As used herein, the term "transformation (genetic modification)" refers to the artificial transformation of a polynucleotide contained in an animal genome in a cell. Transformation includes deleting or substituting a portion of a polynucleotide contained in the animal genome of a cell, and inserting a nucleotide or polynucleotide into the animal genome.

[0108] As used herein, the term "transformation (genetic modification)" includes the addition, modification and deletion of proteins or RNA that can be expressed in a cell.

[0109] "Site-specific transformation" As used herein, the term "site-specific transformation" refers to the transformation that occurs at a specific location of the animal genome in a cell.The specific location can be determined by the nucleotide sequence on the animal genome.For example, RNA-guided endonuclease can recognize the nucleotide sequence on the animal genome that can be complementary to a portion of guide nucleic acid, thereby causing site-specific transformation.

[0110] [Transformed cells (transgenic cells)] As used herein, the term "transgenic cell" refers to a cell that contains within it a transformed portion of an animal's genome.

[0111] A first cell containing a transformed portion in its animal genome may undergo cell division. The animal genome of a second cell obtained through cell division of the first cell may contain a portion having the same nucleotide sequence as the transformed portion of the first cell. As used herein, the term "transformed cell" includes the first cell and the second cell.

[0112] [Transgenic animals (transgenic animals)] As used herein, the term "transgenic animal" refers to an animal that contains at least one transformed cell. The animal F0 may contain the first transformed cell. The animal F0 may produce an offspring F1. At least one transformed cell contained in the F1 and the offspring of the F1 may contain an animal genome that contains a portion having a nucleotide sequence identical to the transformed portion in the animal genome of the first transformed cell. As used herein, the term "transgenic animal" includes the F0, F1, and the offspring of the F1.

[0113] Even if no direct artificial manipulation for transformation is applied during or after the production of the F1 animal, the F1 animal can be considered a transgenic animal if it contains transformed cells.

[0114] In cases where an F1 is derived from an animal F0 after direct artificial manipulation is applied for transformation, the animal F1 can be considered a transgenic animal.

[0115] [Endopolynucleotide] As used herein, the term "endopolynucleotide" refers to a polynucleotide contained in an animal genome in a cell in which transformation has not occurred.

[0116] The first cell that has not undergone transformation may undergo cell division. The second cell obtained through cell division of the first cell may be a transformed cell. The second cell may contain a polynucleotide having the same sequence as the endopolynucleotide contained in the first cell. As used herein, the term "endopolynucleotide" includes the polynucleotide contained in the second cell that has the same sequence as the endopolynucleotide of the first cell.

[0117] [Exopolynucleotide] As used herein, the term "exopolynucleotide" refers to a polynucleotide that is introduced into a cell. The cell may or may not be a transformed cell. The exopolynucleotide may be inserted into the animal genome in the cell or may exist separately from the animal genome in the cell.

[0118] The first cell into which the exopolynucleotide is introduced may undergo cell division. The second cell obtained through cell division of the first cell may contain a polynucleotide having the same sequence as the exopolynucleotide. As used herein, the term "exopolynucleotide" includes a polynucleotide contained in the second cell that has the same sequence as the exopolynucleotide of the first cell.

[0119] An animal (F0) containing cells into which an exopolynucleotide has been introduced can produce offspring (F1). The F1 and F1 offspring can contain cells containing a polynucleotide having the same sequence as the F0 exopolynucleotide. As used herein, the term "exopolynucleotide" includes polynucleotides contained in cells of the F1 and F1 offspring that have the same sequence as the F0 exopolynucleotide.

[0120] Insert As used herein, the term "insertion" includes "nucleotide insertion" and "polynucleotide insertion." As used herein, the term "nucleotide insertion" refers to adding a nucleotide to the center, 5' end, or 3' end of a nucleic acid. As used herein, the term "polynucleotide insertion" refers to adding a polynucleotide to the center, 5' end, or 3' end of a nucleic acid.

[0121] [missing] As used herein, the term "deletion" includes "nucleotide deletion" and "polynucleotide deletion." As used herein, the term "nucleotide deletion" refers to the deletion of a nucleotide contained in a nucleic acid. As used herein, the term "polynucleotide deletion" refers to the deletion of a polynucleotide contained in a nucleic acid.

[0122] [Replace] As used herein, the term "substitution" includes "nucleotide substitution" and "polynucleotide substitution". As used herein, the term "nucleotide substitution" refers to the replacement of a nucleotide contained in a nucleic acid with another nucleotide. As used herein, the term "polynucleotide substitution" refers to the replacement of a polynucleotide contained in a nucleic acid with another polynucleotide.

[0123] [Knock-in (Knock-in)] As used herein, the term "knock-in" refers to the insertion or replacement of an exopolynucleotide containing a gene into an animal genome in a cell.

[0124] For example, an exopolynucleotide containing a human albumin gene can be inserted into a non-human animal genome in a cell, in which case the cell containing the non-human animal genome can be capable of expressing human albumin, a process that can be referred to as knocking-in of the human albumin gene.

[0125] [Knockout (Knock-out)] As used herein, the term "knockout" refers to making a gene present in an animal genome in a cell unable to function. A gene can be knocked out by transformation of a polynucleotide in the corresponding gene located on the animal genome in the cell.

[0126] For example, an exopolynucleotide comprising a human albumin gene can be inserted into a nucleotide sequence corresponding to an exon of a non-human albumin gene present in the genome of a non-human animal in a cell, in which case the cell cannot express non-human albumin, and this process can be referred to as knocking out the non-human albumin gene.

[0127] In another example, a portion of the exon of the non-human albumin gene present on the non-human animal genome in a cell can be deleted using a modified nuclease that targets the portion of the exon of the non-human albumin gene as a target site. In this case, the cell cannot express non-human albumin, and this process can be called knockout of the non-human albumin gene.

[0128] [Modified nuclease] As used herein, the term "modified nuclease" refers to a protein or a complex containing said protein that is capable of site-specific transformation into an animal genome. The protein may be an unmodified protein found in nature or a modified / modified protein.

[0129] Engineered nucleases of the present disclosure can include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / enzyme systems.

[0130] [Target site] A target site of a modified nuclease may refer to a region of a nucleic acid that can be recognized by a component of the modified nuclease.

[0131] For example, the target site of the CRISPR / enzyme system can be at least one nucleotide sequence that is identical to or capable of complementary binding to a partial region of a guide nucleic acid on a nucleic acid.

[0132] As used herein, an example of the term "identical nucleotide sequence" may include the relationship between uracil (U) and thymine (T). As used herein, an example of the term "nucleotide sequence capable of complementary binding" may include the relationship between uracil (U) and adenine (A). The target site may be an endopolynucleotide present in a genome. The target site may be an exopolynucleotide to be inserted into a genome.

[0133] [CRISPR / enzyme system] The CRISPR / enzyme system refers to a complex that includes a protein that can cleave a portion of a target site by interacting with a target site on an animal genome in a cell or a target site of an exopolynucleotide. The CRISPR / enzyme system can include a guide nucleic acid and an RNA-guided endonuclease.

[0134] [Guide nucleic acid] As used herein, the term "guide nucleic acid" refers to a nucleic acid capable of binding to a target site in an animal genome in a cell or a target site in an exopolynucleotide. As used herein, the term "guide nucleic acid" includes single-stranded guide nucleic acids and guide nucleic acids that consist of at least two strands of nucleic acid.

[0135] The single-stranded guide nucleic acid may comprise a gRNA. The gRNA may comprise at least one of a protospacer domain, a first complementary domain, a second complementary domain, a proximal domain, and a tail domain.

[0136] A protospacer domain is a domain that comprises a nucleotide sequence capable of complementary binding to a portion of a target site in an animal genome or a portion of a target site in an exopolynucleotide.

[0137] The first complementary domain and the second complementary domain are domains that can have complementary binding to each other, thereby allowing interaction with RNA-guided endonuclease.The second complementary domain can be located downstream of the first complementary domain.The proximal domain can be located downstream of the second complementary domain.The tail domain can be located at the 3' end of gRNA.

[0138] The guide nucleic acid, which is composed of at least two strands of nucleic acid, may comprise a dual gRNA. The dual gRNA may comprise a crRNA and a tracrRNA. The crRNA may comprise a protospacer domain and a first complementary domain. The tracrRNA may comprise a second complementary domain, a proximal domain, and a tail domain.

[0139] [RNA-guided endonuclease] As used herein, the term "RNA-guided endonuclease" refers to a polypeptide or protein that contains a domain capable of interacting with a polynucleotide and a domain capable of cleaving the middle of the polynucleotide.

[0140] RNA-guided endonucleases can include, but are not limited to, spCas9, CjCas9, StCas9, SaCas9, NmCas9, Cpf1 proteins, and mutants thereof.

[0141] Depending on the purpose of the modification / alteration, the RNA-guided endonuclease may include, but is not limited to, inactive Cas9, Cas9 nickase, eSpCas9 and SpCas9-HF1.

[0142] The RNA-guided endonuclease can cleave a double strand by interacting with a nucleic acid, or can cleave one strand of a double strand by interacting with a nucleic acid. Alternatively, the RNA-guided endonuclease can interact with a nucleic acid but not cleave the nucleic acid.

[0143] When RNA-guided endonuclease interacts with nucleic acid and thereby cuts double strand or one strand of double strand, nucleotide or polynucleotide insertion can occur in the cut region.Alternatively, when RNA-guided endonuclease interacts with nucleic acid and thereby cuts double strand or one strand of double strand, nucleotide or polynucleotide deletion can occur in the cut region.

[0144] [CRISPR / enzyme target site] The target site in the animal genome in the cell or the target site in the exopolynucleotide can be a nucleotide sequence adjacent to the 5' or 3' end of the protospacer adjacent motif (PAM) sequence.

[0145] PAM sequences can include, but are not limited to, NGG, NNGRRT, NNAGAAW, NNNNGATT, NNNVRYAC, and TTN. N can be any one of A, T, U, G, and C. V can be any one of A, C, and G. W can be any one of A and T. Y can be any one of C and T.

[0146] PAM sequence may vary depending on RNA-guided endonuclease. For example, the PAM sequence of spCas9 or its mutants may be NGG. For example, the PAM sequence of SaCas9 or its mutants may be NNGRRT. For example, the PAM sequence of StCas9 or its mutants may be NNAGAAW. For example, the PAM sequence of NmCas9 or its mutants may be NNNGATT. For example, the PAM sequence of CjCas9 or its mutants may be NNNVRYAC. For example, the PAM sequence of Cpf1 and its mutants may be TTN.

[0147] [Transposon system] [Transposon] As used herein, the term "transposon" refers to a polynucleotide that can be transposed in an animal genome in a cell. Additionally, the term "transposon" refers to a polynucleotide that can be transposed between a nucleic acid and an animal genome in a cell.

[0148] Transposons can be divided into class I transposons (retrotransposons) and class 2 transposons (DNA transposons).

[0149] Class I transposons function in a manner in which RNA is transcribed from transposon DNA in a nucleic acid or animal genome in a cell, and then DNA that is reverse transcribed from the RNA is inserted into a different location in the animal's genome.

[0150] Class 2 transposons function in such a way that the transposon DNA in a nucleic acid or animal genome is cut in a cell and the cut transposon DNA is inserted at a different location in the animal genome.

[0151] A class 2 transposon may comprise a first polynucleotide at the 5' end, a second polynucleotide at the 3' end, and a third polynucleotide. The first and second polynucleotides may comprise an inverted terminal repeat (hereinafter, ITR) sequence. The third polynucleotide may be located between the first and second polynucleotides. The third polynucleotide may comprise an exopolynucleotide. The third polynucleotide may comprise a polynucleotide encoding a transposase.

[0152] Unless otherwise specified, the term "transposon" refers to a transposon that is a class 2 transposon. However, when "transposon" is interpreted as a "class I transposon", it is not necessary to interpret the transposon as a class 2 transposon if there is no problem from a technical aspect.

[0153] [Transposase] As used herein, the term "transposase" refers to a protein that can cleave a class 2 transposon or insert a class 2 transposon into an animal genome by interacting with the ITR sequences located at both ends of a class 2 transposon located in a nucleic acid in a cell or an animal genome.

[0154] The transposase may include, but is not limited to, hobo / Ac / Tam, P element, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, and variants thereof. The transposase can cleave transposons present in nucleic acids in cells or animal genomes. The transposase can insert class 2 transposons into animal genomes.

[0155] The location at which the transposase inserts the class 2 transposon into the animal's genome may not be related to a nucleotide sequence on the animal's genome.

[0156] The position where transposase inserts class 2 transposon into animal genome can be determined by the specific nucleotide sequence on animal genome that can be recognized by transposase.For example, in the case of Sleeping Beauty, Sleeping Beauty transposase can insert transposon by recognizing TA sequence on animal genome.In addition, piggyBac transposase can insert transposon by recognizing TTAA sequence on animal genome.

[0157] The transposase and class 2 transposon can be used to insert an exopolynucleotide into an animal genome in a cell. For example, an exopolynucleotide can be inserted into an animal genome in a cell by delivering an exopolynucleotide to a cell that includes an ITR sequence capable of interacting with piggyBac transposase at the 5' end and piggyBac at the 3' end.

[0158] In this case, it is not necessary to determine in advance the position where the exopolynucleotide is to be inserted or to alter the nucleotide sequence of the exopolynucleotide depending on the position where the exopolynucleotide is to be inserted.

[0159] Additionally, there is no limit to the number of exopolynucleotides that can be inserted into the animal genome in a cell, and thus the expression level of a target protein in a cell can be increased by inserting multiple exopolynucleotides encoding the target protein into the animal genome in the cell.

[0160] Additionally, the position at which the exopolynucleotide is inserted by the transposase can be a position at which cellular gene expression is not inhibited, for example, in the case of piggyBac transposase, the transposon can be inserted into an intron, 5'UTR, or 3'UTR in the animal genome.

[0161] [Site-specific recombination] As used herein, the term "site-specific recombination" refers to the phenomenon in which two nucleotide sequences with the same or identical characteristics on nucleic acid or animal genome form a pair, thereby causing mutual exchange between the pair of nucleotide sequences.In this case, the nucleotide sequence where mutual exchange occurs is called recombinase recognition site (RRS).In addition, the protein that interacts with the pair of RRS and promotes site-specific recombination is called site-specific recombinase (SSR).

[0162] [Recombinase recognition site] Recombinase recognition sites (RRS) of the present disclosure may include, but are not limited to, loxp, rox, FRT, attP, attB, and mutants thereof. Loxp mutants may include, but are not limited to, loxp66, loxp71, loxp72, loxp2722, loxp5171, and loxpm2. Rox mutants may include, but are not limited to, rox4R, rox6R, and rox2N. FRT mutants may include, but are not limited to, F3, F5, F10, F11, F12, F13, F14, F15, F16.

[0163] In an RRS, two can form a pair and thereby interact with an SSR. In a loxp or loxp mutant, two of the same RRS can form a pair. In a rox or rox mutant, two of the same RRS can form a pair. In an FRT or FRT mutant, two of the same RRS can form a pair. attP can form a pair with attB.

[0164] [Site-specific recombinase] Site-specific recombinases (SSRs) or recombinases of the disclosure can include, but are not limited to, Cre, Dre, Flp, KD, B2, B3, lambda, HK022, HP1, gamma delta, ParA, Tn3, Hin, Gin, Pin, phiC31, Bxb1, R4, or mutants thereof.

[0165] SSR can interact with a pair of RRS. Cre or a Cre mutant can specifically interact with loxp or a loxp mutant. Dre or a Dre mutant can specifically interact with rox or a rox mutant. Flp or a Flp mutant can specifically interact with FRT or a FRT mutant. PhiC31 or a phiC31 mutant can specifically interact with attP or attB.

[0166] [Type of site-specific recombination] Site-specific recombination of the present disclosure can include insertions, deletions, inversions, and exchanges. Site-specific recombination can be reversible or non-reversible.

[0167] Insert When one of a pair of RRSs is located in a first nucleic acid and the other is located in a second nucleic acid, insertion of the second nucleic acid can occur in the first nucleic acid through interaction with an SSR that is specific for the pair of RRSs.

[0168] When the insertion is reversible, the second nucleic acid can be deleted through interaction with the SSR.

[0169] [missing] When a pair of RRSs are positioned in the same orientation with respect to the nucleic acid, deletion of the polynucleotide located between the pair of RRSs can occur through interaction with an SSR specific to the pair of RRSs.

[0170] When the deletion is reversible, an insertion of a polynucleotide located between the pair of RRSs can occur.

[0171] [Reversed] When a pair of RRSs are located in opposite orientations with respect to the nucleic acid, inversion of the polynucleotide located between the pair of RRSs can occur through interaction with an SSR specific for the pair of RRSs.

[0172] When an inversion is reversible, inversion of the polynucleotide located between the pair of RRSs can occur again.

[0173] [exchange] When RRS A and RRS B form a pair and RRS C and RRS D form a pair, RRS A and RRS C can be located in the first nucleic acid, and RRS B and RRS D can be located in the second nucleic acid. In the first nucleic acid, RRS C can be located further downstream of RRS A, and in the second nucleic acid, RRS D can be located further downstream of RRS B. In this case, interaction with the SSR specific to the first pair of RRSs and interaction with the SSR that forms a pair with the second pair of RRSs can occur. Exchange can occur between a polynucleotide located between RRS A and RRS C and a polynucleotide located between RRS B and RRS D.

[0174] When the exchange is reversible, the exchange can occur again between the polynucleotide located between RRS A and RRS C and the polynucleotide located between RRS B and RRS D.

[0175] [Marker gene] As used herein, the term "marker gene" refers to a gene that is inserted into an animal genome in a cell to select cells in which the intended transformation has been achieved. When an exopolynucleotide containing a polynucleotide encoding a marker gene is introduced into a cell, and then the exopolynucleotide is inserted into the animal genome, the marker gene can be expressed in the cell, which is useful for selecting the cell.

[0176] Marker genes may include, but are not limited to, antibiotic resistance genes, antigen genes, luciferase genes, β-galactosidase genes, genes encoding fluorescent proteins, surface marker genes, suicide genes.

[0177] [Antibiotic resistance genes] When the marker gene is an antibiotic resistance gene, cell selection can be performed by culturing with a combination antibiotic, which can include, but is not limited to, ampicillin, chloramphenicol, tetracycline, and kanamycin.

[0178] [Antigen gene] When the marker gene is a gene encoding an antigen or a gene containing nucleotides that can act as an antigen, cell selection can be performed by culturing with an antibody that can specifically act as the antigen. The antigen can include a surface antigen. The surface antigen can include a CD molecule. The antibody can interact with a magnetic particle or a fluorophore.

[0179] [Luciferase gene] When the marker gene is the luciferase gene, cell selection can be performed by culturing with luciferin.

[0180] [β-galactosidase gene] When the marker gene is a β-galactosidase gene, cell selection can be performed by culturing with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal).

[0181] [Genes that code for fluorescent proteins (fluorescent protein genes)] When the marker gene is a gene encoding a fluorescent protein, cell selection can be performed by measuring the fluorescent signal. The fluorescent protein can include, but is not limited to, green fluorescent protein (hereinafter, GFP), yellow fluorescent protein (hereinafter, YFP) and red fluorescent protein (hereinafter, RFP).

[0182] [Suicide gene] When the marker gene is a suicide gene, cell selection can be performed by culturing with a prodrug that pairs with the suicide gene. The suicide gene can include, but is not limited to, a thymidine kinase gene, a cytosine deaminase gene, a cytochrome P450 gene, a nitroreductase gene, a purine nucleoside phosphorylase gene, and a carboxypeptidase G2 gene. The prodrug can include, but is not limited to, acyclovir, ganciclovir, 5-fluorocytosine, cyclophosphamide, ifosfamide, 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954), 6-methylpurine-2-deoxyriboside (MeP-dR), arabinofuranosyl-2-fluoroadenine monophosphate (F-araA), and N,N-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid) (CMDA).

[0183] [Expression regulator] As used herein, the term "expression regulator" includes transcription regulators, post-transcriptional processing regulators, translation regulators and post-translational modification regulators.

[0184] [Transcriptional regulators] As used herein, the term "transcriptional regulator" refers to a substance or nucleotide sequence that is capable of initiating, promoting, inhibiting, or terminating the synthesis of mRNA from an RNA or polynucleotide that encodes a polypeptide.

[0185] Transcriptional regulators may include, but are not limited to, enhancers, silencers, repressors, activators, inhibitors, promoters, transcription termination codons, and loxp-transcription termination codon-loxp (hereinafter, LTL). Transcription termination codons may include, but are not limited to, polyT sequences (the term "polyT sequence" is used interchangeably with the term "polyA sequence") and AATAAA sequences.

[0186] [Post-transcriptional processing regulators] As used herein, the term "post-transcriptional processing regulator" refers to a chemical compound, polynucleotide, or enzyme capable of causing modifications to the structure of mRNA synthesized from a polynucleotide.

[0187] Post-transcriptional processing regulators can include, but are not limited to, chemical compounds, polynucleotides, or enzymes that can effect 5' capping, 3' cleavage, 3' polyadenylation, or splicing.

[0188] [Translational regulatory factors] As used herein, the term "translational regulator" refers to a substance or nucleotide sequence capable of initiating, promoting, inhibiting, or terminating the synthesis of a polypeptide from mRNA.

[0189] Translational regulatory elements may include, but are not limited to, a 3' untranslated region (hereinafter, 3'UTR), a 5' untranslated region (hereinafter, 5'UTR), an exon, an intron, a start codon, a stop codon, a Kozak sequence IRES, a polynucleotide encoding a 2A peptide, and loxp-stop codon-loxp (hereinafter, LSL).

[0190] [Post-translational modification regulator] As used herein, the term "post-translational modification regulator" refers to a chemical compound, polynucleotide, or enzyme that can cause modifications to the structure of a polypeptide synthesized from mRNA.

[0191] Post-translational modification regulators can include, but are not limited to, chemical compounds or enzymes that can cause glycosylation, folding, ubiquitination, sumoylation, acetylation, phosphorylation of a polypeptide transcribed and translated from a polynucleotide.

[0192] [promoter] A "promoter" in this disclosure is a nucleic acid sequence in a nucleic acid that initiates transcription by interacting with RNA polymerase. A "promoter" in this disclosure includes constitutive promoters, tissue-specific promoters, and inducible promoters.

[0193] [Promoter configuration] The "constitutive promoter" of the present disclosure refers to a promoter that initiates transcription regardless of environmental changes in a cell. Constitutive promoters can include, but are not limited to, CMV promoter, CAG promoter, and U6 promoter.

[0194] [Tissue-specific promoter] A "tissue-specific promoter" of the present disclosure refers to a promoter that is capable of initiating transcription only in a specific tissue of an animal. Tissue-specific promoters can include, but are not limited to, mammary tissue-specific promoters or reproductive organ-specific promoters.

[0195] Mammary tissue specific promoters can include, but are not limited to, an alpha-casein promoter, a beta-casein promoter, a kappa-casein promoter, a mu-casein promoter, and a beta-lactoglobulin promoter.

[0196] Reproductive organ-specific promoters may include, but are not limited to, ovary-specific promoters and testis-specific promoters.

[0197] [Inducible promoter] In the present disclosure, an "inducible promoter" refers to a promoter that initiates transcription in response to changes in the intracellular or extracellular environment. Inducible promoters can include, but are not limited to, chemically inducible promoters, temperature inducible promoters, and light inducible promoters.

[0198] Chemical inducible promoters may include, but are not limited to, antibiotic inducible promoters, alcohol inducible promoters, steroid inducible promoters, and metal inducible promoters. Antibiotic inducible promoters may include, but are not limited to, Tet-on promoters, Tet-off promoters. Steroid inducible promoters may include, but are not limited to, estrogen inducible promoters. Metal inducible promoters may include, but are not limited to, copper inducible promoters.

[0199] Temperature-inducible promoters may include, but are not limited to, high temperature inducible promoters and low temperature inducible promoters. High temperature inducible promoters may include, but are not limited to, Hsp promoters.

[0200] [Delivery] As used herein, the term "delivery" may refer to the introduction of an exopolynucleotide into an organ, tissue, cell or organelle of a living organism.

[0201] As used herein, the term "delivery" can also refer to the introduction of a polypeptide or protein into an organ, tissue, cell or organelle of a living body.

[0202] In this disclosure, the term "delivery" may be used interchangeably with the term "providing." [Non-viral delivery] Delivery can include non-viral delivery.

[0203] Non-viral delivery may use naked nucleic acid vectors. Naked nucleic acid vectors may include circular nucleic acid vectors and linear nucleic acid vectors. Circular nucleic acid vectors may include, but are not limited to, plasmid vectors.

[0204] Non-viral delivery may use non-viral vectors, which may include, but are not limited to, artificial chromosomes, liposomes, polymers, lipid-polymer hybrids, inorganic nanoparticles, and organic nanoparticles.

[0205] Non-viral delivery can include, but is not limited to, microinjection, gene gun, electroporation, sonoporation, photoporation, magnetoporation, hydroporation. [Virus delivery] Gene delivery can include viral delivery.

[0206] Viral delivery may use RNA-based viral vectors. RNA-based viral vectors may include, but are not limited to, oncoretroviral vectors, lentiviral vectors, and human foamy virus vectors.

[0207] Viral delivery may use DNA-based viral vectors, including, but not limited to, adenovirus vectors, adeno-associated virus vectors, Epstein-Barr virus vectors, herpes simplex virus vectors, and poxvirus vectors.

[0208] Viral delivery has the advantage of excellent efficiency in delivery of large genes.

[0209] [Microinjection] As used herein, the term "microinjection" refers to the injection of a substance into an organ, tissue, cell or organelle of a living organism. The substance may include a chemical compound, a polynucleotide or a polypeptide.

[0210] As used herein, the term "microinjection" can include gamete microinjection, zygote microinjection, embryo microinjection and somatic cell microinjection.

[0211] As used herein, the term "gamete microinjection" refers to microinjecting a substance containing a polynucleotide into a gamete. As used herein, the term "gamete microinjection" includes techniques in which a substance containing a polynucleotide is microinjected into a gamete to obtain a transgenic animal through a fertilization stage, a differentiation stage, etc.

[0212] As used herein, the term "zygote microinjection" refers to microinjecting a substance containing a polynucleotide into a zygote. As used herein, the term "zygote microinjection" includes techniques in which a substance containing a polynucleotide is microinjected into a zygote to obtain a transgenic animal through a differentiation stage, etc.

[0213] As used herein, the term "embryo microinjection" refers to microinjecting a material containing a polynucleotide into an embryo. As used herein, the term "embryo microinjection" includes techniques in which a material containing a polynucleotide is microinjected into an embryo to obtain a transgenic animal through a differentiation stage, etc.

[0214] "Somatic cell microinjection" in this disclosure refers to the microinjection of a material, including a polynucleotide, into a somatic cell.

[0215] [Nuclear transfer] As used herein, the term "nuclear transfer" refers to removing the nucleus from an oocyte and introducing donor nucleic acid obtained from another cell into the oocyte. As used herein, the term "somatic cell nuclear transfer (SCNT)" refers to nuclear transfer in which the cell containing the donor nucleic acid is a somatic cell.

[0216] As used herein, the term "nuclear transfer" includes a technique of obtaining an animal containing the same genetic information as the animal cell containing the donor nucleic acid through the steps of removing the nucleus from an animal's oocyte and introducing a donor nucleic acid obtained from an animal cell, a reprogramming step, a differentiation step, and a transplantation step via a surrogate mother. As used herein, when the animal containing the donor nucleic acid is a somatic cell, the term "somatic cell nuclear transfer (SCNT)" includes a technique of obtaining an animal containing the same genetic information as the somatic cell through the steps described above.

[0217] Transgenic animals can be prepared through the delivery of cells containing donor nucleic acids. For example, transgenic animals can be prepared through somatic cell nuclear transfer (SCNT) following somatic cell microinjection. [animal] Animals of the present disclosure may include non-human animals. Animals may include mammals. Mammals may include ungulates.

[0218] Ungulates may include the order Equidae. The order Equidae may include, but is not limited to, horses.

[0219] Ungulates may include even-toed ungulates, which may include, but are not limited to, pigs, cattle, sheep, and goats.

[0220] Mammals may include rodents, which may include, but are not limited to, rats and mice.

[0221] Mammals may include lagomorphs, which may include, but are not limited to, rabbits and hares.

[0222] [Bioreactor] "Bioreactor" in this disclosure refers to an organism capable of causing biological or chemical reactions to occur in a living organism.

[0223] The organism may include a cell, a cell line, or an animal. The organism may include a transgenic cell, a transgenic cell line, or a transgenic animal. For example, the bioreactor may be a transgenic mouse. Alternatively, the bioreactor may be a transgenic rat. Alternatively, the bioreactor may be a transgenic cow.

[0224] The bioreactor can be used to produce a target substance. The target substance can include a target protein. For example, when the bioreactor is a transgenic cow with a human albumin gene knocked in, the transgenic cow can be used to produce human albumin.

[0225] [Part 1 Toolbox] [1. Toolbox definition] As used herein, the term "toolbox" refers to an exopolynucleotide that is or can be inserted into an animal genome in a cell. The cell into which the toolbox can be inserted may or may not be a transformed cell.

[0226] As used herein, the term "toolbox" may refer to an exopolynucleotide for transformation of an animal genome in a cell. For example, the toolbox may include a polynucleotide encoding a target protein in the animal genome in the cell. In another example, the toolbox may include a component of an engineered nuclease, or a polynucleotide encoding a component of an engineered nuclease, capable of performing site-specific transformation in the animal genome in the cell. In another example, the toolbox may include a transposon.

[0227] [2. Toolbox Components] The toolbox may include a first end region at the 5' end, a second end region at the 3' end, and a core domain located between the first end region and the second end region.

[0228] The first end region and the second end region may comprise at least one editable component. The first end region and the second end region may comprise the same polynucleotide. The first end region and the second end region may comprise different polynucleotides.

[0229] The ITR sequence may be included in the first end region and the second end region, and the polynucleotide present between the ITR sequences may be included in the core domain. The core domain may include an editable component. The core domain may include a polynucleotide encoding a protein or RNA. The core domain may include a polynucleotide encoding a non-functional peptide. The core domain may include a polynucleotide encoding an artificial intron. The core domain may include a polynucleotide encoding a non-functional RNA. The core domain may include a non-transcribed polynucleotide. The core domain may include an untranslated polynucleotide. The core domain may include an expression regulator. The core domain may include a promoter.

[0230] [2.1 Editable Components] [2-1-1 Recombinase recognition site] The toolbox may include at least one polynucleotide encoding an RRS. The RRS may include, but is not limited to, loxp, rox, FRT, attP, attB, and mutants thereof. The loxp mutants may include, but are not limited to, loxp66, loxp71, loxp72, loxp2722, loxp5171, and loxpm2. The rox mutants may include, but are not limited to, rox4R, rox6R, and rox2N. The FRT mutants may include, but are not limited to, F3, F5, F10, F11, F12, F13, F14, F15, and F16.

[0231] [2-1-2 Inverted terminal repeat (ITR) sequence] The toolbox may include at least one ITR sequence that can interact with hobo / Ac / Tam, P elements, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, or a mutant thereof.

[0232] [2-1-3 Modified nuclease target site] The toolbox may include at least one modified nuclease target site. The modified nuclease target site may include a target site of a CRISPR / enzyme system. The target site may be a nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence.

[0233] The toolbox may contain at one end a portion of the target site of the modified nuclease, and at its other end another portion of the target site of the modified nuclease and a PAM sequence.

[0234] [2-2. Polynucleotides encoding proteins or RNA] [2-2-1 Target protein] The toolbox may include at least one polynucleotide encoding a protein. The type of target protein is not limited, as long as the target protein can be produced in the cell or animal subject to the transformation technique.

[0235] The target protein may comprise a component of a modified nuclease. For example, the target protein may comprise, but is not limited to, a ZFN, a TALEN, an RNA-guided endonuclease, or modified / altered forms thereof.

[0236] Target proteins can include proteins derived from non-human animals. For example, target proteins can include, but are not limited to, non-human albumin, non-human interleukin, non-human insulin, non-human erythropoietin, non-human antibodies, non-human omega-3, or modified / altered forms thereof.

[0237] Target proteins can include proteins derived from humans. For example, target proteins can include, but are not limited to, human albumin, human interleukins, human insulin, human erythropoietin, human gamma chain, human delta chain, human alpha chain, human mu chain, human epsilon chain, human kappa chain, human lambda chain, or modified / altered forms thereof.

[0238] [2-2-2. Marker genes] The toolbox may include at least one polynucleotide encoding a marker gene, which may include, but is not limited to, an antibiotic resistance gene, an antigen gene, a luciferase gene, a β-galactosidase gene, a fluorescent protein gene, and a suicide gene.

[0239] [2-2-3. Site-specific recombinase] The toolbox may include at least one polynucleotide encoding an SSR, which may include, but is not limited to, Cre, Dre, Flp, KD, B2, B3, lambda, HK022, HP1, gamma delta, ParA, Tn3, Hin, Gin, Pin, phiC31, Bxb1, R4, or a mutant thereof.

[0240] [2-2-4. Transposase] The toolbox may include at least one polynucleotide encoding a transposase, which may include, but is not limited to, hobo / Ac / Tam, P element, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, and variants thereof.

[0241] [2-2-5 Endonuclease] The toolbox may include at least one polynucleotide encoding an endonuclease. The endonuclease may include, but is not limited to, a ZFN, a TALEN, or an RNA-guided endonuclease.

[0242] The toolbox may comprise at least one polynucleotide encoding an RNA-guided endonuclease.The RNA-guided endonuclease may comprise Cas9 or a mutant of Cas9.The RNA-guided endonuclease may comprise, but is not limited to, Cpf1 or a mutant of Cpf1.

[0243] [2-2-6. Guide nucleic acid] The toolbox may include at least one polynucleotide encoding at least one of the crRNA, tracrRNA, and gRNA. [2-3. Polynucleotides encoding non-functional polypeptides] The toolbox may include at least one polynucleotide that encodes a non-functional polypeptide.

[0244] The polynucleotide encoding a non-functional polypeptide may include, but is not limited to, a part of the polynucleotide encoding a target protein, a part of a marker gene, a part of the polynucleotide encoding a site-specific recombinase, a part of the polynucleotide encoding a transposase, or a part of the polynucleotide encoding an endonuclease.The polynucleotide encoding a non-functional polypeptide may include a stop codon.The polynucleotide encoding a non-functional polypeptide may include LSL.

[0245] The polynucleotide encoding the non-functional polypeptide may include a polynucleotide encoding a 2A peptide. The polynucleotide encoding the non-functional polypeptide may include an IRES. [2-4. Polynucleotides encoding untranslated RNA] The toolbox may include at least one polynucleotide encoding an untranslated RNA. The polynucleotide encoding an untranslated RNA may include a guide nucleic acid. The polynucleotide encoding an untranslated RNA may include an AATAAA sequence. The polynucleotide encoding an untranslated RNA may include a polyT. The polynucleotide encoding an untranslated RNA may not include a start codon. The polynucleotide encoding an untranslated RNA may not include a Kozak sequence. [2-5. Non-transcribed polynucleotides] The toolbox may include at least one non-transcribed polynucleotide. The non-transcribed polynucleotide may be a promoter.

[0246] The nucleotide sequence of the non-transcribed polynucleotide can be a nucleotide sequence that is not present in the animal genome in the cell, in which case, when an engineered nuclease is used that targets the non-transcribed polynucleotide as a target site, site-specific transformation can be performed without interacting with the animal genome in the cell.

[0247] The nucleotide sequence of the non-transcribed polynucleotide may be identical to a portion of a polynucleotide that encodes a protein or RNA that can normally be expressed in a cell, in which case the promoter may not be located upstream of the non-transcribed polynucleotide.

[0248] Non-transcribed polynucleotide can be used as modified nuclease target site.For example, when the toolbox containing non-transcribed polynucleotide is inserted into animal genome in cell, site-specific transformation can occur in non-transcribed polynucleotide by introducing Cas9 and gRNA that is identical to or can be complementary to a part of non-transcribed polynucleotide into cell. [2-6. Artificial introns] The toolbox may include at least one artificial intron.

[0249] Artificial introns can be included in the toolbox individually or in combination with a polynucleotide encoding a target protein.

[0250] The artificial intron may be located in a transcription unit of a polynucleotide encoding a target protein.

[0251] The artificial intron may include a splice donor site at the 5' end and a splice acceptor site at the 3' end.

[0252] At least one polynucleotide encoding an RRS may be included between a splice donor and a splice acceptor of an artificial intron. The artificial intron may include a stop codon. The artificial intron may include an enhancer. The artificial intron may be deleted by splicing.

[0253] The artificial intron may be selected from any of the group consisting of: a) introns derived from a natural intron of the target gene itself, b) introns modified by substitution, deletion and / or insertion of nucleotides derived from a natural intron, c) natural introns from a different target gene, d) introns derived from a different intron, e) chimeric introns consisting of a different intron sequence derived from at least one natural intron sequence of the target gene and / or a different gene, f) de novo synthesized synthase introns, and g) combinations thereof.

[0254] The artificial introns can increase or decrease the expression of a polynucleotide encoding a target protein. The artificial introns can increase or decrease the expression of a polynucleotide in a genome. [2-7. Expression Regulatory Factors] The toolbox may include at least one expression regulator.

[0255] The expression regulator may include a transcription regulator, which is the same as described above.

[0256] The expression regulator may include a post-transcriptional processing regulator, which is the same as described above.

[0257] The expression regulator may include a translation regulator, which is the same as described above.

[0258] The expression regulator may include a post-translational modification regulator, which is the same as described above. [2-8. Promoter] The toolbox may include a promoter.

[0259] The promoter may comprise a constitutive promoter, which is the same as described above.

[0260] The promoter may include a tissue-specific promoter, which is the same as described above.

[0261] The promoter may include an inducible promoter, which is the same as described above.

[0262] [2-9. Toolbox Structure] [2-9-1. ITR sequence recombinase recognition site or modified nuclease target site ITR sequence] The first and second end regions of the toolbox may comprise ITR sequences. The core domain of the toolbox may comprise at least one recombinase recognition site.

[0263] The toolbox can provide a location where the insertion or exchange of exopolynucleotides can be achieved without lethal effects on gene expression of the animal genome in the cells.

[0264] [2-9-2. ITR sequence-modified nuclease target site-ITR sequence] The first and second end regions of the toolbox may comprise ITR sequences. The core domain of the toolbox may comprise at least one engineered nuclease target site.

[0265] The toolbox can provide a location where the insertion or exchange of exopolynucleotides can be achieved without lethal effects on gene expression of the animal genome in the cells.

[0266] [2-9-3. ITR sequence polynucleotide encoding modified nuclease-recombinase recognition site-ITR sequence]

[0267] The first and second end regions of the toolbox may comprise an ITR sequence. The core domain of the toolbox may comprise a polynucleotide encoding an RNA-guided endonuclease. The core domain of the toolbox may comprise at least one recombinase recognition site.

[0268] At least one recombinase recognition site can be used to insert a polynucleotide encoding a guide nucleic acid into the toolbox. The recombinase recognition site can be used to alter the target site at which site-specific transformation can occur.

[0269] At least one recombinase recognition site may include recombinase recognition site 1 (RRS1) and recombinase recognition site 2 (RRS2). Recombinase recognition site 1 (RRS1) may be located upstream of the polynucleotide encoding the RNA-guided endonuclease, and recombinase recognition site 2 (RRS2) may be located downstream of the polynucleotide encoding the RNA-guided endonuclease. The polynucleotide encoding the RNA-guided endonuclease in the toolbox may be exchanged with a different exopolynucleotide using recombinase recognition site 1 (RRS1) and recombinase recognition site 2 (RRS2).

[0270] [2-9-4. ITR sequence polynucleotide encoding RNA-guided endonuclease - polynucleotide encoding guide nucleic acid - recombinase recognition site - expression regulator - ITR sequence]

[0271] The first and second end regions of the toolbox may comprise ITR sequences. The toolbox core domain may comprise a polynucleotide encoding an RNA-guided endonuclease. The toolbox core domain may comprise a polynucleotide encoding a guide nucleic acid. The toolbox core domain may comprise at least one recombinase recognition site. The toolbox core domain may comprise at least one of a factor regulating the expression of the RNA-guided endonuclease and a factor regulating the expression of the guide nucleic acid.

[0272] At least one recombinase recognition site can be located upstream or downstream of the polynucleotide encoding the RNA-guided endonuclease. Factors that regulate the expression of the polynucleotide encoding the RNA-guided endonuclease can be inserted or deleted using at least one recombinase recognition site.

[0273] At least one recombinase recognition site may be located upstream or downstream of the polynucleotide encoding the guide nucleic acid. Factors that regulate the expression of the polynucleotide encoding the guide nucleic acid may be inserted or deleted using the at least one recombinase recognition site.

[0274] At least one recombinase recognition site may comprise recombinase recognition site 1 (RRS1) or recombinase recognition site 2 (RRS2). Recombinase recognition site 1 (RRS1) may be located upstream of the polynucleotide encoding the guide nucleic acid, and recombinase recognition site 2 (RRS2) may be located downstream of the polynucleotide encoding the guide nucleic acid. The polynucleotide encoding the guide nucleic acid in the toolbox may be deleted using recombinase recognition site 1 (RRS1) and recombinase recognition site 2 (RRS2).

[0275] [2-9-5. ITR sequence - marker gene - ITR sequence] The first and second end regions of the toolbox may comprise an ITR sequence. The toolbox core domain may comprise a polynucleotide encoding at least one marker gene. The at least one marker gene may comprise a suicide gene.

[0276] A marker gene can be knocked out by site-specific transformation of a polynucleotide encoding the marker gene.

[0277] [2-9-6. Recombinase recognition site-polynucleotide encoding transposase-recombinase recognition site]

[0278] The first end region of the toolbox may comprise recombinase recognition site 1 (RRS1). The second end region of the toolbox may comprise recombinase recognition site 2 (RRS2) that pairs with recombinase recognition site 1 (RRS1). The core domain of the toolbox may comprise a polynucleotide encoding a transposase. The transposase may comprise an excision-only transposase. Transposons contained in an animal genome in a cell may be deleted using the toolbox.

[0279] [2-9-7. Modified nuclease target site-ITR sequence-exopolynucleotide-ITR sequence-modified nuclease target site]

[0280] The first and second end regions of the toolbox may comprise modified nuclease target sites. The core domain of the toolbox may comprise a first ITR sequence, a second ITR sequence and an exopolynucleotide. The polynucleotide encoding the target protein may be located between the first and second ITR sequences.

[0281] The exopolynucleotide can be inserted into the animal genome in the cell using the toolbox while performing site-specific transformation. Additionally, only the exopolynucleotide can be deleted by the transposase while maintaining site-specific transformation in the animal genome in the cell.

[0282] [3. Toolbox Function] Toolbox can be used to express the protein or nucleic acid required for the transformation of animal genome in cell.For example, toolbox can include the polynucleotide encoding Cas9.When gRNA having a part of the nucleotide sequence in animal genome that contains toolbox as target site is introduced into cell, gRNA can induce site-specific transformation at the target site in animal genome by forming a complex with Cas9 expressed in cell.

[0283] The toolbox can be used to knock-in genes into an animal genome in a cell. For example, the toolbox can include a polynucleotide encoding a human interleukin. The toolbox can be inserted into a bovine genome in a cell such that the cell expresses the human interleukin.

[0284] The toolbox can be used to knock out genes present in an animal genome in a cell. For example, the toolbox can be inserted into the middle of a polynucleotide encoding an exon for bovine albumin in the bovine genome in a cell, thereby preventing the cell from expressing bovine albumin.

[0285] The toolbox can provide a site in the animal genome in the cell that can be transformed. For example, the toolbox can include a nucleotide sequence that can be a modified nuclease target site. For example, the toolbox can include a polynucleotide that encodes green fluorescent protein (GFP). When the bovine genome in the cell includes a toolbox, a gRNA that targets a portion of the nucleotide sequence of the polynucleotide that encodes green fluorescent protein (GFP) and Cas9 can be introduced into the cell, thereby directing site-specific transformation to a portion of the nucleotide sequence of the polynucleotide that encodes green fluorescent protein (GFP) in the toolbox, rather than a nucleotide sequence outside the toolbox.

[0286] [Part 2 Toolbox Insertion] 1. Toolbox Delivery Vector The toolbox can be delivered to insert the toolbox into the animal genome in a cell. The nucleic acid delivery vector containing the toolbox can be a naked nucleic acid vector, a non-viral vector, or a viral vector.

[0287] [2. How to insert a toolbox] [2-1. Use of homologous recombination] The first homology arm may be located at the 5' end of the first end region of the toolbox. The second homology arm may be located at the 3' end of the second end region. The first homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell. The second homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell.

[0288] The first homology arm and the second homology arm can interact with a portion of a genome in a cell, and the toolbox can be inserted into the genome through the interaction.

[0289] [2-2 Use of site-specific recombination] A first end region of the toolbox may include RRS1. A second end region of the toolbox may include RRS2.

[0290] At least one recombinase can be provided to introduce the toolbox into cells. The recombinase can include a first recombinase that can interact with RRS1. The recombinase can include a second recombinase that can interact with RRS2. The first recombinase and the second recombinase can be identical to each other. For example, when RRS1 is loxp and RRS2 is a loxp mutant, the first recombinase and the second recombinase can be Cre.

[0291] To introduce at least one recombinase into a cell, a naked nucleic acid vector, a non-viral vector, a viral vector (each of which includes a polynucleotide encoding a recombinase), or a recombinase polypeptide can be used.

[0292] To introduce the toolbox into a cell, the nucleic acid comprising the toolbox and at least one recombinase can be provided in separate forms. For example, the nucleic acid comprising the toolbox can be delivered by a DNA plasmid vector and the recombinase can be delivered by a recombinase polypeptide.

[0293] To introduce the toolbox into a cell, the nucleic acid comprising the toolbox and at least one recombinase can be provided by a single delivery vector. For example, the nucleic acid comprising the toolbox and the polynucleotide encoding the recombinase can be provided by a single DNA plasmid vector. [2-3. Use of transposon systems] The first and second end regions of the toolbox may comprise ITR sequences.

[0294] To introduce the toolbox into a cell, a transposase can be provided, the transposase being capable of interacting with the ITR sequences.

[0295] To introduce a transposase into a cell, a naked nucleic acid vector, a non-viral vector, a viral vector (each of which includes a polynucleotide encoding the transposase), or a transposase polypeptide can be used.

[0296] The nucleic acid comprising the toolbox and the transposase can be provided in individual forms, for example, the nucleic acid comprising the toolbox can be delivered by a DNA plasmid vector and the transposase can be delivered by a polypeptide.

[0297] The nucleic acid comprising the toolbox and the transposase can be provided in individual forms. For example, the nucleic acid comprising the toolbox can be delivered by a DNA plasmid vector, and the transposase can be delivered by a separate DNA plasmid vector comprising a polynucleotide encoding the transposase.

[0298] The nucleic acid comprising the toolbox and the transposase can be introduced into the cell by a single delivery vector. For example, the nucleic acid comprising the toolbox and the polynucleotide encoding the transposase can be provided by a single DNA plasmid vector.

[0299] [2-4. Use of modified nucleases] [2-4-1. Use of Homologous Recombination Repair (HDR)] The first homology arm may be located at the 5' end of the first end region of the toolbox. The second homology arm may be located at the 3' end of the second end region of the toolbox. The first homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell. The second homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell.

[0300] To introduce the toolbox into a cell, any one of the nucleic acid delivery vectors containing the toolbox can be used.

[0301] To introduce the toolbox into a cell, a modified nuclease can be provided. The modified nuclease can specifically act on a modified nuclease target site present in an animal genome. The modified nuclease target site present in an animal genome can be located in a nucleotide sequence that is identical or complementary to a first homology arm, can be located in a nucleotide sequence that is identical or complementary to a second homology arm, or can be located between a nucleotide sequence that is identical or complementary to a first homology arm and a nucleotide sequence that is identical or complementary to a second homology arm.

[0302] At least one modified nuclease may be provided in any one selected from a naked nucleic acid vector, a non-viral vector, and a viral vector, each of which comprises a polynucleotide encoding each component of the modified nuclease, the modified nuclease protein, a complex comprising the modified nuclease protein, or a combination thereof.

[0303] For example, a complex comprising a Cas9 protein and a gRNA can be provided. In another example, a naked nucleic acid vector comprising a polynucleotide encoding Cas9 and a polynucleotide encoding a gRNA can be provided.

[0304] In order to introduce the toolbox into cells, the nucleic acid comprising the toolbox and at least one modified nuclease can be provided in an isolated form.For example, the nucleic acid comprising the toolbox can be provided by a DNA plasmid vector, and the modified nuclease can be provided as a complex comprising Cas9 protein and gRNA.In another example, the nucleic acid comprising the toolbox can be provided by a DNA plasmid vector, and the modified nuclease can be provided by a naked nucleic acid vector comprising the polynucleotide encoding Cas9 and the polynucleotide encoding gRNA.

[0305] To introduce the toolbox into a cell, the nucleic acid comprising the toolbox and the modified nuclease can be provided by a single vector. For example, the nucleic acid comprising the toolbox, the polynucleotide encoding Cas9, and the polynucleotide comprising the gRNA can be provided by a single DNA plasmid vector.

[0306] [2-4-2. Use of Homology-Independent Targeted Insertion (HITI)] A first end region of the toolbox may include a first modified nuclease target site. A second end region of the toolbox may include a second modified nuclease target site. The first modified nuclease target site and the second modified nuclease target site may include identical nucleotide sequences to each other.

[0307] To introduce the toolbox into a cell, at least one modified nuclease may be provided. The modified nuclease may include a first modified nuclease capable of specifically acting on a first modified nuclease target site. The modified nuclease may include a second modified nuclease capable of specifically acting on a second modified nuclease target site. The first modified nuclease and the second modified nuclease may be identical to each other.

[0308] At least one modified nuclease may be provided in any one selected from a naked nucleic acid vector, a non-viral vector, and a viral vector, each of which comprises a polynucleotide encoding each component of the modified nuclease, the modified nuclease protein, a complex comprising the modified nuclease protein, or a combination thereof.

[0309] In order to introduce the toolbox into cells, the nucleic acid comprising the toolbox and at least one modified nuclease can be provided in an isolated form.For example, the nucleic acid comprising the toolbox can be provided by a DNA plasmid vector, and the modified nuclease can be provided as a complex comprising Cas9 protein and gRNA.In another example, the nucleic acid comprising the toolbox can be provided by a DNA plasmid vector, and the modified nuclease can be provided by a naked nucleic acid vector comprising the polynucleotide encoding Cas9 and the polynucleotide encoding gRNA.

[0310] To introduce the toolbox into a cell, the nucleic acid comprising the toolbox and at least one modified nuclease can be provided by a single vector. For example, the nucleic acid comprising the toolbox, the polynucleotide encoding Cas9, and the polynucleotide comprising gRNA can be provided by a single DNA plasmid vector. [3. Toolbox insertion locus] [3-1. Inserting a Single Toolbox] The animal genome in the cell may include one toolbox. One toolbox may be included in any one of the autosomes in the animal genome.

[0311] The toolbox may be included in any one of the sex chromosomes in the animal genome. The toolbox may be included in the X chromosome of the animal genome. The toolbox may be included in the Y chromosome of the animal genome. [3-2. Inserting multiple toolboxes] The animal genome in the cell may contain two or more toolboxes. All of the two or more toolboxes may be identical to one another. One of the two or more toolboxes may be different from the rest.

[0312] The differences between the toolboxes may refer to the differences in sequences between the toolboxes.

[0313] For example, a first toolbox comprising a polynucleotide encoding an RNA-guided endonuclease and a second toolbox comprising a polynucleotide encoding a guide nucleic acid capable of binding to a target site may be considered as two different toolboxes.

[0314] All of the two or more polynucleotides may be contained in one chromosome. The chromosome may be an autosome or a sex chromosome.

[0315] The two or more toolboxes may include at least a first toolbox and a second toolbox. In this case, the first toolbox may be located on a first chromosome, and the second toolbox may be located on a second chromosome different from the first chromosome. The first chromosome may be an autosome. The first chromosome may be a sex chromosome. The second chromosome may be an autosome. The second chromosome may be a sex chromosome.

[0316] [3-3. Characteristics of the inserted gene locus] The toolbox may be located between the nucleotide sequences capable of interacting with the transposase. The nucleotide sequences capable of interacting with the transposase may include, but are not limited to, TA and TTAA.

[0317] The toolbox may be located on a polynucleotide that may be involved in the expression of any protein or RNA in the animal genome in a cell.

[0318] For example, the toolbox can be located in any one of the following: a polynucleotide encoding a protein or RNA, a promoter, a 5'UTR, an intron, an exon, and a 3'UTR of a polynucleotide encoding a protein or RNA. The toolbox can knock out a protein or RNA in an animal genome in a cell. For example, when the toolbox is located in an exon of the beta-lactoglobulin gene in a bovine genome, it can prevent the expression of beta-lactoglobulin in a cell containing a bovine genome.

[0319] The toolbox may be located in a polynucleotide in the animal genome in the cell that is not involved in the expression of protein or RNA. Additionally, the toolbox may be located within a safe harbor in the animal genome in the cell.

[0320] When the animal genome is a mouse genome, the safe harbor for the mouse genome may include the already widely known rosa26 locus.

[0321] When the animal genome is a bovine genome, the bovine genome safe harbor may include locations already widely known in the bovine genome. The bovine genome safe harbor may include, but is not limited to, the loci shown in Table 1 below. Each of the loci listed in Table 1 below may be located between the gene located closest to the 5' end (5' gene) and the gene located closest to the 3' end (3' gene). [Table 1] [Table 1]

[0322] When the toolbox is located on a polynucleotide that is not involved in the expression of protein or RNA in the animal genome in the cell, or is located within a safe harbor in the animal genome in the cell, the toolbox can be used as an artificial safe harbor for additional transformation.For example, when the toolbox located in the safe harbor in the bovine genome contains loxp, the exopolynucleotide can be inserted into the toolbox by delivering an exopolynucleotide that contains loxp and Cre recombinase without affecting the expression of any protein or RNA in the animal genome in the cell.

[0323] [4. Transformed cells into which the toolbox is inserted] [4-1. Single cells] [4-1-1. Polyploidy] The transformed cell comprising at least one toolbox may be a diploid cell. The diploid cell may include a stem cell, a somatic cell, an oogonial stem cell, an oogonia, a primary oocyte, a spermatogonial stem cell, a spermatogonia, a primary spermatocyte, and a zygote. The transformed cell comprising at least one toolbox may be a haploid cell. The haploid cell may include a secondary oocyte, an egg, a secondary spermatocyte, and a sperm.

[0324] [4-1-2. Zygosity] The transformed cell may comprise at least one pair of homologous chromosomes. The at least one pair of homologous chromosomes may comprise chromosome 1 and chromosome 2 in a homologous chromosomal relationship. The transformed cell containing two or more toolboxes may be homozygous.

[0325] In homozygous transformed cells, the types, numbers, and locations of toolboxes contained in chromosomes 1 and 2 can all be identical.

[0326] In homozygous transformed cells, both the type and number of toolboxes contained on chromosome 1 and chromosome 2 can be identical.

[0327] In homozygous transformed cells, the types of toolboxes contained in chromosome 1 and chromosome 2 may be identical.

[0328] In transformed cells that are homozygous, both chromosomes 1 and 2 may be absent of any toolbox. Transformed cells that contain more than one toolbox may be heterozygous.

[0329] In a transformed cell that is heterozygous, chromosome 1 may contain no toolboxes and chromosome 2 may contain at least one toolbox.

[0330] Alternatively, in a heterozygous transformed cell, the second chromosome may not contain a toolbox that is identical to that contained in the first chromosome.

[0331] [4-2. Cell colonies] The transformed cells containing at least one toolbox can form cell colonies, which can be cell populations cultured from a single cell.

[0332] [4-2-1. Homologous cell colonies] Each cell contained in the homologous cell colony may contain a single toolbox. The single toolbox carried by each cell may be identical. In each cell, the single toolbox may be located at the same location.

[0333] Each cell in the homologous cell colony may contain two or more toolboxes. The two or more toolboxes may include an nth toolbox (n≧1, n is an integer). In each cell, both the type and location of the nth toolbox may be identical.

[0334] The two or more toolboxes may be identical to one another, or alternatively, one of the two or more toolboxes may be different from the other of the remaining toolboxes.

[0335] Hereinafter, unless otherwise specified, the expression "the toolbox contained in a first cell is identical to that contained in a second cell" means that the type, number, and location of the toolboxes contained in the first cell are completely identical to the type, number, and location of the toolboxes contained in the second cell.

[0336] [4-2-2. Chimeric cell colonies] A chimeric cell colony refers to a cell colony other than a homologous cell colony. A chimeric cell colony can include both cells having a genome that does not include a toolbox and cells having a genome that includes at least one toolbox.

[0337] The chimeric cell colony may include a first cell and a second cell, each having a genome that includes at least one toolbox, where at least one of the type, number, and locus of the toolboxes included in the genome of the first cell may not be identical to the type, number, and locus of the toolboxes included in the genome of the second cell.

[0338] For example, in the case of a cell colony in which a first toolbox is included in the genome of a first cell and the same type of first toolbox is included in the genome of a second cell, the cell colony may be a chimeric cell colony if i) the number of first toolboxes included in the genome of the first cell is different from the number of first toolboxes included in the genome of the second cell, or ii) the locus of the first toolbox included in the genome of the first cell is different from the number of first toolboxes included in the genome of the second cell.

[0339] In another example, in the case of a cell colony in which a first toolbox is included in the genome of a first cell and a second toolbox of a different type is included in the genome of a second cell, the cell colony can also be a chimeric cell colony when i) the number of first toolboxes included in the genome of the first cell is identical to the number of second toolboxes included in the genome of the second cell, and ii) all loci of the first toolbox included in the genome of the first cell are identical to the number of second toolboxes included in the genome of the second cell.

[0340] Additionally, in the case of a cell colony in which a first toolbox is included in the genome of a first cell and a second toolbox of a different type is included in the genome of a second cell, the cell colony can be a chimeric cell colony when i) the number of first toolboxes included in the genome of the first cell is different from the number of second toolboxes included in the genome of the second cell, or ii) the locus of the first toolbox included in the genome of the first cell is different from the number of second toolboxes included in the genome of the second cell.

[0341] As used herein, the term "locus" may be designated in relation to a toolbox by one or more of the endogenous gene located closest to the 5' end and the endogenous gene located closest to the 3' end.

[0342] As used herein, the term "toolbox locus" may be designated by one or more of the endogenous gene located closest to the 5' end and the endogenous gene located closest to the 3' end with respect to the toolbox.

[0343] That is, a locus of a first toolbox differs from a second toolbox when the endogenous gene located closest to the 5' end of the first toolbox differs from the one located closest to the 5' end of the second toolbox. Additionally, a locus of a first toolbox differs from a second toolbox when the endogenous gene located closest to the 3' end of the first toolbox differs from the one located closest to the 3' end of the second toolbox.

[0344] [5. Selection of transformed cells into which the toolbox is inserted] [5-1. Selection of transformed cells using antibiotic resistance genes] The core domain of the toolbox can contain an antibiotic resistance gene. Animal cells containing the toolbox can survive when the cells are treated with an antibiotic. Thus, animal cells containing the toolbox can be separated from those that do not contain the toolbox.

[0345] [5-2. Selection of transformed cells using antigen-antibody reactions] The core domain of the toolbox can contain a polynucleotide that encodes an antigen or a nucleotide that can act as an antigen. Animal cells that contain the toolbox can interact with an antibody specific to the antigen. Thus, animal cells that contain the toolbox can be distinguished from those that do not contain the toolbox.

[0346] [5-3. Selection of transformed cells using fluorescent proteins] The core domain of the toolbox can include a polynucleotide encoding a fluorescent protein. The animal cells containing the toolbox can measure the fluorescent signal. Thus, the animal cells containing the toolbox can be distinguished from those that do not contain the toolbox.

[0347] [5-4. Selection of transformed cells using surface marker genes] The core domain of the toolbox can include a polynucleotide encoding a surface marker. The animal cells that contain the toolbox can interact with an antibody specific to the surface marker. The antibody can interact with a magnetic particle or a fluorophore. Thus, the animal cells that contain the toolbox can be distinguished from those that do not contain the toolbox through magnetic properties or fluorescent signals.

[0348] [6. Transgenic animals into which the toolbox is inserted] [6-1. Individual transgenic animals into which the toolbox is inserted] A transgenic animal can contain one or more transformed cells, each transformed cell containing at least one toolbox.

[0349] [6-1-1.Homology] Each of the cells contained in the homologous transgenic animal may individually contain a single toolbox. The single toolbox carried by each cell may be identical. In each cell, the single toolbox may be located on the same chromosome.

[0350] Each of the cells contained in the homologous transgenic animal may contain two or more toolboxes. The two or more toolboxes may include an nth toolbox (n≧1, n is an integer). In each cell, all of the chromosomes on which the nth toolbox is located may be identical.

[0351] The two or more toolboxes may be identical to each other. Alternatively, one of the two or more toolboxes may be different from the other of the remaining toolboxes. A homologous transgenic animal may include transformed cells that are homozygous. A homologous transgenic animal may include transformed cells that are heterozygous.

[0352] [6-1-2. Chimera] A chimeric transgenic animal refers to a transgenic animal other than a homologous transgenic animal. A chimeric transgenic animal may comprise homozygous transformed cells. A chimeric transgenic animal may comprise heterozygous transformed cells.

[0353] A chimeric transgenic animal can include both cells having a genome that does not include a toolbox and cells having a genome that includes at least one toolbox.

[0354] A chimeric transgenic animal may include a first cell and a second cell, each having a genome including at least one toolbox. In this case, at least one of the type, number, and locus of the toolboxes included in the genome of the first cell may not be the same as the type, number, and locus of the toolboxes included in the genome of the second cell in the chimeric transgenic animal.

[0355] For example, in the case of a transgenic animal comprising a first cell having a genome including a first toolbox, and a second cell having a genome including the first toolbox, the transgenic animal can be a chimeric transgenic animal when i) the number of first toolboxes included in the genome of the first cell is different from the number of first toolboxes included in the genome of the second cell, or ii) at least one of the loci of the first toolboxes included in the genome of the first cell is different from the number of first toolboxes included in the genome of the second cell.

[0356] In another example, in the case of a transgenic animal comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox that is a different type than the first toolbox, the transgenic animal can be a chimeric transgenic animal if i) the number of first toolboxes included in the genome of the first cell is identical to the number of second toolboxes included in the genome of the second cell, and ii) all loci of the first toolbox included in the genome of the first cell are identical to the number of second toolboxes included in the genome of the second cell.

[0357] Additionally, in the case of a transgenic animal comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox that is a different type than the first toolbox, the transgenic animal may be a chimeric transgenic animal when i) the number of first toolboxes included in the genome of the first cell is different from the number of second toolboxes included in the genome of the second cell, or ii) at least one of the loci of the first toolbox included in the genome of the first cell is different from the number of second toolboxes included in the genome of the second cell.

[0358] [6-2. Method for preparing individual transgenic animals into which the toolbox is inserted] Methods for preparing transgenic animals into which the toolbox is inserted include methods for preparing transgenic animals from animal cells, methods for preparing transgenic animals via delivery of the toolbox to animal tissues or organs, and methods for preparing transgenic animals by mating between transgenic animals. Methods for preparing transgenic animals from animal cells include methods for preparing transgenic animals from wild-type animal cells and methods for preparing transgenic animals from cells of transgenic animals.

[0359] The transgenic animals produced by any one of the above methods may be any one of chimeric or homologous transgenic animals.

[0360] [6-2-1. Methods for preparing transgenic animals from animal cells] Transgenic animals can be prepared that contain the process of delivering the toolbox into wild-type animal cells.

[0361] For example, transgenic animals can be prepared by somatic cell nuclear transfer (hereinafter SCNT) followed by injection of a polynucleotide into a wild-type somatic cell by somatic cell microinjection. The transgenic animal can be a homologous transgenic animal.

[0362] For example, the transgenic animal may be prepared by gamete microinjection into wild type gametes. The transgenic animal may be a chimeric transgenic animal.

[0363] For example, the transgenic animals can be prepared by zygote microinjection into a wild type zygote. The transgenic animals can be chimeric transgenic animals.

[0364] For example, the transgenic animal can be prepared by embryonic microinjection into a wild type embryo. The transgenic animal can be a chimeric transgenic animal. The transgenic animal can be prepared using transgenic animal cells.

[0365] For example, a transgenic animal can be prepared via SCNT using transgenic somatic cells. The transgenic animal can be a homologous transgenic animal.

[0366] For example, transgenic animals can be prepared via SCNT following somatic cell microinjection into transgenic somatic cells. The transgenic animals can be homologous transgenic animals.

[0367] For example, the transgenic animal can be prepared by gamete microinjection into the transgenic gamete. The transgenic animal can be a chimeric transgenic animal.

[0368] For example, the transgenic animal may be prepared by zygote microinjection into a transgenic zygote. The transgenic animal may be a chimeric transgenic animal.

[0369] For example, the transgenic animal can be prepared by embryonic microinjection into a transgenic embryo. The transgenic animal can be a chimeric transgenic animal.

[0370] [6-2-2. Method for preparing transgenic animals via delivery of toolbox into animal tissues or organs] Transgenic animals can be prepared by toolbox delivery into tissues or organs of the animal.

[0371] For example, the transgenic animal may be prepared by microinjection into the mammary tissue of the animal. The transgenic animal may be a chimeric transgenic animal.

[0372] For example, transgenic animals can be prepared by microinjection into the reproductive tract of an animal. Offspring resulting from the gametes of a transgenic animal can also be transgenic animals. The transgenic animal can be a chimeric transgenic animal.

[0373] [6-2-3. Mating of transgenic animals] Transgenic animals can be prepared by mating between transgenic animals and wild-type animals.

[0374] Alternatively, transgenic animals can be prepared by breeding between a first transgenic animal and a second transgenic animal.

[0375] The second transgenic animal may be the offspring of the first transgenic animal or may be related to the first transgenic animal. Alternatively, the second transgenic animal may be unrelated to the first transgenic animal.

[0376] The transgenic animal resulting from the cross may contain a toolbox that is identical to a portion of the toolbox contained in the animal genome of the first transgenic animal, in the same location.

[0377] The transgenic animal obtained by mating can contain a toolbox that is identical to a portion of the toolbox contained in the animal genome of the second transgenic animal, at the same location. The transgenic animal obtained by mating can be a homologous transgenic animal.

[0378] The transgenic animals obtained by mating may contain transformed cells that are homozygous. The transgenic animals obtained by mating may contain transformed cells that are heterozygous.

[0379] 7. Use of transgenic animals into which the toolbox is inserted [7-1. Improved Animals] The transgenic animal into which the toolbox is inserted can be used as an improved animal.The improved animal can include, but is not limited to, cattle with the polynucleotide encoding beta-lactoglobulin knocked out, cattle with the polynucleotide encoding omega-3 knocked in.

[0380] [7-2. Animal models of disease] The transgenic animals into which the toolbox is inserted can be used as disease animal models, including, but not limited to, cattle in which a polynucleotide encoding a tumor suppressor protein has been knocked out.

[0381] [7-3. Disease-resistant animals] The transgenic animals into which the toolbox is inserted can be used as disease-resistant animals, which can include, but are not limited to, cattle in which the polynucleotide encoding the prion protein has been knocked out.

[0382] [7-4. Use of by-products] The parts of the transgenic animal into which the toolbox is inserted may include, but are not limited to, organs, flesh, skin, hair, and body fluids.

[0383] [7-5. Bioreactor] The transgenic animal into which the toolbox is inserted may be used as a bioreactor. A body fluid of the transgenic animal may be obtained. The body fluid may include breast milk, blood, or urine. A biomolecule may be obtained from the body fluid of the transgenic animal. The biomolecule may include a protein. The protein may include a target protein.

[0384] [Part 3: Transformation using RRS in the Toolbox] [1. Structure for inserting RRS into the toolbox] The transformed cell can contain at least one toolbox in the animal genome, any one of which can contain at least one RRS.

[0385] [1-1. When one RRS is included] A toolbox may include one RRS. For example, a first end region of a toolbox may include one RRS. In another example, a second end region of a toolbox may include one RRS. In yet another example, a core domain of a toolbox may include one RRS.

[0386] [If RRS is 1-2.2 or higher] The toolbox may include two or more RRSs. The two or more RRSs may include RRS1 and RRS2. RRS1 may be located in any one of the first end region, the second end region, and the core domain of the toolbox. RRS2 may be located in any one of the first end region, the second end region, and the core domain of the toolbox.

[0387] For example, RRS1 and RRS2 can both be located in the core domain. In a different example, RRS1 can be located in the first end region and RRS2 can be located in the second end region.

[0388] RRS1 and RRS2 may be identical to each other. RRS1 and RRS2 may be located in the same direction as each other. Alternatively, RRS1 and RRS2 may be located in opposite directions to each other.

[0389] RRS1 and RRS2 may be different from each other. RRS1 and RRS2 may be located in the same direction as each other. Alternatively, RRS1 and RRS2 may be located in the opposite direction to each other.

[0390] RRS1 and RRS2 can be different from each other. SSR1 capable of specifically interacting with RRS1 and SSR2 capable of specifically interacting with RRS2 can be identical to each other or different from each other.

[0391] RRS1 and RRS2 may be different from each other. RRS1 and RRS2 may form a pair, thereby resulting in mutual exchange. Alternatively, RRS1 and RRS2 may not form a pair, thereby resulting in no mutual exchange.

[0392] [2. SSR for use of RRS in the toolbox] [2-1. SSRs provided by animal genomes] The animal genome in the cell may comprise a polynucleotide encoding an SSR specific to any one of the RRSs contained in the toolbox. The polynucleotide encoding the SSR may be included in the toolbox that contains the RRS. The polynucleotide encoding the SSR may be included in different toolboxes.

[0393] [2-2. SSRs provided by sources other than animal genomes] When a polynucleotide encoding an SSR that can interact with any one of the RRSs included in the toolbox is not present in the animal genome in the cell, the SSR can be provided. The form in which the SSR is provided can be a naked nucleic acid vector, a non-viral vector, or a viral vector, each of which includes a polynucleotide encoding the SSR or a recombinase polypeptide.

[0394] 3. Site-specific recombination using RRS in the toolbox [3-1. Polynucleotide insertion using RRS] The toolbox can include at least one RRS. An exopolynucleotide that includes an RRS that pairs with any one of the RRSs included in the toolbox can be provided to a cell that includes the toolbox.

[0395] In this case, insertion of the exopolynucleotide into the toolbox can occur via interaction with an SSR specific for the pair of RRSs. [3-2. Deletion of polynucleotides using RRS] The toolbox may include two or more RRSs. The two or more RRSs may include RRS1 and RRS2.

[0396] RRS1 and RRS2 may form a pair and be located in the same direction. For example, in the core domain of the toolbox, two loxps may be located in the same direction.

[0397] In this case, the polynucleotide located between the pair of RRSs can be deleted through interaction with the SSR specific to the pair of RRSs.For example, when the core domain of the toolbox comprises the polynucleotide encoding the gRNA between two loxps, the polynucleotide encoding the gRNA can be deleted from the toolbox by providing Cre recombinase. [3-3. Polynucleotide inversion using RRS] The toolbox may include two or more RRSs. The two or more RRSs may include RRS1 and RRS2. RRS1 and RRS2 may form a pair and may be located in opposite directions.

[0398] In this case, a polynucleotide located between the pair of RRSs can undergo inversion via interaction with an SSR specific to the pair of RRSs. [3-4. Exchange of polynucleotides using RRS] A toolbox may include two or more RRSs. The two or more RRSs may include RRS1 and RRS2.

[0399] RRS1 and RRS2 may not form a pair. For example, the core domain of the toolbox may include loxp(RRS1) and loxp2722(RRS2). Loxp may be located upstream of loxp2722.

[0400] An exopolynucleotide, each of which includes RRS3 that pairs with RRS1 and RRS4 that pairs with RRS2, can be provided to a cell that includes a toolbox. For example, the exopolynucleotide can include loxp(RRS3) and loxp2722(RRS4). Loxp can be located upstream of loxp2722.

[0401] In this case, an interaction with the SSR specific for RRS1 and RRS3, as well as an interaction with the SSR specific for RRS2 and RRS4, may occur. Additionally, exchange may occur between a polynucleotide located between RRS1 and RRS2 and a polynucleotide located between RRS3 and RRS4.

[0402] For example, when a non-transcribed polynucleotide is located between loxp and loxp2722 of the toolbox and a polynucleotide encoding a gRNA is located between loxp and loxp2722 of the exopolynucleotide, the non-transcribed polynucleotide in the toolbox and the polynucleotide encoding a gRNA of the exopolynucleotide can be exchanged by providing Cre recombinase. [3-5. Site-specific recombination using two or more RRSs in one toolbox] The toolbox may include two or more RRSs. The two or more RRSs may include RRS1 and RRS2.

[0403] RRS1 and RRS2 may not form a pair. For example, the core domain of the toolbox may include loxp(RRS1) and loxp2722(RRS2). Loxp may be located upstream of loxp2722.

[0404] A first exopolynucleotide comprising an RRS3 that pairs with RRS1, and a second exopolynucleotide comprising an RRS4 that pairs with RRS2 can be provided to a cell that contains the toolbox.

[0405] For example, the first exopolynucleotide may comprise loxp(RRS3) and the second exopolynucleotide may comprise loxp2722(RRS4).

[0406] In this case, interactions with SSRs specific for RRS1 and RRS3, as well as interactions with SSRs specific for RRS2 and RRS4 may occur. Additionally, a first exopolynucleotide may be inserted at the position of RRS1, and a second exopolynucleotide may be inserted at the position of RRS2. That is, two or more types of exopolynucleotides can all be inserted into a cell having an animal genome that includes a toolbox that includes two or more RRSs.

[0407] As described above, not only can two or more types of exopolynucleotides be inserted into the toolbox at any desired time, but also can two or more types of exopolynucleotides already present in the toolbox be deleted or replaced. In an embodiment, the toolbox can include loxp, loxp mutants, rox and attP.

[0408] For example, a first exopolynucleotide consisting of a rox variant (that pairs with a rox contained in the toolbox) and Cas9 can be provided to a cell that contains the toolbox, in which case insertion of the first exopolynucleotide into the toolbox can occur via interaction with rox and Dre specific for that rox variant.

[0409] In another example, a first exopolynucleotide consisting of a rox variant (paired with rox contained in the toolbox) and Cas9, and a second exopolynucleotide consisting of attB and gRNA (paired with attP contained in the toolbox) can be provided to a cell containing the toolbox. In this case, the insertion of the first exopolynucleotide and the second exopolynucleotide into the toolbox can occur through interaction with Dre (specific for rox and its rox variant) and with PhiC31 (specific for attP and attB). In this case, Cas9 and gRNA can be expressed simultaneously in a cell containing the toolbox, so that the CRISPR / enzyme system can function even when Cas9 or gRNA is not delivered separately.

[0410] Additionally, in another example, a first exopolynucleotide consisting of a rox variant (paired with rox contained in the toolbox) and Cas9, and a second exopolynucleotide present between loxp or a loxp variant paired with loxp and a loxp variant contained in the toolbox can be provided to a cell containing the toolbox. In this case, the insertion of the first exopolynucleotide into the toolbox can occur through interaction with rox and Dre specific to the rox variant, and the exchange of the second exopolynucleotide can occur through interaction with loxp and Cre specific to the loxp variant.

[0411] [3-6. Site-specific recombination in a desired toolbox among multiple toolboxes] As described above, when an RRS contained in any one toolbox present in an animal genome has a different sequence from an RRS contained in a different toolbox in the same animal genome, site-specific recombination can occur in a toolbox located at a desired locus.

[0412] For example, an exopolynucleotide containing RRS1 or its variant that pairs with RRS1 of a first toolbox can be provided to a transformed cell containing an animal genome that contains a first toolbox containing RRS1 and a second toolbox containing RRS2, in which case insertion of the exopolynucleotide into the first toolbox can occur through interaction with SSR1 specific for RRS1 or its variant.

[0413] In another example, an exopolynucleotide located between RRS1 or its RRS1 variant paired with RRS1 of a toolbox and RRS2 or its RRS2 variant paired with RRS2 of a first toolbox can be provided to a transformed cell having an animal genome including a first toolbox containing RRS1 and RRS2 and a second toolbox containing RRS1 and RRS3. In this case, exchange between the polynucleotide located between RRS1 and RRS2 of the first toolbox and the exopolynucleotide located between RRS1 or its RRS1 variant and RRS2 or its RRS2 variant can occur through interaction with SSR1 specific to RRS1 or its RRS1 variant and interaction with SSR2 specific to RRS2 or its RRS2 variant.

[0414] Additionally, in another example, an exopolynucleotide containing an RRS1 of a first toolbox, or an RRS1 that pairs with an RRS1 variant thereof, can be provided to a transformed cell having an animal genome that includes a first toolbox containing two or more RRS1s and a second toolbox containing an RRS2.

[0415] In this case, RRS1 or its variant can interact with SSR1 specific to RRS1 or its variant. Additionally, after the polynucleotide located between two RRS1 in the first toolbox is deleted, an insertion of an exopolynucleotide containing RRS1 or its variant into the first toolbox can occur.

[0416] 4. Transformed cells with edited toolboxes The transformed cell may comprise at least one toolbox. Any one of the at least one toolbox may comprise at least one RRS. The at least one RRS may be used to enable transformation via site-specific recombination.

[0417] The transformed cell may comprise an edited toolbox, which refers to a toolbox in which site-specific recombination has occurred.

[0418] [4-1. Single cells] [4-1-1. Polyploidy] The transformed cell containing at least one edited toolbox may be a diploid cell. The diploid cell is as described above.

[0419] The transformed cell comprising at least one edited toolbox can be a haploid cell. A haploid cell is as described above. [4-1-2. Zygosity] A transformed cell containing two or more edited toolboxes can be homozygous. A transformed cell containing two or more edited toolboxes can be heterozygous. [4.2 Cell colonies] The transformed cells containing at least one edited toolbox are capable of forming cell colonies.

[0420] [4-2-1. Homologous cell colonies] A characteristic of homologous cell colonies is that the toolboxes contained in each cell are identical to each other, and the edited toolboxes contained in each cell are also identical to each other. [4-2-2. Chimeric cell colonies] A chimeric cell colony refers to a cell colony other than a homologous cell colony. [5. Selection of transformed cells into which the edited toolbox is inserted] [5-1. Selection of transformed cells using fluorescent proteins] The compiled toolbox may include a polynucleotide encoding a fluorescent protein.

[0421] For example, an exopolynucleotide encoding a fluorescent protein can be inserted into a toolbox containing at least one RRS using site-specific recombination.

[0422] Thus, animal cells containing an edited toolbox can be distinguished from animal cells that do not contain an edited toolbox.

[0423] [5-2. Selection of transformed cells using antibiotic resistance genes] The edited toolbox can include an antibiotic resistance gene. The animal genome can include the edited toolbox.

[0424] For example, an exopolynucleotide encoding an antibiotic resistance gene can be inserted into a toolbox containing at least one RRS using site-specific recombination.

[0425] The animal cells containing the edited toolbox can survive when the animal cells are treated with an antibiotic, and therefore the animal cells containing the edited toolbox can be separated from the animal cells that do not contain the edited toolbox.

[0426] [5-3. Selection of transformed cells using antigen-antibody reactions] The edited toolbox may comprise a polynucleotide encoding an antigen or a nucleotide capable of acting on an antigen. The animal genome may comprise the edited toolbox.

[0427] For example, an exopolynucleotide containing nucleotides capable of acting as an antigen can be inserted into a toolbox containing at least one RRS using site-specific recombination.

[0428] Animal cells containing the edited toolbox can interact with an antibody specific to the antigen, and therefore can be distinguished from animal cells that do not contain the edited toolbox.

[0429] [5-4. Selection of transformed cells using surface marker genes] The edited toolbox can comprise a polynucleotide encoding a surface marker. The animal cell can comprise the edited toolbox.

[0430] For example, site-specific recombination can be used to insert a polynucleotide encoding a surface marker into a toolbox that contains at least one RRS.

[0431] The animal cells containing the edited toolbox can interact with an antibody specific to the surface marker. The antibody can interact with a magnetic particle or a fluorophore. Thus, the animal cells containing the edited toolbox can be distinguished from the animal cells not containing the edited toolbox through magnetic properties or fluorescent signals.

[0432] [5-5. Selection of transformed cells using suicide genes] The toolbox may include a polynucleotide encoding a suicide gene. The edited toolbox may not include a polynucleotide encoding a suicide gene.

[0433] For example, the core domain of the toolbox may comprise loxp, a suicide gene, and a loxp variant in succession, in which case an exopolynucleotide comprising a loxp at its 5' end and a loxp variant at its 3' end may be exchanged for the suicide gene by site-specific recombination.

[0434] Since the animal cells containing the edited toolbox do not contain the suicide gene, apoptosis does not occur in these animal cells even when the prodrug is provided, and therefore the animal cells containing the edited toolbox can be distinguished from the animal cells not containing the edited toolbox.

[0435] [6. Transgenic animals into which the edited toolbox is inserted] [6-1. Individual transgenic animals into which the edited toolbox is inserted] [6-1-1.Homology] Each cell contained in the homologous transgenic animal can include at least one toolbox. The at least one toolbox can include at least one edited toolbox.

[0436] [6-1-2. Chimera] A chimeric transgenic animal refers to a transgenic animal other than a homologous transgenic animal.

[0437] [6-2. Method for preparing transgenic animals with edited toolboxes inserted] Methods for preparing a transgenic animal having an edited toolbox inserted therein may include methods for preparing a transgenic animal from an animal cell, methods for preparing a transgenic animal by delivery of an exopolynucleotide to an animal tissue or organ, or introduction of an SSR, and methods for preparing a transgenic animal by mating between transgenic animals. Methods for preparing a transgenic animal from an animal cell may include methods for preparing a transgenic animal from a cell of a transgenic animal into which a toolbox comprising at least one RRS is inserted.

[0438] The transgenic animals produced by any one of the above methods may be any one of chimeric or homologous transgenic animals.

[0439] [6-2-1. Methods for preparing transgenic animals from animal cells] A transgenic animal in which an edited toolbox is inserted can be prepared from cells of a transgenic animal in which a toolbox containing at least one RRS has been inserted.

[0440] For example, the transgenic animal can be prepared by introducing an SSR into a somatic cell into which a toolbox containing at least one RRS is inserted, followed by SCNT. In another example, the transgenic animal can be prepared by introducing a polynucleotide containing an SSR and at least one RRS into a somatic cell into which a toolbox containing at least one RRS is inserted by somatic cell microinjection, followed by SCNT. The transgenic animal can be a homologous transgenic animal.

[0441] For example, a transgenic animal can be prepared by introducing a polynucleotide comprising an SSR into a gamete into which a toolbox comprising at least one RRS is inserted via gamete microinjection. In another example, a transgenic animal can be prepared by introducing a polynucleotide comprising an SSR and a polynucleotide comprising at least one RRS into a gamete into which a toolbox comprising at least one RRS is inserted via gamete microinjection. The transgenic animal can be a chimeric transgenic animal.

[0442] For example, a transgenic animal can be prepared by introducing an SSR into a zygote into which a toolbox containing at least one RRS is inserted. In another example, a transgenic animal can be prepared by introducing a polynucleotide containing at least one RRS into a zygote into which a toolbox containing at least one RRS is inserted via zygote microinjection while treating the zygote with an SSR. The transgenic animal can be a chimeric transgenic animal.

[0443] For example, a transgenic animal can be prepared by introducing an SSR into an embryo into which a toolbox containing at least one RRS is inserted. In another example, a transgenic animal can be prepared by introducing a polynucleotide encoding an SSR and a polynucleotide containing at least one RRS via embryo microinjection into an embryo into which a toolbox containing at least one RRS is inserted. The transgenic animal can be a chimeric transgenic animal.

[0444] [6-2-2. Methods for preparing transgenic animals by delivery of exopolynucleotides or introduction of SSRs into animal tissues or organs]

[0445] The animal tissue or organ can contain cells into which a toolbox containing at least one RRS is inserted. Transgenic animals containing edited toolboxes can be prepared by introducing SSRs or by delivery of exopolynucleotides into the animal tissue or organ.

[0446] For example, a transgenic animal containing an edited toolbox can be prepared by microinjecting a polynucleotide encoding an SSR and a polynucleotide containing at least one RRS into the mammary tissue of the animal. The transgenic animal can be a chimeric transgenic animal.

[0447] For example, a transgenic animal in which an edited toolbox is inserted can be prepared by microinjecting an SSR into the reproductive organs of an animal. The offspring obtained from the gametes of the transgenic animal can be a transgenic animal. The transgenic animal can be a chimeric transgenic animal.

[0448] [6-2-3. Mating of transgenic animals] A transgenic animal can be prepared through breeding between a first transgenic animal and a second transgenic animal.

[0449] For example, a transgenic animal containing an edited toolbox can be prepared by mating between a first transgenic animal into which a toolbox containing at least one RRS is inserted and a second transgenic animal into which a toolbox containing a polynucleotide encoding an SSR is inserted.

[0450] The second transgenic animal may be the offspring of the first transgenic animal or may be related to the first transgenic animal. Alternatively, the second transgenic animal may be unrelated to the first transgenic animal.

[0451] The transgenic animal obtained by mating may contain a toolbox that is identical to a portion of the toolbox contained in the animal genome of the first transgenic animal, in the same location.

[0452] The transgenic animal obtained by mating may contain a toolbox that is identical to a part of the toolbox contained in the animal genome of the second transgenic animal at the same location. The transgenic animal obtained by mating may be a homologous transgenic animal.

[0453] The transgenic animals obtained by mating may contain transformed cells that are homozygous. The transgenic animals obtained by mating may contain transformed cells that are heterozygous.

[0454] 7. Use of transgenic animals into which edited toolboxes are inserted [7-1. Improved Animals] The transgenic animals into which the edited toolbox is inserted can be used as bred animals.

[0455] [7-2. Animal models of disease] Transgenic animals into which the edited toolbox is inserted can be used as animal models of disease.

[0456] [7-3. Disease-resistant animals] The transgenic animals into which the edited toolbox is inserted can be used as disease-resistant animals.

[0457] [7-4. Use of by-products] The parts of the transgenic animal into which the edited toolbox is inserted may include, but are not limited to, organs, flesh, skin, hair, and body fluids. [7-5. Bioreactor] Transgenic animals into which the edited toolbox has been inserted can be used as bioreactors.

[0458] [Part 4 Transformation using CRISPR / enzyme system components] 1. Introduction of RNA-guided endonucleases and guide nucleic acids into cells containing the toolbox RNA-guided endonucleases and guide nucleic acids can be introduced into animal genomes in cells that contain a toolbox for site-specific transformation.

[0459] The RNA-guided endonuclease may include, but is not limited to, Cas9. The guide nucleic acid may include, but is not limited to, a gRNA. [1-1. Introduction of isolated forms] Cas9 and gRNA can be introduced into cells that contain the toolbox in an isolated form.

[0460] The form in which Cas9 is provided may include DNA plasmids, DNA linear fragments, RNA linear fragments, and proteins. The RNA linear fragments may include the mRNA of Cas9.

[0461] The form in which the gRNA is provided may include a DNA plasmid, a DNA linear fragment, or an RNA linear fragment.

[0462] The form in which Cas9 and gRNA are provided together may include a ribonucleoprotein (RNP).

[0463] [1-2. Single delivery vector] Cas9 and gRNA can be provided to the cell containing the toolbox as a single delivery vector.

[0464] The form in which the Cas9 and gRNA are provided may include a DNA linear fragment and an RNA linear fragment.

[0465] [2. CRISPR / enzyme system components in the toolbox] The toolbox contained in the animal genome in the cell may include at least one of an RNA-guided endonuclease and a guide nucleic acid.

[0466] The cells in which site-specific transformation occurs can express all or some of the components of the CRISPR / enzyme system, facilitating site-specific transformation.

[0467] [2-1. RNA-guided endonucleases in the toolbox] The toolbox may include at least one polynucleotide encoding an RNA-guided endonuclease. The RNA-guided endonuclease may include, but is not limited to, Cas9 or a Cas9 mutant. [2-1-1. Cas9 and promoter combination] The toolbox may comprise at least one polynucleotide encoding Cas9.

[0468] The toolbox may include a promoter capable of initiating Cas9 transcription. The promoter may be any one selected from a constitutive promoter, a tissue-specific promoter, and an inducible promoter.

[0469] [2-1-2. Combination of Cas9, promoter and RRS] The toolbox may include at least one polynucleotide encoding Cas9. The toolbox may include a promoter capable of initiating transcription of Cas9. The toolbox may include at least one RRS.

[0470] The toolbox can include at least one RRS at the 5' end of a promoter capable of initiating transcription of Cas9.

[0471] The toolbox may include at least one RRS between a promoter capable of initiating transcription of Cas9 and a polynucleotide encoding Cas9. The toolbox may include an RRS at the 3' end of the polynucleotide encoding Cas9.

[0472] The toolbox may include at least one RRS at the 5' end of the polynucleotide encoding Cas9, and at least one RRS at the 3' end of the polynucleotide encoding Cas9. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can interact with the same SSR. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can be identical to each other.

[0473] [2-2. Guide nucleic acids in the toolbox] The toolbox may include at least one polynucleotide encoding a guide nucleic acid. The guide nucleic acid may include, but is not limited to, a gRNA. [2-2-1. gRNA and promoter combination] The toolbox may include at least one polynucleotide encoding a gRNA.

[0474] The toolbox may include a promoter capable of initiating the transcription of gRNA. The promoter may be any one selected from a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The constitutive promoter capable of initiating the transcription of gRNA may include, but is not limited to, the U6 promoter. [2-2-2. Combination of gRNA, promoter, and RRS] The toolbox may include at least one polynucleotide encoding a gRNA.

[0475] The toolbox may include a promoter capable of initiating transcription of the gRNA. The toolbox may include at least one RRS.

[0476] The toolbox may include at least one RRS at the 5' end of a promoter capable of initiating transcription of a gRNA.

[0477] The toolbox may include at least one RRS between a promoter capable of initiating transcription of the gRNA and a polynucleotide encoding the gRNA. The toolbox may include an RRS at the 3' end of the polynucleotide encoding the gRNA.

[0478] The toolbox may comprise at least one RRS at the 5' end of the polynucleotide encoding the gRNA, and at least one RRS at the 3' end of the polynucleotide encoding the gRNA. In this case, one of the RRSs at the 5' end and one of the RRSs at the 3' end can interact with the same SSR. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can be identical to each other.

[0479] [2-3. RNA-guided endonucleases and guide nucleic acids in the toolbox] The toolbox can include at least one polynucleotide that encodes an RNA-guided endonuclease, and can include at least one polynucleotide that encodes a guide nucleic acid.The RNA-guided endonuclease can include, but is not limited to, Cas9.The guide nucleic acid can include, but is not limited to, gRNA. [2-3-1. Combination of Cas9, gRNA and promoter] The toolbox may comprise at least one polynucleotide encoding Cas9.

[0480] The toolbox may include a promoter capable of initiating the transcription of Cas9. The promoter may be any one selected from a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The toolbox may include at least one polynucleotide encoding a gRNA.

[0481] The toolbox may include a promoter capable of initiating the transcription of gRNA. The promoter may be any one selected from a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The constitutive promoter capable of initiating the transcription of gRNA may include, but is not limited to, the U6 promoter.

[0482] [2-3-2. Combination of Cas9, gRNA, promoter and RRS] The toolbox may comprise at least one polynucleotide encoding Cas9. The toolbox may comprise a promoter capable of initiating the transcription of Cas9. The toolbox may comprise at least one polynucleotide encoding gRNA. The toolbox may comprise a promoter capable of initiating the transcription of gRNA. The toolbox may comprise at least one RRS.

[0483] The toolbox can include at least one RRS at the 5' end of a promoter capable of initiating transcription of Cas9.

[0484] The toolbox may include at least one RRS between a promoter capable of initiating transcription of Cas9 and a polynucleotide encoding Cas9. The toolbox may include an RRS at the 3' end of the polynucleotide encoding Cas9.

[0485] The toolbox may include at least one RRS at the 5' end of the polynucleotide encoding Cas9, and at least one RRS at the 3' end of the polynucleotide encoding Cas9. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can interact with the same SSR. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can be identical to each other.

[0486] The toolbox may include at least one RRS at the 5' end of a promoter capable of initiating transcription of a gRNA.

[0487] The toolbox may include at least one RRS between a promoter capable of initiating transcription of the gRNA and a polynucleotide encoding the gRNA. The toolbox may include an RRS at the 3' end of the polynucleotide encoding the gRNA.

[0488] The toolbox may comprise at least one RRS at the 5' end of the polynucleotide encoding the gRNA, and at least one RRS at the 3' end of the polynucleotide encoding the gRNA. In this case, one of the RRSs at the 5' end and one of the RRSs at the 3' end can interact with the same SSR. In this case, any one of the RRSs at the 5' end and any one of the RRSs at the 3' end can be identical to each other.

[0489] [3. Regulation of CRISPR / enzyme function] [3.1 Regulation of RNA-guided endonuclease expression] The toolbox may include at least one polynucleotide encoding an RNA-guided endonuclease. The RNA-guided endonuclease may include, but is not limited to, Cas9. [3-1-1. Regulation of Cas9 transcription using promoters] The toolbox may comprise a polynucleotide encoding Cas9.

[0490] The toolbox can include a promoter that initiates transcription of a polynucleotide encoding Cas9. The promoter can include a tissue-specific promoter or an inducible promoter.

[0491] If the promoter is a tissue-specific promoter, transcription of the polynucleotide encoding Cas9 contained in the toolbox can be initiated when the toolbox is contained in cells of a particular tissue.

[0492] If the tissue-specific promoter is a mammary tissue-specific promoter, transcription of a polynucleotide encoding Cas9 contained in the toolbox can be initiated when the toolbox is contained in a cell of the mammary tissue. Mammary tissue-specific promoters can include, but are not limited to, an alpha-casein promoter, a beta-casein promoter, a kappa-casein promoter, a mu-casein promoter, and a beta-lactoglobulin promoter.

[0493] When the tissue-specific promoter is a reproductive organ-specific promoter, the transcription of the polynucleotide encoding Cas9 contained in the toolbox can be initiated when the toolbox is contained in a gamete.Reproductive organ-specific promoters can include, but are not limited to, ovary-specific promoters and testis-specific promoters.

[0494] If the promoter that initiates transcription of the polynucleotide encoding Cas9 is a tissue-specific promoter, the site at which site-specific transformation occurs in the transgenic animal containing the toolbox can be restricted. Additionally, unwanted site-specific transformation in other tissues of the transgenic animal can be prevented.

[0495] If the promoter is an inducible promoter, transcription can be initiated when certain conditions are met. Inducible promoters can include, but are not limited to, chemically inducible promoters, temperature inducible promoters, and light inducible promoters.

[0496] A chemically inducible promoter can initiate transcription when a particular chemical compound is present. Chemically inducible promoters can include, but are not limited to, antibiotic inducible promoters, alcohol inducible promoters, steroid inducible promoters, and metal inducible promoters. Antibiotic inducible promoters can include, but are not limited to, Tet-on promoters, Tet-off promoters. Steroid inducible promoters can include, but are not limited to, estrogen inducible promoters. Metal inducible promoters can include, but are not limited to, copper inducible promoters.

[0497] When the temperature condition is met, the temperature-inducible promoter can start transcription.The temperature-inducible promoter can include, but is not limited to, a high-temperature-inducible promoter and a low-temperature-inducible promoter.The high-temperature-inducible promoter can include, but is not limited to, an Hsp promoter.

[0498] When the light wavelength conditions are met, the light-inducible promoter can initiate transcription.

[0499] If the promoter that initiates transcription of the polynucleotide encoding Cas9 is an inducible promoter, the time at which site-specific transformation occurs in transformed cells or animals containing the toolbox can be controlled. [3-1-2. Insertion of a promoter using RRS] The toolbox may comprise a polynucleotide encoding Cas9. The toolbox may comprise an RRS at the 5' end of the polynucleotide encoding Cas9.

[0500] The nucleic acid may comprise a polynucleotide encoding a promoter. The promoter may comprise a constitutive promoter, a tissue-specific promoter, or an inducible promoter. The nucleic acid may comprise an RRS at one or both ends of the polynucleotide encoding the promoter. The RRS may be paired with an RRS included in the toolbox.

[0501] The SSR and RRS that can interact with nucleic acid can be provided in the toolbox.The polynucleotide that codes for promoter can be inserted into the toolbox.In this case, the transcription of the polynucleotide that codes for Cas9 can not be initiated before the polynucleotide that codes for promoter is inserted, and can be initiated after the polynucleotide that codes for promoter is inserted.

[0502] [3-1-3. Regulation of Cas9 transcription using transcription termination codons] The toolbox can include a polynucleotide encoding Cas9. The toolbox can include a promoter that initiates transcription of the polynucleotide encoding Cas9. The promoter can be a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0503] The toolbox may include RRS1, a transcription termination codon, and RRS2. RRS1, a transcription termination codon, and RRS2 may be located consecutively between the polynucleotide encoding Cas9 and the promoter that starts the transcription of the polynucleotide encoding Cas9. RRS1 and RRS2 may be identical to each other. RRS1 and RRS2 may include, but are not limited to, loxp.

[0504] SSR that can interact with RRS1 and RRS2 can be provided in the toolbox. SSR can include but is not limited to Cre. In the toolbox, the polynucleotide that comprises the transcription stop codon located between RRS1 and RRS2 can be deleted. In this case, the mRNA that is transcribed from the polynucleotide that codes Cas9 can not be transcribed before the transcription stop codon is deleted, and can be transcribed after the transcription stop codon is deleted. [3-1-4. Regulation of Cas9 translation using stop codons] The toolbox may comprise a polynucleotide encoding Cas9.

[0505] The toolbox can include a promoter that initiates transcription of a polynucleotide encoding Cas9. The promoter can be a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0506] The toolbox may include RRS1, a stop codon, and RRS2. RRS1, a transcription stop codon, and RRS2 may be located consecutively between the promoter that starts the transcription of the polynucleotide that codes for Cas9 and the polynucleotide that codes for Cas9. RRS1 and RRS2 may be identical to each other. RRS1 and RRS2 may include, but are not limited to, loxp.

[0507] SSR that can interact with RRS1 and RRS2 can be provided in the toolbox. SSR can include but is not limited to Cre recombinase. In the toolbox, the polynucleotide that includes the stop codon located between RRS1 and RRS2 can be deleted. In this case, the mRNA that is transcribed from the polynucleotide that codes Cas9 can not be transcribed before the stop codon is deleted, and can be transcribed after the stop codon is deleted.

[0508] [3-2. Regulation of guide nucleic acid expression] The toolbox may include at least one polynucleotide encoding a guide nucleic acid. The guide nucleic acid may include, but is not limited to, a gRNA. [3-2-1. Promoter insertion using RRS] The toolbox may include a polynucleotide encoding a gRNA. The toolbox may include an RRS at the 5' end of the polynucleotide encoding the gRNA.

[0509] The nucleic acid may comprise a polynucleotide encoding a promoter. The promoter may comprise a constitutive promoter, a tissue-specific promoter, or an inducible promoter. The constitutive promoter may comprise a U6 promoter. The nucleic acid may comprise an RRS at one or both ends of the polynucleotide encoding the promoter. The RRS may be paired with an RRS included in the toolbox.

[0510] The SSR and RRS that can interact with nucleic acid can be provided in the toolbox.The polynucleotide that codes for a promoter can be inserted into the toolbox.In this case, the transcription of the polynucleotide that codes for gRNA can not be initiated before the promoter is inserted, and can be initiated after the promoter is inserted. [3-2-2. Regulation of gRNA transcription] The toolbox may include a polynucleotide encoding a gRNA.

[0511] The toolbox may include a promoter that initiates transcription of a polynucleotide encoding the gRNA. The promoter may be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. The constitutive promoter may include a U6 promoter.

[0512] The toolbox may include RRS1, a transcription termination codon, and RRS2. The transcription termination codon may include a polyT sequence. The transcription termination codon may include an AATAAA sequence. RRS1, a transcription termination codon, and RRS2 may be located consecutively between a promoter that initiates transcription of a polynucleotide encoding a gRNA and a polynucleotide encoding a gRNA. RRS1 and RRS2 may be identical to each other. RRS1 and RRS2 may include, but are not limited to, loxp.

[0513] The SSR that can interact with RRS1 and RRS2 can be provided in the toolbox. The SSR can include, but is not limited to, Cre recombinase. In the toolbox, the polynucleotide that includes the transcription termination codon located between RRS1 and RRS2 can be deleted.

[0514] In this case, the polynucleotide encoding the gRNA may not be expressed before the transcription stop codon is deleted, and may be expressed after the transcription stop codon is deleted. [4. CRISPR / enzyme target site] The animal genome in the cell may comprise at least one toolbox.

[0515] The at least one toolbox may include a first toolbox. The first toolbox may include a polynucleotide encoding a guide nucleic acid.

[0516] Alternatively, the first toolbox may comprise a polynucleotide encoding an RNA-guided endonuclease but not a polynucleotide encoding a guide nucleic acid, in which case the guide nucleic acid or a polynucleotide encoding the guide nucleic acid may be introduced into the cell by a separate delivery vector.

[0517] The guide nucleic acid may comprise a protospacer domain. The protospacer domain may comprise a nucleotide sequence that is identical to or capable of complementary binding to a target site located in the animal genome or exopolynucleotide in the cell.

[0518] The RNA-guided endonuclease may include, but is not limited to, Cas9. The guide nucleic acid may include, but is not limited to, a gRNA.

[0519] [4-1. Animal genomes] The animal genome in the cell can include a target site of the gRNA. The target site can be a nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence. The PAM sequence can include, but is not limited to, NGG.

[0520] The target site may be located in a polynucleotide that is involved in the expression of a polypeptide or in an RNA in the animal genome in the cell.

[0521] The target site may be located in any one of the promoter, 5'UTR, exon, intron, 3'UTR of a polynucleotide encoding a polypeptide or RNA in the animal genome in the cell.

[0522] In this case, the amount of polypeptide or RNA expression in the cell can be affected via the CRISPR / enzyme system. The target site can be located in a safe harbor of the animal genome in the cell.

[0523] When the animal genome is a mouse genome, the safe harbor for the mouse genome may include the already widely known rosa26 locus.

[0524] Where the animal genome is a bovine genome, the bovine genome safe harbor may include, but is not limited to, the loci in Table 1.

[0525] In this case, through the CRISPR / enzyme system it is possible to perform site-specific transformation that has no lethal effects on the above cells and on the transgenic animals containing these cells. [4-2. Exopolynucleotides] At least one toolbox contained in the animal genome may include a target toolbox.

[0526] As used herein, the term "target toolbox" may refer to a toolbox that contains as components target sites that can be recognized by components of an engineered nuclease.

[0527] The target toolbox may include at least one target site for the gRNA. The target site may be a nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence. The PAM sequence may include, but is not limited to, NGG. The target toolbox may be the same toolbox as the first toolbox. The target toolbox may be a toolbox different from the first toolbox.

[0528] The target site may be located in any one of the promoter, 5'UTR, exon, intron, 3'UTR of a polynucleotide encoding a polypeptide or RNA in the target toolbox.

[0529] The polypeptide may comprise a portion of a target protein, in which case the amount of expression of the target protein in cells containing the targeting toolbox, and in transgenic animals containing these cells, may be affected through the CRISPR / enzyme system.

[0530] The polypeptide may include the polypeptide expressed by a marker gene. In this case, the marker gene can be knocked out through CRISPR / enzyme system, and can be used to select transformed cells. For example, when the targeting toolbox contained in the animal genome in the cell contains a polynucleotide encoding thymidine kinase, the thymidine kinase can be knocked out through site-specific transformation by CRISPR / enzyme system, which targets a part of the exon of thymidine kinase as a targeting site. The cells that have undergone site-specific transformation can be selected by treating the cells that contain the targeting toolbox with ganciclovir.

[0531] The polypeptide may comprise a portion of a recombinase, in which case site-specific recombination in a cell can be inhibited by knocking out the polynucleotide encoding the recombinase through the CRISPR / enzyme system.

[0532] The polypeptide can include a transposase, in which case transposon insertion into or deletion from a cell can be inhibited by knocking out the polynucleotide encoding the transposase through a CRISPR / enzyme system.

[0533] The polypeptide may comprise a part of an RNA-guided endonuclease. In this case, the site-specific transformation in the cell can be prevented by knocking out the RNA-guided endonuclease through the CRISPR / enzyme system. For example, when the target toolbox contained in the animal genome in the cell comprises a polynucleotide encoding Cas9, Cas9 can be knocked out through site-specific transformation by the CRISPR / enzyme system, which targets a part of the exon of Cas9 as the target site. In the cell that comprises the toolbox, the site-specific transformation in the target toolbox is simultaneously performed, and the occurrence of site-specific transformation at a position other than the target site (off-target activity) can be prevented by reducing Cas9 expression in the cell.

[0534] The RNA may comprise a portion of the guide nucleic acid. In this case, site-specific transformation in the cell can be prevented by knocking out the guide nucleic acid through the CRISPR / enzyme system. For example, when the target toolbox contained in the animal genome in the cell comprises a polynucleotide encoding a gRNA, the gRNA can be knocked out through site-specific transformation by the CRISPR / enzyme system that targets the protospacer domain of the gRNA as the target site. In a cell that comprises a toolbox, it is possible to prevent the occurrence of site-specific transformation at a position other than the target site (off-target activity) by reducing gRNA expression in the cell while simultaneously performing site-specific transformation in the target toolbox. Additionally, when the cell that comprises the target toolbox can express an RNA-guided endonuclease, it is possible to prevent off-target activity without affecting the expression of the RNA-guided endonuclease.

[0535] The target site may be located in a polynucleotide encoding a non-functional polypeptide in the target toolbox. Alternatively, the target site may be located in a polynucleotide encoding an untranslated RNA in the target toolbox. Alternatively, the target site may be located in a non-transcribed polynucleotide in the target toolbox.

[0536] When the animal genome in a cell contains two or more of the target toolboxes that are identical to each other, it is possible to carry out multiple identical site-specific transformations through a single CRISPR / enzyme system.For example, when the target toolbox containing the bovine genome in a cell contains a non-transcribed polynucleotide, it is possible to carry out site-specific insertion of a polynucleotide encoding omega-3 through a CRISPR / enzyme system that targets a part of the non-transcribed polynucleotide as a target site.When the bovine genome contains two or more of the target toolboxes, it is possible to prepare cells or transgenic bovines with high expression of omega-3, since a polynucleotide encoding omega-3 can be inserted into each target toolbox.

[0537] When the animal genome in the cell contains two or more of the targeting toolboxes that are different from each other, the guide nucleic acid can carry out multiple different site-specific transformations through different CRISPR / enzyme systems. For example, the bovine genome in the cell can contain a first targeting toolbox and a second targeting toolbox. The first targeting toolbox can contain a first target site, and the second targeting toolbox can contain a second target site. The nucleotide sequence of the first target site can be different from the second target site. A polynucleotide encoding a human immunoglobulin heavy chain can be knocked in at the first toolbox through a CRISPR / enzyme system that includes a first gRNA that is identical to or can be complementary to the first target site. A polynucleotide encoding a human immunoglobulin light chain can be knocked in at the second toolbox through a CRISPR / enzyme system that includes a second gRNA that is identical to or can be complementary to the second target site. A cell containing both the first and second targeting toolboxes, or a transgenic cow containing the cells, can produce human antibodies by expressing both a human immunoglobulin heavy chain and a human immunoglobulin light chain.

[0538] [5. Site-specific transformation using CRISPR / enzyme system] The animal genome in the cell may include at least one toolbox. The at least one toolbox may include at least one of a first toolbox, a second toolbox, and a third toolbox.

[0539] The first toolbox may comprise at least one polynucleotide encoding an RNA-guided endonuclease, in which case site-specific transformation can be performed by the CRISPR / enzyme system via delivery of a guide nucleic acid, or a polynucleotide encoding the guide nucleic acid, to a cell containing the toolbox.

[0540] The second toolbox may comprise at least one polynucleotide encoding a guide nucleic acid. In this case, it is possible to carry out site-specific transformation through the CRISPR / enzyme system by introducing an RNA-guided endonuclease or by delivering a polynucleotide encoding an RNA-guided endonuclease into the cell comprising the toolbox.

[0541] The third toolbox may comprise at least one polynucleotide encoding an RNA-guided endonuclease and at least one polynucleotide encoding a guide nucleic acid, in which case site-specific transformation can be performed in the cell through the CRISPR / enzyme system.

[0542] The animal genome in the cell comprising at least one toolbox can include a target site for the guide nucleic acid. The target site can be a nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence.

[0543] At least one toolbox may include a target toolbox. The target toolbox may include a target site of the guide nucleic acid. The target site may be a nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence. The target toolbox may be the same toolbox as the first toolbox. Alternatively, the target toolbox may be a toolbox different from the first toolbox.

[0544] The RNA-guided endonuclease may include, but is not limited to, Cas9. The guide nucleic acid may include, but is not limited to, a gRNA.

[0545] [5-1. Non-homologous end joining (NHEJ)] When both strands of DNA are broken, i.e., a double-stranded break occurs, the rejoining of the broken DNA strands by DNA ligase is called non-homologous end joining (NHEJ).

[0546] The animal genome in the cell can include at least one of the first toolbox, the second toolbox, and the third toolbox.Cas9 can be expressed in the cell or delivered to the cell.gRNA can be expressed in the cell or delivered to the cell.

[0547] The gRNA can complementarily bind to a target site in the animal genome or targeting toolbox in a cell. Cas9 can interact with the gRNA, thereby causing a double-stranded break at the target site.

[0548] In this case, nucleotide or polynucleotide can be inserted during non-homologous end joining (NHEJ) process. Alternatively, nucleotide or polynucleotide can be deleted during non-homologous end joining (NHEJ) process. Due to insertion or deletion, modification can occur in the nucleotide sequence at the target site.

[0549] [5-2. Homologous recombination] The animal genome in the cell can include at least one of the first toolbox, the second toolbox, and the third toolbox.Cas9 can be expressed in the cell or delivered to the cell.gRNA can be expressed in the cell or delivered to the cell.

[0550] The donor polynucleotide or the donor can be delivered to the cell. The first homology arm can be located at the 5' end of the donor. The second homology arm can be located at the 3' end of the donor.

[0551] The first homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell, and the second homology arm may have a nucleotide sequence identical to a portion of the animal genome in the cell, in which case the donor polynucleotide may be inserted in the animal genome in the cell between the nucleotide sequence identical to the first homology arm and the nucleotide sequence identical to the second homology arm.

[0552] Alternatively, the first homology arm may have a nucleotide sequence identical to a portion of the target toolbox in the cell, and the second homology arm may have a nucleotide sequence identical to a portion of the target toolbox in the cell. In this case, the donor may be inserted in the target toolbox in the cell between the nucleotide sequence identical to the first homology arm and the nucleotide sequence identical to the second homology arm. The donor may include a toolbox.

[0553] [5-3.Homology-independent targeted insertion (HITI)] The animal genome in the cell can include at least one of the first toolbox, the second toolbox, and the third toolbox.Cas9 can be expressed in the cell or delivered to the cell.gRNA can be expressed in the cell or delivered to the cell.Donor can be delivered to the cell.

[0554] The target site having the same nucleotide sequence as that located at the target site in the animal genome in the cell can be located at the 5' end and 3' end of the donor.In this case, the CRISPR / enzyme system can generate double-strand breaks at the target site located at the animal genome in the cell, at the target site at the 5' end of the donor, and at the target site at the 3' end of the donor.The donor can be inserted between the double-strand breaks of the animal genome through non-homologous end joining (NHEJ).

[0555] Alternatively, the target site having the same nucleotide sequence as that located in the target toolbox in the cell can be located at the 5'-end and 3'-end of the donor.In this case, the CRISPR / enzyme system can generate double-strand breaks at the target site located in the target toolbox in the cell, at the target site at the 5'-end of the donor, and at the target site at the 3'-end of the donor.Through non-homologous end joining (NHEJ), the donor can be inserted between the double-strand breaks of the target toolbox.The donor can include a toolbox.

[0556] [5-4. Knock-in (Knock-in)] The animal genome in the cell can include at least one of the first toolbox, the second toolbox, and the third toolbox.Cas9 can be expressed in the cell or delivered to the cell.gRNA can be expressed in the cell or delivered to the cell.

[0557] The donor polynucleotide can be delivered to the cell. In this case, the donor can be inserted into the animal genome or into the target toolbox in the cell through homologous recombination. Alternatively, the donor can be inserted into the animal genome or into the target toolbox in the cell through HITI.

[0558] For example, at least one nucleotide of a sequence present at a target site in an animal genome or within a target toolbox in a cell can be deleted and a donor polynucleotide can be added.

[0559] In another example, a donor polynucleotide can be added to a sequence present at a target site in an animal genome or at a target site within a target toolbox in a cell.

[0560] If the donor comprises a polynucleotide encoding a protein or RNA, the polynucleotide encoding the protein or RNA can be knocked in to the cell into which the donor is inserted, thereby allowing the protein or RNA to be expressed.

[0561] [5-5. Knockout (Knockout)] The animal genome in the cell can include at least one of the first toolbox, the second toolbox, and the third toolbox.Cas9 can be expressed in the cell or delivered to the cell.gRNA can be expressed in the cell or delivered to the cell.

[0562] A donor polynucleotide can be delivered to a cell. In this case, the donor can be inserted into a target site in an animal genome or into a target site within a target toolbox in a cell through homologous recombination. Alternatively, the donor can be inserted into a target site in an animal genome or into a target site within a target toolbox in a cell through HITI.

[0563] The donor polynucleotide may not be delivered to the cell, in which case a nucleotide insertion, polynucleotide insertion, nucleotide deletion, or polynucleotide deletion may occur at the target site in the animal genome or at a target site within the targeting toolbox in the cell.

[0564] For example, at least one nucleotide present at a target site in an animal genome or within a target toolbox in a cell can be deleted.

[0565] In another example, at least one nucleotide present at a target site in an animal genome or within a target toolbox in a cell can be deleted and at least one additional nucleotide can be added thereto.

[0566] If a target site in an animal genome or within a target toolbox in a cell is located in a polynucleotide that encodes a protein or RNA, the protein or RNA can be knocked out, thereby reducing expression.

[0567] [6. Site-specific transformation of cells using the CRISPR / enzyme system] The animal genome of the transformed cell may include at least one toolbox. The at least one toolbox may include any one of a first toolbox, a second toolbox, and a third toolbox. The first toolbox may include at least one polynucleotide encoding an RNA-guided endonuclease. The second toolbox may include at least one polynucleotide encoding a guide nucleic acid. The third toolbox may include at least one polynucleotide encoding an RNA-guided endonuclease and may include at least one polynucleotide encoding a guide nucleic acid.

[0568] A site-specific transformed cell can include a cell in which site-specific transformation has occurred at a target site in the animal genome.

[0569] A site-specific transformed cell can include a cell in which site-specific transformation has occurred at a target site within a target toolbox in the cell. [6-1. Single cells] [6-1-1. Polyploidy] The site-specifically transformed cell may be a diploid cell. A diploid cell is as described above. The site-specifically transformed cell may be a haploid cell. A haploid cell is as described above.

[0570] [6-1-2. Zygosity] The site-specific transformed cells may comprise at least one pair of homologous chromosomes. The at least one pair of homologous chromosomes may comprise chromosome 1 and chromosome 2 in a homologous chromosomal relationship. The site-specific transformed cells may be homozygous.

[0571] In homozygous site-specific transformed cells, the type, number, and location of the site-specific transformations contained in chromosome 1 and chromosome 2 can all be identical.

[0572] In homozygous site-specific transformed cells, both the type and number of site-specific transformations contained on chromosome 1 and chromosome 2 can be identical.

[0573] In homozygous site-specific transformed cells, the types of site-specific transformation contained in chromosome 1 and chromosome 2 may be the same.

[0574] In homozygous site-specific transformed cells, the type, number, and location of toolboxes and site-specific transformations contained on chromosomes 1 and 2 can all be identical.

[0575] In homozygous site-specific transformed cells, both the type and number of toolboxes and site-specific transformations contained on chromosomes 1 and 2 can be identical.

[0576] In homozygous site-specific transformed cells, the type of toolbox and site-specific transformation contained in chromosome 1 and chromosome 2 may be identical.

[0577] Alternatively, in a homozygous site-specific transformed cell, both chromosome 1 and chromosome 2 may not contain any toolbox, and both chromosome 1 and chromosome 2 may not contain any site-specific transformation. A site-specific transformed cell may be heterozygous.

[0578] In a site-specific transformed cell that is heterozygous, chromosome 1 may contain no toolboxes and chromosome 2 may contain at least one toolbox.

[0579] In a heterozygous site-specific transformed cell, chromosome 1 may contain zero site-specific transformations and chromosome 2 may contain at least one site-specific transformation.

[0580] In site-specifically transformed cells that are heterozygous, the second chromosome may not contain a toolbox that is identical to that contained in the first chromosome.

[0581] In a heterozygous site-specifically transformed cell, the second chromosome may not contain the same site-specific transformation as that contained in the first chromosome. [6-2. Cell colonies] The site-specific transformed cells are capable of forming cell colonies.

[0582] [6-2-1. Homologous cell colonies] A characteristic of homologous cell colonies is that the toolboxes carried by each cell are identical to each other, and the types, numbers, and positions of site-specific transformations contained in each cell are all identical to each other. [6-2-2. Chimeric cell colonies] A chimeric cell colony refers to a cell colony other than a homologous cell colony.

[0583] For example, a chimeric cell colony may comprise both a first cell having a genome that has been site-specifically transformed at a first target site by the CRISPR / enzyme system, and a second cell that does not have a genome that has been site-specifically transformed at the first target site by the CRISPR / enzyme system.

[0584] [7. Selection of site-specific transformed cells using the CRISPR / enzyme system] [7-1. Selection of transformed cells using fluorescent proteins] The transformed cell can include at least one target toolbox. The transformed cell can include at least one toolbox. Any one of the at least one toolbox can be a target toolbox that includes a polynucleotide that encodes a fluorescent protein. For example, the polynucleotide that encodes a target protein can be inserted into the transformed cell through site-specific transformation using an exon of the polynucleotide that encodes a fluorescent protein as a target site.

[0585] Site-specifically transformed cells can differ in terms of fluorescent signal from cells in which site-specific transformation has not occurred, and therefore site-specifically transformed cells can be distinguished.

[0586] Site-specific transformed cell can contain a polynucleotide that encodes a fluorescent protein.For example, through site-specific transformation, a donor that contains a polynucleotide that encodes a fluorescent protein can be inserted into a transformed cell that contains at least one target toolbox.The donor can contain a polynucleotide that encodes a target protein.

[0587] Since the site-specifically transformed cells express the fluorescent protein, a fluorescent signal can be measured, and therefore the site-specifically transformed cells can be distinguished from animal cells in which site-specific transformation has not occurred. [7-2. Selection of transformed cells using antibiotic resistance genes] The site-specifically transformed cells may contain an antibiotic resistance gene.

[0588] For example, a donor comprising a polynucleotide encoding an antibiotic resistance gene can be inserted into a transformed cell comprising at least one target toolbox through site-specific transformation. The donor can comprise a polynucleotide encoding a target protein.

[0589] Site-specifically transformed cells can express antibiotic resistance genes, allowing the cells to survive even when treated with multiple antibiotics, and therefore can be separated from animal cells in which site-specific transformation has not occurred.

[0590] [7-3. Selection of transformed cells using antigen-antibody reactions] The site-specific transformed cell may comprise at least one target toolbox. The site-specific transformed cell may comprise at least one toolbox. Any one of the at least one toolbox may be a target toolbox that comprises a polynucleotide encoding an antigen or a nucleotide that can act as an antigen. For example, a polynucleotide encoding a target protein can be inserted into a transformed cell that comprises a target toolbox that comprises a polynucleotide encoding an antigen through site-specific transformation, in which an exon of the polynucleotide encoding an antigen is used as a target site.

[0591] Site-specifically transformed cells can differ from cells in which site-specific transformation has not occurred in terms of the amount of antigen expression, and therefore can be distinguished from site-specifically transformed cells through antigen-antibody reactions.

[0592] The site-specific transformed cell may contain a polynucleotide that encodes an antigen or a nucleotide that can act as an antigen. For example, through site-specific transformation, a donor that contains a nucleotide that can act as an antigen can be inserted into a transformed cell that contains a target site in an animal genome. The donor may contain a polynucleotide that encodes a target protein.

[0593] The nucleotides inserted into the site-specifically transformed cells that can act as antigens can interact with specific antibodies, thus allowing the site-specifically transformed cells to be distinguished from animal cells in which site-specific transformation has not occurred.

[0594] [7-4. Selection of transformed cells using surface marker genes] Site-specific transformed cells can include at least one target toolbox. Site-specific transformed cells can include at least one toolbox. Any one of the at least one toolbox can be a target toolbox that includes a polynucleotide that encodes a surface marker gene. For example, a polynucleotide that encodes a target protein can be inserted into transformed cells through site-specific transformation, in which the exon of the polynucleotide that encodes a surface marker gene is used as a target site.

[0595] Site-specific transformed cells can differ from cells in which site-specific transformation has not occurred in terms of the amount of surface markers expressed on the surface. Surface markers can interact with specific antibodies. Antibodies can interact with magnetic particles or fluorophores. Thus, site-specific transformed cells can be distinguished from cells in which site-specific transformation has not occurred based on magnetic properties or fluorescent signals.

[0596] Site-specific transformed cells can contain a polynucleotide that encodes a surface marker.For example, through site-specific transformation, a donor that contains a polynucleotide that encodes a surface marker can be inserted into a transformed cell that contains at least one target toolbox.The donor can contain a polynucleotide that encodes a target protein.

[0597] Site-specifically transformed cells can express a surface marker on the cell surface. The surface marker can interact with a specific antibody. The antibody can interact with a magnetic particle or a fluorophore. Thus, site-specifically transformed cells can be distinguished from cells in which site-specific transformation did not occur based on magnetic properties or fluorescent signals.

[0598] [7-5. Selection of transformed cells using suicide genes] The site-specific transformed cells may contain a polynucleotide encoding a suicide gene within the animal genome or toolbox. Site-specific transformed cells can be prepared in which the suicide gene is knocked out through site-specific transformation.

[0599] For example, when a transformed cell contains a toolbox that includes a polynucleotide encoding thymidine kinase, a donor can be inserted through site-specific transformation in which a portion of the nucleotide sequence of an exon of the polynucleotide encoding thymidine kinase is used as a target site.

[0600] A characteristic of site-specifically transformed cells is that they do not undergo apoptosis when treated with a prodrug because the suicide gene is knocked out, and therefore can be distinguished from animal cells in which site-specific transformation has not occurred.

[0601] [8. Site-specific transgenic animals using the CRISPR / enzyme system] [8-1. Individual site-specific transgenic animals using the CRISPR / enzyme system] [8-1-1.Homology] Each cell in the homology site-specific transgenic animal may comprise at least one toolbox. The at least one toolbox may comprise at least one of a first toolbox, a second toolbox, and a third toolbox. The first toolbox may comprise at least one polynucleotide encoding an RNA-guided endonuclease. The second toolbox may comprise at least one polynucleotide encoding a guide nucleic acid. The third toolbox may comprise at least one polynucleotide encoding an RNA-guided endonuclease and may comprise at least one polynucleotide encoding a guide nucleic acid.

[0602] The RNA-guided endonuclease may include, but is not limited to, Cas9. The guide nucleic acid may include, but is not limited to, a gRNA.

[0603] Each cell contained in the homologous site-specific transgenic animal can contain at least one site-specific transformation at a target site in the animal's genome.

[0604] Each cell contained in the homologous site-specific transgenic animal can contain at least one site-specific transformation at a target site within the toolbox.

[0605] [8-1-2. Chimera] A chimeric site-specific transgenic animal refers to a transgenic animal other than a homologous transgenic animal.

[0606] For example, a chimeric site-specific transgenic animal may comprise both a first cell having a genome that has been site-specifically transformed at a first target site by a CRISPR / enzyme system, and a second cell that does not have a genome that has been site-specifically transformed at the first target site by a CRISPR / enzyme system.

[0607] [8-2. Method for preparing site-specific transgenic animals using the CRISPR / enzyme system] Methods for preparing site-specific transgenic animals include methods for preparing site-specific transgenic animals from animal cells, methods for preparing site-specific transgenic animals by delivering exopolynucleotides to animal tissues or organs, and methods for preparing site-specific transgenic animals by mating between transgenic animals. Methods for preparing site-specific transgenic animals from animal cells include methods for preparing site-specific transgenic animals from cells of a transgenic animal into which at least one toolbox is inserted.

[0608] The site-specific transgenic animal prepared by any one of the above methods can be any one of a chimeric site-specific transgenic animal or a homologous site-specific transgenic animal.

[0609] [8-2-1. Methods for preparing site-specific transgenic animals from animal cells] Site-specific transgenic animals can be prepared from transformed cells containing at least one toolbox.

[0610] For example, a site-specific transgenic animal can be prepared by somatic cell microinjection of a gRNA into a somatic cell into which a first toolbox is inserted, followed by SCNT to perform site-specific transformation at a target site within the animal genome or the target toolbox in the somatic cell. The site-specific transgenic animal can be a homologous site-specific transgenic animal.

[0611] For example, a site-specific transgenic animal in which a donor polynucleotide is inserted into a target site within the animal genome or a target toolbox of a gamete that includes a third toolbox can be prepared via gamete microinjection of the donor polynucleotide into the gamete. The site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0612] For example, a site-specific transgenic animal in which NHEJ has occurred at a target site within the animal genome or a target toolbox of a zygote that includes a second toolbox can be prepared via zygote microinjection of a polynucleotide encoding Cas9 into a zygote. The site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0613] For example, by inducing NHEJ at the target site present inside the target toolbox through embryo microinjection of Cas9-encoding polynucleotide and gRNA-encoding polynucleotide into the embryo containing the target toolbox, site-specific transgenic animal can be prepared.Site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0614] [8-2-2. Method for preparing site-specific transgenic animals by delivery of exopolynucleotides to animal tissues or organs]

[0615] The animal tissue or organ may comprise cells that contain at least one toolbox. Site-specific transgenic animals may be prepared by introducing Cas9, gRNA or donor polynucleotides into the animal tissue or organ.

[0616] For example, when the mammary tissue of an animal contains the cell into which the first toolbox is inserted, a site-specific transgenic animal can be prepared in which the protein specifically expressed in the mammary gland is knocked out by microinjecting a gRNA that targets a part of the nucleotide sequence of the polynucleotide that encodes the protein specifically expressed in the mammary gland in the animal genome as a target site.The site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0617] For example, when the mammary tissue of an animal contains cells in which the targeting toolbox is inserted, a site-specific transgenic animal in which the donor has inserted the targeting toolbox can be prepared by microinjecting a polynucleotide encoding Cas9, a polynucleotide encoding a gRNA, and a donor polynucleotide into the mammary tissue. The site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0618] For example, when the reproductive organs of an animal contain cells in which a second toolbox is inserted, a polynucleotide encoding Cas9 can be microinjected into the reproductive organs of the animal to induce site-specific transformation at a target site within the animal genome or the target toolbox in the cells of the reproductive organs. The offspring of the gametes of the site-specific transgenic animal can also be site-specific transgenic animals. The site-specific transgenic animal can be a chimeric site-specific transgenic animal.

[0619] [8-2-3. Method for preparing transgenic animals through breeding] Site-specific transgenic animals can be prepared through breeding between a first transgenic animal and a second transgenic animal.

[0620] For example, offspring resulting from site-specific transformation at a target site within the animal genome or within a target toolbox can be prepared through breeding between a first transgenic animal containing a first toolbox and a second transgenic animal containing a second toolbox.

[0621] The second transgenic animal may be the offspring of the first transgenic animal or may be related to the first transgenic animal. Alternatively, the second transgenic animal may be unrelated to the first transgenic animal.

[0622] The site-specific transgenic animal obtained through breeding may contain a toolbox that is identical to a portion of the toolbox contained in the animal genome of the first transgenic animal, at the same location.

[0623] The site-specific transgenic animal obtained through breeding can contain a toolbox that is identical to a portion of a toolbox contained in the animal genome of a second transgenic animal, at the same location.

[0624] The site-specific transgenic animals obtained through breeding can be homologous site-specific transgenic animals.

[0625] The site-specific transgenic animals obtained through mating may contain homozygous site-specific transformed cells. The transgenic animals obtained through mating may contain heterozygous site-specific transformed cells.

[0626] [9. Use of site-specific transgenic animals using the CRISPR / enzyme system] [9-1. Improved Animals] The site-specific transgenic animals can be used as breeding animals. [9-2. Animal models of disease] The site-specific transgenic animals can be used as animal models of disease. [9-3. Disease-resistant animals] The site-specific transgenic animals can be used as disease-resistant animals.

[0627] [9-4. Use of by-products] The parts of the site-specific transgenic animal that can be used include, but are not limited to, organs, flesh, skin, hair, and body fluids of the site-specific transgenic animal. [9-5. Bioreactor] The site-specific transgenic animals can be used as bioreactors.

[0628] [Part 5: Toolbox Removal] [1. Toolbox excision by transposase] The animal genome in the cell may comprise at least one toolbox. A transposon toolbox of the at least one toolbox may comprise an ITR polynucleotide at a first end region and a second end region.

[0629] [1-1. Use of transposases in animal genomes] [1-1-1. Structure of toolbox resection] The animal genome may comprise at least one polynucleotide encoding a transposase that can interact with the ITR polynucleotide. The transposase may comprise an excision-limited transposase. The polynucleotide encoding the transposase may be located in a transposon toolbox.

[0630] As used herein, the term "transposon toolbox" may refer to a toolbox that includes a polynucleotide encoding a transposon as a component.

[0631] A polynucleotide encoding a transposase can be located in a toolbox other than the transposon toolbox. A polynucleotide encoding a transposase can be located outside of a toolbox.

[0632] A promoter that controls the transcription of the polynucleotide encoding the transposase can be located upstream of the polynucleotide encoding the transposase. The promoter can include any one of a constitutive promoter, a tissue-specific promoter, and an inducible promoter.

[0633] The LSL can be located between the promoter that regulates transcription of the transposase and the polynucleotide encoding the transposase.

[0634] [1-1-2. Toolbox Removal Mechanism] The transposase expressed in the cell can delete the transposon toolbox present in the cell from the animal genome.

[0635] When the promoter that controls the transcription of the polynucleotide encoding the transposase is a tissue-specific promoter, the transposase can be expressed in cells of a specific tissue of the transgenic animal that contains the animal genome. In this case, the transposon toolbox in the animal genome in the cell contained in the specific tissue can be deleted through the interaction of the transposon toolbox with the ITR polynucleotide.

[0636] If the promoter regulating the transcription of the polynucleotide encoding the transposase is an inducible promoter, the transposase can be expressed only when certain conditions are met. In this case, the transposon toolbox in the animal genome in cells that meet the certain conditions can be deleted through the interaction of the transposon toolbox with the ITR polynucleotide.

[0637] When LSL is located between a promoter regulating transcription of the transposase and a polynucleotide encoding the transposase, the transposase can be expressed only when the stop codon present in LSL is deleted via site-specific recombination by introducing Cre recombinase into the cell, in which case the transposon toolbox in the animal genome in the cell containing Cre recombinase can be deleted through interaction of the transposon toolbox with the ITR polynucleotide.

[0638] [1-2. Use of transposase outside the animal genome] [1-2-1. Structure of toolbox resection] The animal genome may not contain a polynucleotide encoding a transposase capable of interacting with an ITR polynucleotide.

[0639] In this case, a polynucleotide encoding a transposase can be delivered to the cell. A promoter regulating the transcription of the polynucleotide encoding the transposase can be located upstream of the polynucleotide encoding the transposase. The promoter can include any one of a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The LSL can be located between the promoter regulating the transcription of the transposase and the polynucleotide encoding the transposase. The transposase itself can be introduced into the cell. The transposase can include an excision-limited transposase.

[0640] [1-2-2. Toolbox Removal Mechanism] The transposase introduced into the cell can delete the transposon toolbox present in the cell from the animal genome.

[0641] If the promoter regulating the transcription of the polynucleotide encoding the transposase is a tissue-specific promoter, the transposase to be introduced can be expressed in a specific tissue of the transgenic animal that contains the animal genome. In this case, the transposon toolbox in the animal genome in the cell into which the transposase is introduced can be deleted through the interaction of the transposon toolbox with the ITR polynucleotide.

[0642] If the promoter regulating the transcription of the polynucleotide encoding the transposase is an inducible promoter, the transposase introduced into the cell can be expressed only when a certain condition is met. In this case, the transposon toolbox in the animal genome in the cell that meets the certain condition can be deleted through the interaction of the transposon toolbox with the ITR polynucleotide.

[0643] When LSL is located between a promoter regulating transcription of the transposase and a polynucleotide encoding the transposase, the transposase can be expressed only when the stop codon present in LSL is deleted via site-specific recombination by co-introduction with Cre recombinase. In this case, the transposon toolbox in the animal genome in cells containing Cre recombinase can be deleted through interaction of the transposon toolbox with the ITR polynucleotide.

[0644] [2. Toolbox excision by site-specific recombinase] The animal genome in the cell may include at least one toolbox, wherein a first end region of the RRS toolbox may include RRS1 and a second end region of the RRS toolbox may include RRS2.

[0645] As used herein, the term "RRS toolbox" may refer to a toolbox that includes an RRS as a component. RRS1 and RRS2 may be identical to or different from each other. RRS1 and RRS2 may be paired with each other.

[0646] SSR1 capable of interacting with RRS1 and SSR2 capable of interacting with RRS2 may be identical SSRs to each other. [2-1. Use of site-specific recombinases in animal genomes] [2-1-1. Structure of toolbox resection] The animal genome in the cell can comprise at least one polynucleotide that encodes SSR.The polynucleotide that encodes SSR can be located in the SSR toolbox.The polynucleotide that encodes SSR can be located in a toolbox other than the SSR toolbox.The polynucleotide that encodes SSR can be located outside the toolbox.

[0647] The promoter that regulates the transcription of the polynucleotide encoding the SSR may be located upstream of the polynucleotide encoding the SSR. The promoter may include any one of a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The LSL may be located between the promoter that regulates the transcription of the polynucleotide encoding the SSR and the polynucleotide encoding the SSR.

[0648] [2-1-2. Toolbox Removal Mechanism] SSRs expressed in cells can delete the RRS toolbox from the animal genome through interactions with RRS1 and RRS2.

[0649] When the promoter that regulates the transcription of the polynucleotide encoding the SSR is a tissue-specific promoter, the SSR can be expressed in a specific tissue of the transgenic animal that comprises the animal genome.In this case, the RRS toolbox in the animal genome in the cell contained in the specific tissue can be deleted through the interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0650] If the promoter regulating the transcription of the polynucleotide encoding the SSR is an inducible promoter, the SSR can be expressed only when certain conditions are met. In this case, the RRS toolbox in the animal genome in cells that meet the certain conditions can be deleted through the interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0651] When LSL is located between a promoter that regulates the transcription of an SSR and a polynucleotide that encodes the SSR, the SSR can be expressed only when the stop codon present in LSL is deleted through site-specific recombination by introducing Cre recombinase into the cell. In this case, the RRS toolbox in the animal genome in the cell containing Cre recombinase can be deleted through interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0652] [2-2. Use of site-specific recombinases outside the animal genome] [2-2-1. Structure of toolbox resection] The animal genome may not contain a polynucleotide encoding the SSR. In this case, the polynucleotide encoding the SSR may be delivered to the cell. The promoter that regulates the transcription of the polynucleotide encoding the SSR may be located upstream of the polynucleotide encoding the SSR. The promoter may include any one of a constitutive promoter, a tissue-specific promoter, and an inducible promoter. The LSL may be located upstream of the polynucleotide encoding the SSR. Alternatively, the SSR itself may be introduced into the cell.

[0653] [2-2-2. Toolbox Removal Mechanism] SSRs introduced into cells can delete the RRS toolbox from the animal genome through interactions with RRS1 and RRS2.

[0654] If the promoter that regulates the transcription of the polynucleotide encoding the SSR is a tissue-specific promoter, the SSR introduced into the cells of a specific tissue of the transgenic animal containing the animal genome can be expressed. In this case, the RRS toolbox in the animal genome can be deleted in the cell into which the SSR is introduced through the interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0655] If the promoter regulating the transcription of the polynucleotide encoding the SSR is an inducible promoter, the SSR introduced into the cell can be expressed only when a certain condition is met. In this case, the RRS toolbox in the animal genome in the cell that meets the certain condition can be deleted through the interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0656] When LSL is located between a promoter that regulates the transcription of an SSR and a polynucleotide that encodes the SSR, the SSR can be expressed only when the stop codon present in LSL is deleted through site-specific recombination by introducing it together with Cre recombinase. In this case, the RRS toolbox in the animal genome in cells containing Cre recombinase can be deleted through interaction with RRS1 and RRS2 at both ends of the RRS toolbox.

[0657] Specific embodiments according to the details disclosed in the present disclosure are described below.

[0658] [A toolbox including at least one of an RNA-guided endonuclease and a guide nucleic acid] [1. Toolbox Structure]

[0659] The toolbox disclosed by some embodiments of the present disclosure may include a first ITR sequence, at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid, and a second ITR sequence.

[0660] The first ITR sequence, the polynucleotide encoding the RNA-guided endonuclease, the polynucleotide encoding the guide nucleic acid, and the second ITR sequence have been described above, and therefore will not be described in detail. The structure of the toolbox will be described in detail with reference to FIG. 1.

[0661] The toolbox (100) may include polynucleotides encoding components of an engineered nuclease (110) between the first ITR sequence (101) and the second ITR sequence (107).

[0662] FIG. 2 shows various embodiments of polynucleotides encoding components of the engineered nuclease (110).

[0663] The polynucleotides encoding the components of the engineered nuclease (110) may include at least one of a polynucleotide encoding an RNA-guided endonuclease (102) and a polynucleotide encoding a guide nucleic acid (104).

[0664] The RNA-guided endonuclease can be a Cas9 protein or a Cpf1 protein that constitutes a modified nuclease complex. The guide nucleic acid can be a gRNA that constitutes a modified nuclease complex.

[0665] Additionally, the polynucleotides encoding the components of the modified nuclease (110) may further include a polynucleotide encoding an RNA-guided endonuclease and / or a promoter (106) for expression of the guide nucleic acid. The toolbox (100) may further include a recombinase recognition site (RRS). The type and number of the recombinase recognition site (RRS) may be one or more.

[0666] There may be various positions where the recombinase recognition site (RRS) may be present within the toolbox 100. For example, the recombinase recognition site (RRS) may be located at one or more of positions 1-24 shown in FIG.

[0667] The type, number, and / or location of recombinase recognition sites (RRS) can be designed to allow for changes in the structure of the toolbox.

[0668] The structural change may include the replacement or deletion of a polynucleotide included in the toolbox, and may include the insertion into the toolbox of a polynucleotide not included in the toolbox.

[0669] Using FIG. 2, several embodiments are described below in which the structure of the toolbox is modified according to the structure of the toolbox containing recombinase recognition sites (RRS).

[0670] For example, when a toolbox is designed to include recombinase recognition sites (RRS) in the 5' and 3' directions with respect to a polynucleotide encoding an RNA-guided endonuclease (102), the polynucleotide encoding the RNA-guided endonuclease included in the toolbox can be exchanged with a different type of polynucleotide through site-specific recombination. Specifically, when different types of recombinase recognition sites (RRS) are included in positions 4 and 5 of FIG. 2, the polynucleotide encoding the RNA-guided endonuclease included in the toolbox can be exchanged with a different type of polynucleotide through site-specific recombination.

[0671] In another example, when a toolbox is designed to include recombinase recognition sites (RRS) that can pair in the 5' and 3' directions with respect to a polynucleotide encoding an RNA-guided endonuclease (102), the polynucleotide encoding the RNA-guided endonuclease included in the toolbox can be deleted through site-specific recombination. Specifically, when a polynucleotide encoding loxp is included in positions 4 and 5 of FIG. 2, the polynucleotide encoding the RNA-guided endonuclease included in the toolbox can be deleted through site-specific recombination.

[0672] In yet another example, if the toolbox is designed to contain a recombinase recognition site (RRS) in the 5' and / or 3' direction with respect to a polynucleotide encoding an RNA-guided endonuclease (102), a polynucleotide not included in the toolbox can be inserted into the toolbox through site-specific recombination. Specifically, if the recombinase recognition site (RRS) is contained at position 5 in Figure 2, a polynucleotide not included in the toolbox can be inserted into the toolbox through site-specific recombination.

[0673] Additionally, the type, number and / or location of recombinase recognition sites (RRS) may be designed taking into consideration the regulation of expression of RNA or proteins encoded by the polynucleotides constituting the toolbox.

[0674] Using FIG. 2, several embodiments are described below in which RNA or protein expression is regulated according to a toolbox structure that contains recombinase recognition sites (RRS).

[0675] Modulation of expression of the RNA or protein encoded by the polynucleotides constituting the toolbox may involve, at a later stage, the insertion of a polynucleotide encoding a promoter using recombinase recognition sites (RRS).

[0676] For example, if the polynucleotide encoding the RNA-guided endonuclease contains a recombinase recognition site (RRS) toward the 5' end, it is possible to regulate expression of the RNA-guided endonuclease by inserting, through site-specific recombination, a promoter for transcription and / or translation of the polynucleotide encoding the RNA-guided endonuclease.

[0677] Specifically, when a recombinase recognition site (RRS) is contained at 17 in FIG. 2, it is possible to regulate expression of the RNA-guided endonuclease by inserting a promoter for transcription and / or translation of a polynucleotide encoding the RNA-guided endonuclease through site-specific recombination.

[0678] Modulation of expression of an RNA or protein encoded by a polynucleotide constituting a toolbox may include temporal termination of expression by a polynucleotide encoding a promoter already present in the toolbox using a recombinase recognition site (RRS).

[0679] For example, when a polynucleotide encoding a first recombinase recognition site (RRS1)-stop codon-first recombinase recognition site (RRS1) (hereinafter, RSR1) or a polynucleotide encoding a first recombinase recognition site (RRS1)-transcription stop codon-first recombinase recognition site (RRS1) (hereinafter, RTR1) is included between a polynucleotide encoding an RNA-guided endonuclease (102) and a promoter (106) for transcription and / or translation of the polynucleotide encoding the RNA-guided endonuclease, the RNA-guided endonuclease cannot be expressed and ultimately cannot form a modified nuclease complex until the polynucleotides encoding RSR1 and / or RTR1 are deleted through site-specific recombination.

[0680] Specifically, when the polynucleotide encoding RSR1 or RTR1 is included in 21 of FIG. 2, the RNA-guided endonuclease cannot be expressed and ultimately the modified nuclease complex cannot be formed until the polynucleotide encoding RSR1 and / or RTR1 is deleted through site-specific recombination.

[0681] As described above, toolboxes can have a variety of configurations, and the effects they can exert in cells or animals having a genome into which the toolbox has been inserted can also vary according to the structure of the toolbox.

[0682] Hereinafter, cells and fertilized eggs having genomes into which the toolbox has been inserted will be described. [2. Cells and fertilized eggs containing genomes with toolbox insertions] [2-1. Construction of cells and fertilized eggs containing genomes with toolbox insertions] [2-1-1. Genome or chromosome into which the toolbox has been inserted] The toolboxes provided by the present disclosure can be inserted into the genome of a cell. The position of the genome where the toolbox can be inserted can be random. The number of toolboxes that can be inserted into the genome can be one. The number of toolboxes that can be inserted into the genome can be two or more. The type of toolbox that can be inserted into the genome can be one or more.

[0683] For example, a first toolbox and a second toolbox can be inserted into a genome, where the sequence of the first toolbox can be the same or different from the sequence of the second toolbox.

[0684] When the cell is a eukaryotic cell, the toolbox provided by the present disclosure can be inserted into a chromosome. The position of the chromosome into which the toolbox can be inserted can be random. The number of toolboxes that can be inserted into a chromosome can be one. The number of toolboxes that can be inserted into a chromosome can be two or more. The type of toolbox that can be inserted into a chromosome can be one or more.

[0685] For example, a first toolbox and a second toolbox can be inserted into a chromosome, where the sequence of the first toolbox can be the same or different from the sequence of the second toolbox.

[0686] Hereinafter, assuming that one type of toolbox is inserted into a chromosome, the location of the toolbox on the chromosome where the toolbox is inserted will be described in detail.

[0687] For ease of explanation, the following discussion assumes that there are four chromosomes formed from the genome present in one cell. Figure 3 shows the four chromosomes formed from the genome present in one cell.

[0688] As shown in FIG. 3, chromosome 1 (210) and chromosome 2 (220) shown in FIG. 3 are in a homologous chromosome relationship, and chromosome 3 (230) and chromosome 4 (240) are also in a homologous chromosome relationship.

[0689] Chromosome 1 (210) consists of the first (CT1) and second (CT2) chromatids, chromosome 2 (220) consists of the third (CT3) and fourth (CT4) chromatids, additionally chromosome 3 (230) consists of the fifth (CT5) and sixth (CT6) chromatids, and chromosome 4 (240) consists of the seventh (CT7) and eighth (CT8) chromatids.

[0690] FIG. 4 shows several embodiments of the toolbox (100) in which the toolbox is inserted into any one of the first through eighth chromatids (CT1 through CT8).

[0691] The toolbox (100) can be inserted into only one of the chromatids 1 to 8 (CT1 to CT8).

[0692] The toolbox (100) can be inserted into two or more of the first through eighth chromatids (CT1 through CT8).

[0693] For example, the toolbox (100) can be inserted only into the first chromatid (CT1) of a cell's chromosome (see FIG. 4(a)).

[0694] In another example, the toolbox (100) can be inserted into the first chromatid (CT1) and the second chromatid (CT2) of chromosome 1 (210) of a cell's chromosomes (see FIG. 4(b)).

[0695] In yet another example, the toolbox (100) can be inserted into the first chromatid (CT1) of chromosome 1 (210) and the third chromatid (CT3) of chromosome 2 (220) of the cell's chromosomes (see FIG. 4(c)). In this case, chromosome 1 (210) and chromosome 2 (220) can be in a homologous chromosome relationship.

[0696] In yet another example, the toolbox (100) can be inserted into the first chromatid (CT1) of chromosome 1 (210) and the fifth chromatid (CT5) of chromosome 3 (230) of the cell (see FIG. 4(d)). In this case, chromosome 1 (210) and chromosome 3 (230) may not be related as homologous chromosomes.

[0697] [2-1-2. Types of cells that have genomes or chromosomes with toolboxes inserted] The toolbox can be inserted into the genome and / or chromosome of a somatic cell, gamete or stem cell.

[0698] For ease of explanation, the following description assumes that there is only one type of toolbox inserted into the genome and / or chromosomes of a somatic cell, gamete, or stem cell, and that the toolbox is the toolbox (100) described above.

[0699] Figure 5 shows a somatic cell (301) and a gamete, each having a genome with the toolbox (100) inserted into it. The gametes can be eggs (303) and sperm (305).

[0700] The genome and / or chromosome into which the toolbox (100) has been inserted may be contained in the nucleus of a somatic cell (302), the nucleus of an egg (304), and the nucleus of a sperm (306).

[0701] The toolbox (100) into which the genome of a cell is inserted may be transcribed and / or translated according to the expression machinery of each cell, in which case expression of endopolynucleotides already present in the genome of the cell may not be affected.

[0702] [2-1-3. Fertilized eggs containing genomes with toolbox insertions] The toolbox can be inserted into the genome or chromosome of a zygote (fertilized egg) or embryo.

[0703] According to one embodiment of the present specification, a transformed zygote or transgenic embryo may be provided, the genome of which includes a polynucleotide encoding an RNA-guided endonuclease contained between the first and second ITR sequences. Specifically, the embryo may be an artiodactyl embryo.

[0704] Additionally, a transformed fertilized egg or transgenic embryo may be provided, further comprising a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the genome of the fertilized egg or embryo between the first and second ITR sequences. In particular, the expression regulator may be included in at least one of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid.

[0705] FIG. 6 shows a fertilized egg having a genome into which the toolbox (100) has been inserted, as well as 2-cell, 4-cell, 8-cell and 16-cell embryos produced by cell division from a fertilized egg into which the toolbox (100) has been inserted.

[0706] When the stage at which the toolbox (100) begins to be inserted into the genome of the fertilized egg is the one-cell stage fertilized egg, the one-cell stage fertilized egg (401) into which the toolbox (100) is inserted may be homologous (see FIG. 6).

[0707] When a one-cell zygote (401) having a genome with the toolbox (100) inserted therein undergoes cell division, the zygote (401) can develop into a two-cell embryo (403), a four-cell embryo (405), an eight-cell embryo (407), a sixteen-cell embryo (409), a morula, a blastocyst, or a gastrula, each of which is composed of cells having a genome with the toolbox (100) inserted therein.

[0708] However, 2 n The number and location of toolboxes (100) inserted into the genome of 2-cell stage embryos is n+1The number and position of the toolbox (100) inserted into the genome of the 2-cell stage embryo (n is an integer equal to or greater than 0) may be different. n+1 Constituting the 2-cell embryo n+1 The locations and numbers of toolboxes (100) inserted into the genome of a cell can vary from one another.

[0709] This is because, although the positions and number of toolboxes inserted into the nucleus of the 1-cell stage fertilized egg (401) can be maintained in the 2-cell stage embryo (403), as described above, more toolboxes may be additionally inserted into the 2-cell stage embryo 403 due to interactions between the first plasmid vector, the transposase, and the genome of the 2-cell stage embryo (403). Furthermore, this is because a part of the toolbox (100) already inserted into the genome may be deleted due to interactions between the genome of the 2-cell stage embryo (403) and the transposase.

[0710] That is, according to one embodiment provided herein, a transgenic embryo may be provided, comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox. In the transgenic embryo, the first toolbox may be present at a first locus, and the second toolbox may be present at a second locus different from the first locus. Each of the first toolbox and the second toolbox may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the embryo or an exopolynucleotide contained in the genome of the embryo, and may be included between the first ITR sequence and the second ITR sequence. In particular, the sequence of the first toolbox may be the same as or different from the sequence of the second toolbox.

[0711] According to another embodiment provided herein, a transgenic embryo may be provided, comprising a first cell having a genome including a first toolbox and a second cell having a genome including a second toolbox. In the transgenic embryo, the first toolbox may be present at a first locus, and the second toolbox may be present at a second locus, and the first locus and the second locus are identical to each other. Each of the first toolbox and the second toolbox may include at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the embryo or an exopolynucleotide contained in the genome of the embryo, and may be included between the first ITR sequence and the second ITR sequence. In particular, the sequence of the first toolbox is different from the sequence of the second toolbox.

[0712] For example, the genome of the first cell may further comprise a third toolbox, wherein the sequence of the third toolbox is identical to the first toolbox.

[0713] In another example, the genome of the second cell may further comprise a fourth toolbox, wherein the sequence of the fourth toolbox is identical to the sequence of the second toolbox.

[0714] The transgenic embryo may further comprise a third cell having a genome that does not include a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid.

[0715] Figure 7 shows a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, and a 16-cell embryo in which the toolbox (100) has been inserted into the genome of each of several cells. In Figure 7, a first nucleus (501) represents a nucleus having a genome into which the toolbox (100) has been inserted, and a second nucleus (503) represents a nucleus having a genome into which the toolbox (100) has not been inserted.

[0716] The toolbox (100) started to be inserted into the genome at step 2.m In the case of a 2-cell stage embryo, the toolbox (100) m After the cellular stage, the toolbox may be inserted into the genome of only some of the cells that make up the embryo (m is a natural number equal to or greater than 1). Even in this case, the toolbox may be inserted into the genome of all of the cells that make up the embryo. However, this can be explained from the above, and therefore, an explanation is provided that is limited to the case where the toolbox is inserted into the genome of only some of the cells that make up the embryo.

[0717] For example, when the stage at which the toolbox (100) begins to be inserted into the genome of a fertilized egg is a two-cell embryo, the toolbox (100) can be inserted only into the genome of the first cell (511) among the cells that make up the two-cell embryo (411).

[0718] When a 2-cell embryo (411), in which the toolbox (100) is inserted into the genome of one cell, undergoes cell division, the 2-cell embryo (411) can develop into a 4-cell embryo (413), an 8-cell embryo (415), a 16-cell embryo (417), a morula, a blastula, or a gastrula, in which the toolbox is inserted into the genome of only some of the cells.

[0719] According to some embodiments provided herein, a chimeric transgenic embryo may be provided, the chimeric transgenic embryo may comprise a first cell having a genome that includes a toolbox and a second cell having a genome that does not include the toolbox.

[0720] Specifically, a transgenic embryo can be provided, the transgenic embryo comprising a first cell having a genome that includes a first toolbox and a target site, and a second cell having a genome that includes the target site.

[0721] The first toolbox may comprise at least one of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to a target site, and the genome of the second cell may not comprise a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to a target site. Additionally, ITR sequences may be further comprised at the 5' and 3' ends of the first toolbox.

[0722] In particular, the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be identical or different from each other, and the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be identical or different from each other. The target site may be an endopolynucleotide.

[0723] Specifically, the target site can be a nucleotide sequence of 18 bp to 25 bp present in the genome of the transgenic embryo. The target site can be an exopolynucleotide.

[0724] For example, the target site can be a sequence adjacent to the 5' or 3' end of the PAM sequence. The target site and PAM sequence can be contained between the first and second ITR sequences.

[0725] However, even in this case, for the same reasons as above, m The number and location of toolboxes (100) inserted into the genome of 2-cell stage embryos is m+1 The number and location of the toolboxes (100) inserted into the genome of the 2-cell stage embryo may be different. m+1 Constituting the 2-cell embryo m+1 The locations and numbers of toolboxes (100) inserted into the genome of a cell can vary from one another.

[0726] The stage where the toolbox (100) begins to be inserted into the genome is 2 m If it is a 2-cell embryo, m For each genome of a cell constituting an embryo after the 3-cell stage, the toolbox (100) can be inserted at a different locus (m is a natural number equal to or greater than 1).

[0727] [2-2. Methods for producing cells and / or zygotes containing genomes with toolboxes inserted]

[0728] Below, we describe several methods for producing cells having a genome with a toolbox (100) inserted. For ease of explanation, we will assume that there is one type of toolbox that can be inserted into a genome.

[0729] One method for preparing a cell into which the toolbox (100) is inserted may include delivering the toolbox (100) to the cell. In particular, the toolbox (100) may include a transposon gene and a polynucleotide encoding a component of a modified nuclease. There may be various methods for delivering the toolbox (100) to a cell.

[0730] For example, the toolbox can be delivered to a cell by introducing a polynucleotide encoding the toolbox into the cell. Introducing a polynucleotide encoding the toolbox into a cell can include a method in which a polynucleotide encoding the toolbox is incorporated into a plasmid vector and then introduced into the cell. Additionally, introducing a polynucleotide encoding the toolbox into a cell can include a method in which a polynucleotide encoding the toolbox is incorporated into a viral vector and then transduced into the cell.

[0731] One method for preparing a cell into which the toolbox 100 is inserted may include delivering a transposase to the cell. There may be a variety of methods for delivering the transposase to the cell.

[0732] For example, the transposase can be delivered to the cell by introduction of the transposase in the form of a protein or polypeptide.

[0733] In another example, the transposase can be delivered to a cell by introducing a polynucleotide encoding the transposase into the cell.

[0734] Introducing a polynucleotide encoding a transposase into a cell can include incorporating the polynucleotide encoding the transposase into a plasmid vector and then introducing the resultant into the cell. Additionally, introducing a polynucleotide encoding the transposase into a cell can include incorporating the polynucleotide encoding the transposase into a viral vector and then transducing the resultant into the cell.

[0735] The polynucleotide encoding the transposase may be incorporated into a vector containing a polynucleotide encoding the toolbox and then delivered to the cell. Additionally, the polynucleotide encoding the transposase may be incorporated into a vector different from the vector containing the polynucleotide encoding the toolbox and then delivered to the cell. In particular, the vector may be a non-viral vector or a viral vector.

[0736] When a polynucleotide encoding a transposase is incorporated into a plasmid vector and then introduced into a cell, the plasmid can be transported into the nucleus, and then the mRNA encoding the transposase can be produced in the nucleus through transcription. The produced mRNA can be transported into the cytoplasm and interact with ribosomes and tRNA in the cytoplasm, and the transposase can be expressed in the cytoplasm through the interaction. The transposase expressed by the above mechanism can be introduced into the nucleus.

[0737] Hereinafter, the mechanism by which the toolbox and transposase delivered to the cell insert the toolbox into the genome of the cell will be described. The toolbox (100) and transposase delivered into the cell by the above method can be introduced into the cytoplasm of the cell. The toolbox (100) and transposase introduced into the cytoplasm can be transported into the nucleus through the nuclear pores.

[0738] Inside the nucleus, the transposase can interact with the first ITR sequence (101) and the second ITR sequence (107), which are components of the toolbox (100), and the toolbox (100) can be inserted into the genome through the interaction. For the structure of the toolbox (100), see Figure 1.

[0739] When the toolbox (100) is incorporated into a plasmid vector and then delivered into a cell, the toolbox (100) can be deleted from the plasmid vector and the deleted toolbox (100) can be inserted into the genome of the cell.

[0740] The cells can be isolated cells or non-isolated cells contained in an organ or tissue of an animal.

[0741] One method for preparing a non-isolated cell having a genome with the toolbox (100) inserted therein may comprise delivering the toolbox (100) and transposase by direct injection into a tissue or organ of an individual.

[0742] In this case, the toolbox (100) can still be inserted into the genome of a non-isolated cell in a manner similar to the mechanism by which the toolbox (100) is inserted into the genome of an isolated cell.

[0743] Where the tissue or organ is a reproductive tissue or organ, there is an advantage that transformed gametes can be obtained on a continuous basis.

[0744] Below, several methods are described for producing zygotes and / or embryos having genomes with toolboxes (100) inserted therein. As mentioned above, for ease of explanation, it is assumed that there is one type of toolbox that can be inserted into the genome of a zygote.

[0745] One method for producing zygotes and / or embryos having a genome with the toolbox (100) inserted therein may include microinjecting the toolbox (100) into the zygotes and / or embryos.

[0746] One method for producing zygotes and / or embryos having a genome with the toolbox (100) inserted therein may include microinjecting the transposase into the zygotes and / or embryos.

[0747] Methods for delivering the toolbox and transposase to cells have been described above, and specific details will not be given here.

[0748] An exemplary method for preparing a transgenic embryo provided herein may include microinjecting a vector containing a polynucleotide encoding a transposon gene and a component of a modified nuclease into a fertilized egg or embryo. Additionally, the method may include microinjecting a transposase capable of interacting with the transposon gene into the fertilized egg or embryo.

[0749] The transposase can be in the form of a protein, polypeptide, or a polynucleotide encoding the transposase. The polynucleotide can be incorporated into a plasmid vector or a viral vector. Furthermore, the polynucleotide encoding the transposase can be incorporated into a single vector together with the polynucleotide encoding the transposon gene and the modified nuclease component, and then microinjected into a fertilized egg or embryo.

[0750] In an exemplary embodiment of a transgenic embryo that can be prepared by the above method, the transgenic embryo can comprise a first cell having a genome in which a polynucleotide encoding a first modified nuclease component is included at a first locus, and a second cell having a genome in which a polynucleotide encoding a second modified nuclease component is included at a second locus that is different from the first locus. In particular, the sequence of the polynucleotide encoding the first modified nuclease component and the sequence of the polynucleotide encoding the second modified nuclease component can be identical to or different from each other.

[0751] In another exemplary embodiment of a transgenic embryo that can be prepared by the above method, the transgenic embryo can comprise a first cell having a genome in which a polynucleotide encoding a first modified nuclease component is included at a first locus, and a second cell having a genome in which a polynucleotide encoding a second modified nuclease component is included at the first locus. In particular, the sequence of the polynucleotide encoding the first modified nuclease component and the sequence of the polynucleotide encoding the second modified nuclease component can be identical or different from each other.

[0752] One method for preparing fertilized eggs and / or embryos having a genome with the toolbox (100) inserted therein may include performing somatic cell nuclear transfer (SCNT) using the genome with the toolbox (100) inserted therein.

[0753] Exemplary methods for preparing a transgenic embryo provided herein may include a method for preparing a transgenic donor cell in which a component of a modified nuclease is expressed, and a method for transferring the nucleus of the transgenic donor cell into an enucleated egg.

[0754] Preparing the transgenic donor cell may include transforming the cell with a vector comprising a polynucleotide encoding a transposon gene and a component of the modified nuclease. Additionally, preparing the transgenic donor cell may further include transforming the cell with a transposase capable of interacting with the transposon gene.

[0755] The transposase can be in the form of a protein, polypeptide, or a polynucleotide encoding the transposase. The polynucleotide can be incorporated into a plasmid vector or a viral vector. Furthermore, the polynucleotide encoding the transposase can be incorporated into a single vector together with the polynucleotide encoding the transposon gene and the modified nuclease components, and then microinjected into fertilized eggs or embryos.

[0756] The mechanism by which the toolbox (100) is inserted into the genome of the cells that compose the fertilized egg is similar to the mechanism by which the toolbox (100) is inserted into the genome of an isolated cell.

[0757] [3. Transgenic animals containing the toolbox inserted genome] [3-1. Transgenic animals containing genomes with toolboxes inserted] According to some embodiments provided herein, a transgenic animal may be provided that comprises a genome into which the toolbox has been inserted.

[0758] According to an exemplary embodiment provided herein, a transgenic animal may be provided having a genome comprising a polynucleotide encoding an RNA-guided endonuclease, the polynucleotide being included between a first ITR sequence and a second ITR sequence. Specifically, the animal may be an artiodactyl.

[0759] Additionally, a transgenic animal may be provided that further comprises a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the animal between the first and second ITR sequences. In particular, the expression regulator may be comprised in one or more of the 5' end of the polynucleotide encoding the RNA-guided endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid.

[0760] In this disclosure, a transgenic animal having a genome into which a toolbox has been inserted refers to an animal having at least one cell having a genome into which a toolbox has been inserted. Transformation can include both transient and permanent transformation.

[0761] Furthermore, hereinafter, among the cells carried by the transgenic animal, cells into which the toolbox is inserted are referred to as "toolbox-modified cells," and cells into which the toolbox is not inserted are referred to as "non-toolbox-modified cells."

[0762] However, in the present disclosure, the term "non-toolbox modified cells" refers only to cells having a genome in which the toolbox is not inserted, and the term should not be understood as a term for cells whose genome is genetically modified for other purposes. That is, cells that do not have the toolbox inserted but have other genetically engineered genomes may also be the so-called "non-toolbox modified cells" of the present disclosure. The transgenic animals into which the toolbox is inserted may be chimeric or homologous animals.

[0763] For example, a chimeric animal can refer to an animal that has "non-toolbox modified cells" in addition to "toolbox modified cells."

[0764] That is, according to some exemplary embodiments provided herein, a chimeric transgenic animal may be provided comprising a first cell having a genome that includes a toolbox and a second cell having a genome that does not include the toolbox.

[0765] Specifically, a transgenic animal can be provided that comprises a first cell having a genome that includes a first toolbox and a target site, and a second cell having a genome that includes the target site.

[0766] The first toolbox may comprise at least one of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to a target site, and the genome of the second cell may not comprise a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to a target site. Additionally, ITR sequences may be further comprised at the 5' and 3' ends of the first toolbox.

[0767] In particular, the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be identical or different from each other, and the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be identical or different from each other. The target site may be an endopolynucleotide.

[0768] Specifically, the target site can be a nucleotide sequence of 18 bp to 25 bp present in the genome of the transgenic animal. The target site can be an exopolynucleotide.

[0769] For example, the target site can be a sequence adjacent to the 5' or 3' end of the PAM sequence. The target site and PAM sequence can be contained between the first and second ITR sequences.

[0770] In another example, a chimeric animal can refer to an animal that has only "toolbox-modified cells," but the locus at which the toolbox is inserted into the genome of each "toolbox-modified cell" that makes up the animal is different.

[0771] That is, according to exemplary embodiments provided herein, a transgenic animal may be provided comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, the first toolbox being present at a first locus and the second toolbox being present at a second locus, the first locus and the second locus being different. The first toolbox and the second toolbox may comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the animal or an exopolynucleotide located between a first ITR sequence and a second ITR sequence contained in the genome of the animal. In particular, the sequence of the first toolbox and the sequence of the second toolbox may be the same or different.

[0772] According to another exemplary embodiment provided herein, a transgenic animal may be provided, comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox is present at a first locus and the second toolbox is present at a second locus, and the first locus and the second locus are identical. The first toolbox and the second toolbox may comprise at least one of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site. The target site may be an endopolynucleotide of the animal or an exopolynucleotide located between a first ITR sequence and a second ITR sequence contained in the genome of the animal. In particular, the sequence of the first toolbox and the sequence of the second toolbox are different.

[0773] For example, the genome of the first cell may further include a third toolbox whose sequence is identical to the sequence of the first toolbox.

[0774] In another example, the genome of the second cell can further include a fourth toolbox whose sequence is identical to the sequence of the second toolbox.

[0775] The transgenic animal may further comprise a third cell having a polynucleotide encoding an RNA-guided endonuclease and a genome that does not include a polynucleotide encoding a guide nucleic acid.

[0776] A homologous animal may refer to an animal in which the locus at which the toolbox is inserted into the genome of each "toolbox-modified cell" that constitutes the animal is identical.

[0777] According to some exemplary embodiments disclosed herein, gametes or zygotes (and / or embryos) having a genome with a toolbox inserted therein can be obtained from an animal comprising a cell having a genome with a toolbox inserted therein, and the gametes can be sperm or eggs.

[0778] According to some exemplary embodiments disclosed herein, offspring comprising cells having a genome in which the toolbox has been inserted can be produced from an animal comprising cells having a genome in which the toolbox has been inserted.

[0779] According to some exemplary embodiments disclosed herein, additional gene editing can occur in the genome of an animal comprising a cell having a genome into which the toolbox has been inserted.

[0780] [3-2. Method for preparing transgenic animals with genomes containing toolboxes inserted] One method for producing an animal comprising a "toolbox-modified cell," according to some exemplary embodiments provided in the present disclosure, may comprise implanting a fertilized egg or embryo having a genome with the toolbox inserted into the uterus of a surrogate mother. After implanting the fertilized egg or embryo into the uterus of the surrogate mother and undergoing a gestational period, an animal having a genome with the toolbox inserted may be produced.

[0781] One method for producing zygotes or embryos with a genome inserted with the toolbox may include microinjection (MI) of the toolbox and / or transposase.

[0782] Fertilized eggs and / or embryos produced by microinjection (MI) may be chimeric or homozygous. Additionally, animals obtained by implanting fertilized eggs and / or embryos into the uterus of a surrogate mother may be chimeric or homozygous.

[0783] Another method for producing a zygote or embryo having a genome with a toolbox inserted therein may comprise performing somatic cell nuclear transfer (SCNT).

[0784] Somatic cell nuclear transfer may involve transferring the nucleus of a somatic cell (301) having a genome into which the toolbox (100) of Figure 5 has been inserted, into an enucleated oocyte. Animals containing "toolbox modified cells" can be produced by transferring a fertilized egg or embryo produced by somatic cell nuclear transfer (SCNT) into the uterus of a surrogate mother.

[0785] The fertilized eggs and / or embryos obtained by somatic cell nuclear transfer (SCNT) may be homologous. Additionally, animals produced using the fertilized eggs and / or embryos may also be homologous. In this case, the genome of the fertilized eggs, embryos and / or animals produced by somatic cell nuclear transfer (SCNT) that are homologous may have sequences that are identical to the genome of the somatic cell (301).

[0786] Another method for producing a fertilized egg or embryo having a genome with a toolbox inserted therein may comprise a method for in vitro fertilization between an egg (303) and a sperm (305) having a genome with a toolbox (100) inserted therein, or a method for in vitro fertilization between an egg (303) or sperm (305) and a wild type (WT) sperm or egg.

[0787] The zygote and / or embryo produced by in vitro fertilization may be chimeric or homologous. Additionally, the animals obtained by implanting the zygote and / or embryo into the uterus of a surrogate mother may be chimeric or homologous.

[0788] More specifically, one exemplary method for preparing a transgenic animal provided herein may comprise preparing a transgenic embryo in which a component of a modified nuclease is expressed, and implanting the transgenic embryo into the uterus of a surrogate mother.

[0789] An exemplary method for preparing a transgenic embryo may comprise microinjecting a vector comprising a polynucleotide encoding a transposon gene and a component of a modified nuclease into a fertilized egg or embryo. Additionally, preparing a transgenic embryo may further comprise microinjecting a transposase capable of interacting with the transposon gene into the fertilized egg or embryo.

[0790] Another exemplary method for preparing a transgenic embryo may comprise preparing a transgenic donor cell in which a component of a modified nuclease is expressed, and transferring the nucleus of the transgenic donor cell into an enucleated egg of an animal.

[0791] Preparing the transgenic donor cell can include transforming the cell with a vector comprising a polynucleotide encoding a transposon gene and a component of the modified nuclease. Additionally, preparing the transgenic donor cell can further include transforming the cell with a transposase capable of interacting with the transposon gene.

[0792] The transposase may be in the form of a protein, polypeptide, or a polynucleotide encoding the transposase. The polynucleotide may be contained in a plasmid or viral vector. Additionally, the polynucleotide encoding the transposase may be in the form of a polynucleotide contained in a vector together with a transposon gene and a polynucleotide encoding a component of a modified nuclease.

[0793] A method for producing an animal comprising "toolbox-modified cells" according to some exemplary embodiments provided in the present disclosure may comprise injecting the toolbox and transposase into a tissue of the animal. In this case, only a portion of the injected tissue may become "toolbox-modified cells". The animal produced through the above-mentioned method for injecting into a tissue may be a chimeric animal.

[0794] One method for producing animals comprising "toolbox-modified cells" according to some exemplary embodiments provided in the present disclosure may involve natural mating between males having testes that contain "toolbox-modified cells" or females having ovaries that contain "toolbox-modified cells."

[0795] For example, the method may comprise natural mating between a male having testes that contain the "toolbox-modified cells" and a female having ovaries that contain the "toolbox-modified cells."

[0796] In another example, the method may include natural mating between a male having testes containing the "toolbox modification" and a wild type (WT) female, or natural mating between a female having ovaries containing the "toolbox modification" and a wild type (WT) male.

[0797] The animals produced through the above-mentioned natural breeding may be chimeric or homologous animals.

[0798] [Second gene editing using the toolbox] In this disclosure, when the toolbox is inserted into a genome and transformed as described above, it is referred to as the first gene edit.

[0799] Additional second gene editing can occur using the toolbox inserted into the genome by the first gene editing. Additionally, n+1 gene editing can occur using the genome in which n gene editing occurred (n is a natural number of 2 or more). The n gene editing and n+1 gene editing can occur in the genome of one cell or in the genome of another cell.

[0800] The "nth gene edit" can be a gene edit using a recombinase recognition site (RRS) included as a component of the toolbox.

[0801] Additionally, the "nth gene edit" can be a gene edit using a modified nuclease component expressed from a toolbox.

[0802] Furthermore, the "nth gene edit" can be a gene edit using a polynucleotide having a PAM sequence included as a component of the toolbox.

[0803] For ease of explanation, the following describes a second gene edit using a polynucleotide encoding a recombinase recognition site (RRS) and / or an engineered nuclease component included as a component of the toolbox.

[0804] [1. Secondary gene editing using recombinase recognition sites (RRS) included in the toolbox] The following describes a second gene edit using a recombinase recognition site (RRS) present in the toolbox. A second gene edit using a recombinase recognition site (RRS) can occur within the toolbox.

[0805] [1-1. Second gene exchange editing using recombinase recognition sites (RRS)] [1-1-1. Toolbox for second gene exchange editing using recombinase recognition sites (RRS)] According to some example embodiments of the present disclosure, a toolbox for swap editing may be provided.

[0806] The toolbox for exchange editing may include a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0807] The first polynucleotide may be included between a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a second recombinase recognition site (RRS2). The first polynucleotide may be one or more selected from, but not limited to, a polynucleotide encoding a protein or RNA, a non-transcribed polynucleotide, a polynucleotide encoding an untranslated RNA, and a non-functional polynucleotide. Hereinafter, some exemplary embodiments of the toolbox for exchange editing will be described.

[0808] For example, a toolbox for exchange editing can be a toolbox that includes a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first RNA-guided endonuclease, a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0809] In another example, a toolbox for exchange editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding an RNA-guided endonuclease, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first guide nucleic acid, a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0810] In yet another example, a toolbox for exchange editing can be a toolbox including a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a fluorescent protein gene, a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0811] In yet another example, a toolbox for exchange editing can be a toolbox including a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first target protein, a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0812] Hereinafter, a second gene exchange editing method using the above-mentioned toolbox for exchange editing will be described, that is, a method for exchanging a specific polynucleotide will be described.

[0813] [1-1-2. The second gene exchange editing method using recombinase recognition sites (RRS)] In a genome into which a toolbox for exchange editing has been inserted, a first polynucleotide present between a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a second recombinase recognition site (RRS2) can be exchanged for a second polynucleotide.

[0814] One method for exchanging a first polynucleotide with a second polynucleotide may include delivering the second polynucleotide to a cell into which a toolbox for exchange editing is inserted. There may be various methods for delivering the second polynucleotide to a cell.

[0815] For example, the second polynucleotide can be delivered to the cell by introducing the second polynucleotide into the cell. Introducing the polynucleotide encoding the second polynucleotide into the cell can include incorporating the second polynucleotide into a plasmid vector and then introducing the resultant into the cell. Additionally, introducing the second polynucleotide into the cell can include incorporating the second polynucleotide into a viral vector and then transducing the resultant into the cell.

[0816] The second polynucleotide is located between a polynucleotide encoding a third recombinase recognition site (RRS3) and a polynucleotide encoding a fourth recombinase recognition site (RRS4). In this case, the third recombinase recognition site (RRS3) can be paired with the first recombinase recognition site (RRS1). The fourth recombinase recognition site (RRS4) can be paired with the second recombinase recognition site (RRS2).

[0817] Additionally, a method for exchanging a first polynucleotide with a second polynucleotide can include delivering a first recombinase and a second recombinase to a cell.

[0818] In this case, the first recombinase can interact with the first recombinase recognition site (RRS1) and / or the third recombinase recognition site (RRS3), and the second recombinase can interact with the second recombinase recognition site (RRS2) and / or the fourth recombinase recognition site (RRS4). There can be various ways to deliver recombinases to cells.

[0819] For example, the recombinase can be delivered to the cell by introducing the recombinase into the cell as a protein.

[0820] In another example, the recombinase can be delivered to a cell by introducing a polynucleotide encoding the recombinase into the cell. Introducing a polynucleotide encoding the recombinase into a cell can include incorporating the polynucleotide encoding the recombinase into a plasmid vector and then introducing the resultant into the cell. Additionally, introducing a polynucleotide encoding the recombinase into a cell can include incorporating the polynucleotide encoding the recombinase into a viral vector and then transducing the resultant into the cell.

[0821] The first recombinase delivered to a cell by the methods described above is capable of interacting with a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a third recombinase recognition site (RRS3).

[0822] Additionally, the second recombinase delivered to the cell can interact with a polynucleotide encoding a second recombinase recognition site (RRS2) and a polynucleotide encoding a fourth recombinase recognition site (RRS4).

[0823] Through the interaction, a first polynucleotide can be deleted from the toolbox and a second polynucleotide can be inserted into the toolbox.

[0824] Hereinafter, various exemplary embodiments for exchange editing by the above method and the effects of exchange editing will be described, which may vary according to the type of the first polynucleotide and / or the second polynucleotide.

[0825] According to some exemplary embodiments provided in the present disclosure, a polynucleotide encoding a first RNA-guided endonuclease included in a toolbox for exchange editing can be subjected to exchange editing for a polynucleotide encoding a second RNA-guided endonuclease and a second guide nucleic acid.

[0826] In this case, without any additional further treatment to the cell having a genome into which the exchange-edited toolbox has been inserted, the second RNA-guided endonuclease and the second guide nucleic acid can be expressed, thereby forming an engineered nuclease complex, and a third gene edit can occur in the genome.

[0827] In some exemplary embodiments of the toolbox for exchange editing provided in the present disclosure, a polynucleotide encoding a first guide nucleic acid may be subjected to exchange editing for a polynucleotide encoding a second guide nucleic acid. The sequence of the first guide nucleic acid and the sequence of the second guide nucleic acid may differ from each other.

[0828] In this case, the target site for gene editing using the modified nuclease can be altered by exchange editing.

[0829] The fluorescent protein genes included in the toolbox for exchange editing according to some exemplary embodiments provided in the present disclosure can be subjected to exchange editing of a gene encoding a target protein (target protein gene).

[0830] In this case, the fluorescent protein is not expressed in the cell by exchange editing. Such a property can be used to select whether the target protein gene is inserted into the genome of the cell.

[0831] A first target protein gene included in the toolbox for exchange editing according to some exemplary embodiments provided in the present disclosure can be subjected to exchange editing of a second target protein gene. By such second gene editing, the target protein can be expressed in a cell at a desired time.

[0832] A first target protein gene included in the toolbox for exchange editing according to some exemplary embodiments provided in the present disclosure may be subjected to exchange editing for a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid.

[0833] Such a second gene edit can allow an RNA-guided endonuclease and a guide nucleic acid to be expressed in the cell. The RNA-guided endonuclease and the guide nucleic acid can form a modified nuclease complex, and the modified nuclease complex can cause an additional third gene edit in the genome of the cell.

[0834] [1-2. Second gene insertion editing using recombinase recognition sites (RRS)] [1-2-1. Toolbox for insertion editing using recombinase recognition sites (RRS)] According to some example embodiments of the present disclosure, a toolbox for insert editing may be provided.

[0835] The toolbox for insertional editing may include a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), and a second ITR sequence. Hereinafter, several exemplary embodiments of the toolbox for insertional editing will be described.

[0836] For example, a toolbox for insertion editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first RNA-guided endonuclease, a second ITR sequence.

[0837] In another example, a toolbox for insertional editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding a first RNA-guided endonuclease, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first guide nucleic acid, and a second ITR sequence.

[0838] In yet another example, a toolbox for insertional editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first RNA-guided endonuclease, a polynucleotide encoding a first guide nucleic acid, and a second ITR sequence.

[0839] In yet another example, a toolbox for insertion editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding a first RNA-guided endonuclease, a polynucleotide encoding a first recombinase recognition site (RRS1), and a second ITR sequence.

[0840] In yet another example, a toolbox for insertional editing can be a toolbox comprising a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first guide nucleic acid, and a second ITR sequence.

[0841] Hereinafter, a method for second gene insertion editing using the above-mentioned toolbox for insertion editing will be described, i.e., a method for inserting a specific polynucleotide will be described.

[0842] [1-2-2. Method for second gene insertion editing using recombinase recognition sites (RRS)] In a genome into which the toolbox for insertional editing has been inserted, a first polynucleotide can be inserted at a position where a polynucleotide encoding a first recombinase recognition site (RRS1) is located.

[0843] One method for inserting the first polynucleotide may include delivering the first polynucleotide to a cell into which the toolbox for insertional editing is inserted. There may be various methods for delivering the first polynucleotide to a cell.

[0844] For example, the first polynucleotide can be delivered to a cell by introducing the first polynucleotide into the cell. Introducing the polynucleotide encoding the first polynucleotide into a cell can include incorporating the first polynucleotide into a plasmid vector and then introducing the resultant into the cell. Additionally, introducing the first polynucleotide into a cell can include incorporating the first polynucleotide into a viral vector and then transducing the resultant into the cell.

[0845] A polynucleotide encoding a second recombinase recognition site (RRS2) is further included in the vector containing the first polynucleotide, where the second recombinase recognition site (RRS2) is capable of pairing with the first recombinase recognition site (RRS1).

[0846] Additionally, a method for inserting a first polynucleotide may include delivering a first recombinase to a cell.

[0847] In this case, the first recombinase can interact with the first recombinase recognition site (RRS1) and / or the second recombinase recognition site (RRS2).

[0848] Methods for delivering recombinases to cells have been described above and will not be described in detail here.

[0849] The recombinase delivered to a cell by the above-described method can interact with a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a second recombinase recognition site (RRS2), whereby the first polynucleotide can be inserted into the toolbox.

[0850] Hereinafter, various exemplary embodiments for insertion editing according to the above method and their effects of insertion editing will be described, which may vary according to the type of toolbox for insertion editing and / or the first polynucleotide.

[0851] When a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a first RNA-guided endonuclease are included in a toolbox for insertional editing according to some exemplary embodiments provided in the present disclosure, a polynucleotide encoding a guide nucleic acid can be inserted into the toolbox.

[0852] In this case, an engineered nuclease complex can be formed in the cell by transcription and / or translation of a polynucleotide encoding the first RNA-guided endonuclease and a polynucleotide encoding the insertion guide nucleic acid. The engineered nuclease complex formed in the cell can allow an additional third gene edit to occur after the second gene edit (insertion edit) without further processing.

[0853] When a polynucleotide encoding a first RNA-guided endonuclease, a polynucleotide encoding a first recombinase recognition site (RRS1), and a polynucleotide encoding a first guide nucleic acid are included in a toolbox for insertional editing according to some exemplary embodiments provided in the present disclosure, a polynucleotide encoding a promoter for transcription of the polynucleotide encoding the first guide nucleic acid may be inserted into the toolbox.

[0854] In this case, the first guide nucleic acid can be expressed at the time when the polynucleotide encoding the promoter is inserted, and the modified nuclease complex can be formed in the cell through the expression. That is, by adjusting the time when the polynucleotide encoding the promoter is inserted, the first guide nucleic acid can be expressed at the desired time. Finally, the time of the third gene editing can be adjusted through the second gene editing (insertion editing).

[0855] When a polynucleotide encoding a first recombinase recognition site (RRS1), a polynucleotide encoding a first RNA-guided endonuclease, and a polynucleotide encoding a first guide nucleic acid are included in a toolbox for insertional editing according to some exemplary embodiments provided in the present disclosure, a polynucleotide encoding a promoter for transcription and / or translation of the polynucleotide encoding the first RNA-guided endonuclease may be inserted into the toolbox.

[0856] In this case, when the polynucleotide encoding the promoter is inserted, the RNA-guided endonuclease can be expressed, and through expression, modified nuclease complex can be formed in the cell.That is, by adjusting the time of inserting the polynucleotide encoding the promoter, the first RNA-guided endonuclease can be expressed at the desired time.Finally, the time of the third gene editing can be adjusted through the second gene editing (insertion editing).

[0857] When a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first recombinase recognition site (RRS1) are included in a toolbox for insertional editing according to some exemplary embodiments provided in the present disclosure, a fluorescent protein gene can be inserted into the toolbox.

[0858] In this case, it is possible to select whether a polynucleotide encoding the first RNA-guided endonuclease, a component of the toolbox, is inserted into the genome by insertion of a fluorescent protein gene.

[0859] When a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a first guide nucleic acid are included in a toolbox for insertion editing according to some exemplary embodiments provided in the present disclosure, a polynucleotide encoding a second guide nucleic acid can be inserted into the toolbox. In particular, the sequence of the second guide nucleic acid can be the same or different from the sequence of the first guide nucleic acid.

[0860] When the sequence of the second guide nucleic acid is identical to the sequence of the first guide nucleic acid, greater amounts of the guide nucleic acid can be expressed in the cell, and gene editing efficiency using the toolbox can be increased.

[0861] When the sequence of the second guide nucleic acid differs from the first guide nucleic acid, the target sites for gene editing using the toolbox can be diverse.

[0862] [1-3. Second gene deletion editing using recombinase recognition sites (RRS)] [1-3-1. Toolbox for deletion editing using recombinase recognition sites (RRS)] According to some exemplary embodiments of the present disclosure, a toolbox for deletion editing may be provided.

[0863] The toolbox for deletion editing can include a first ITR sequence, a polynucleotide encoding a first recombinase recognition site (RRS1), a first polynucleotide, a polynucleotide encoding a second recombinase recognition site (RRS2), and a second ITR sequence.

[0864] The first polynucleotide may include, but is not limited to, one or more selected from a polynucleotide encoding a protein or RNA, a non-transcribed polynucleotide, a polynucleotide encoding an untranslated RNA, and a non-functional polynucleotide.

[0865] The first recombinase recognition site (RRS1) and the second recombinase recognition site (RRS2) can form a pair. For convenience of explanation, hereinafter, it is assumed that the sequence of the first recombinase recognition site (RRS1) is identical to the sequence of the second recombinase recognition site (RRS2). Hereinafter, several exemplary embodiments of the toolbox for deletion editing are described.

[0866] For example, a toolbox for deletion editing can be a toolbox that includes a first ITR sequence, a polynucleotide encoding a constitutive promoter, a polynucleotide encoding a first recombinase recognition site (RRS1), a stop codon, a polynucleotide encoding a second recombinase recognition site (RRS2), a polynucleotide encoding a first RNA-guided endonuclease, and a second ITR sequence.

[0867] In another example, a toolbox for deletion editing can be a toolbox including a first ITR sequence, a polynucleotide encoding a constitutive promoter, a polynucleotide encoding a first recombinase recognition site (RRS1), a transcription stop codon, a polynucleotide encoding a second recombinase recognition site (RRS2), a polynucleotide encoding a first guide nucleic acid, and a second ITR sequence.

[0868] Hereinafter, a method for second gene deletion editing using the above-mentioned toolbox for deletion editing is described, i.e., a method for deleting a specific polynucleotide is described.

[0869] [1-3-2. Method for editing a second gene deletion using recombinase recognition sites (RRS)] In a genome into which a toolbox for deletion editing has been inserted, a first polynucleotide located between a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a second recombinase recognition site (RRS2) may be deleted.

[0870] One method for deleting a first polynucleotide can include delivering a first recombinase to a cell into which the toolbox for deletion editing is inserted.

[0871] The first recombinase can interact with the first recombinase recognition site (RRS1) and / or the second recombinase recognition site (RRS2).

[0872] Methods for delivering recombinase to cells have been described above and so details of the methods will not be provided here.

[0873] The first recombinase delivered to the cell by the above-described method can interact with a polynucleotide encoding a first recombinase recognition site (RRS1) and a polynucleotide encoding a second recombinase recognition site (RRS2). The first polynucleotide can be deleted from the toolbox by the interaction.

[0874] Hereinafter, various exemplary embodiments of the deletion editing according to the above method and the effect of the deletion editing will be described, which may vary according to the type of the first polynucleotide and / or the toolbox for deletion editing.

[0875] When a polynucleotide encoding a constitutive promoter, a polynucleotide encoding a first recombinase recognition site (RRS1), a stop codon, a polynucleotide encoding a second recombinase recognition site (RRS2), and a polynucleotide encoding a first RNA-guided endonuclease are included in a toolbox for deletion editing according to some exemplary embodiments provided in the present disclosure, the stop codon can be deleted from the toolbox.

[0876] In this case, the first RNA-guided endonuclease can be expressed by deletion of a stop codon, i.e., the time point of transcription and / or translation of a particular polynucleotide contained in the toolbox can be regulated by deletion of a partial component of the toolbox.

[0877] When a polynucleotide encoding a constitutive promoter, a polynucleotide encoding a first recombinase recognition site (RRS1), a transcription stop codon (polyT), a polynucleotide encoding a second recombinase recognition site (RRS2), and a polynucleotide encoding a first guide nucleic acid are included in a toolbox for deletion editing according to some exemplary embodiments provided in the present disclosure, the transcription stop codon (polyT) can be deleted from the toolbox.

[0878] In this case, the first guide nucleic acid can be expressed by deletion of a transcription termination codon (polyT), i.e., the time of transcription and / or translation of a particular polynucleotide included in the toolbox can be regulated by deletion of partial components of the toolbox.

[0879] 2. Second gene editing using engineered nuclease components expressed from the toolbox Hereinafter, a second gene edit is described that uses engineered nuclease components expressed from a polynucleotide contained in the toolbox that is inserted into the genome.

[0880] The second gene edit using a component of the engineered nuclease can occur outside the toolbox or inside the toolbox.

[0881] [2-1. Secondary gene editing using engineered nuclease components expressed from a toolbox that does not contain expression regulators] [2-1-1. Toolboxes that do not include expression regulators]

[0882] According to some exemplary embodiments of the present disclosure, a toolbox that does not include expression regulators may be provided.

[0883] The toolbox without expression regulators may comprise a first ITR sequence, a polynucleotide encoding a component of the modified nuclease, and a second ITR sequence.

[0884] Hereinafter, several exemplary embodiments of the toolbox that do not include expression regulators are described.

[0885] For example, the toolbox can be a toolbox that includes one polynucleotide that encodes an RNA-guided endonuclease.

[0886] In another example, the toolbox may be a toolbox comprising two or more polynucleotides encoding an RNA-guided endonuclease, where the sequences of the two or more polynucleotides encoding the RNA-guided endonuclease may be identical or different from each other.

[0887] When the sequences of two or more polynucleotides encoding RNA-guided endonucleases included in the toolbox are identical, the expression amount of the same RNA-guided endonuclease can be increased in a cell, thereby increasing the second gene editing efficiency.

[0888] In yet another example, the toolbox can be a toolbox that includes one polynucleotide encoding a guide nucleic acid.

[0889] In yet another example, the toolbox can be a toolbox that includes two or more polynucleotides encoding guide nucleic acids, where the sequences of the two or more polynucleotides encoding the guide nucleic acids can be the same or different.

[0890] When at least one sequence of two or more polynucleotides encoding guide nucleic acids included in the toolbox is identical, the expression amount of the guide nucleic acid having the identical sequence can be increased in the cell, thereby increasing the second gene editing efficiency.

[0891] When at least one sequence of two or more polynucleotides encoding guide nucleic acids included in the toolbox differs, various types of guide nucleic acids can be expressed in the cell, in which case a second gene edit can occur at a larger number of target sites.

[0892] In yet another example, the toolbox can be a toolbox that includes one or more types of polynucleotides encoding an RNA-guided endonuclease and one or more types of polynucleotides encoding a guide nucleic acid.

[0893] In this case, the first gene edit can insert the toolbox into the genome of the cell without further processing, and then the second gene edit can occur at a target site present in the genome of the cell, where one or more of the guide nucleic acids expressed from the toolbox can bind complementary to the target site.

[0894] That is, when a toolbox including a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid is inserted into a genome without including an expression control element, an nth gene edit (n is a natural number equal to or greater than 2) can occur in the genome without further processing.

[0895] The above-mentioned toolboxes that do not contain expression regulators can be inserted into the genome and / or chromosomes of a cell.

[0896] Each toolbox having various configurations described above can be inserted into the genome and / or chromosome of a cell through various combinations.

[0897] According to some exemplary embodiments provided in the present disclosure, the first toolbox and the second toolbox can be inserted into the genome of a single cell.

[0898] For example, a first toolbox may include a first ITR sequence, one or more polynucleotides encoding an RNA-guided endonuclease, and a second ITR sequence, a second toolbox may include a third ITR sequence, one or more polynucleotides encoding a guide nucleic acid, and a fourth ITR sequence.

[0899] According to some exemplary embodiments provided in the present disclosure, each of the first toolbox and the second toolbox can be separately inserted into the genome (and / or chromosome) of a different cell.

[0900] For example, the first toolbox can be inserted into the genome of a first cell present in a single subject, and the second toolbox can be inserted into the genome of a second cell present in the subject. The first toolbox can include one or more types of polynucleotides encoding RNA-guided endonucleases. The second toolbox can include one or more types of polynucleotides encoding guide nucleic acids. Each of the different cells can be a non-isolated cell contained in a single subject.

[0901] Each of the different cells can be a non-isolated cell contained in a fertilized egg and / or an embryo.

[0902] Hereinafter, we describe a method for the second gene editing using the above-mentioned toolbox that encodes the components of an engineered nuclease without including expression regulators.

[0903] [2-1-2. Second gene editing method] In the present disclosure, a method for the second gene editing in cells is provided, in which the toolbox is inserted without containing expression regulator and comprises the polynucleotide encoding the component of modified nuclease.According to the structure of the toolbox inserted into the genome of cells, various methods for the second gene editing can be provided.

[0904] For example, one method of second gene editing in a cell having a genome into which a polynucleotide encoding an RNA-guided endonuclease has been inserted can include delivering a guide nucleic acid to the cell.

[0905] Additionally, a method for second gene editing in a cell having a genome into which a toolbox comprising a polynucleotide encoding an RNA-guided endonuclease has been inserted can include delivering a donor polynucleotide to the cell.

[0906] Delivering a guide nucleic acid to a cell includes introducing a polynucleotide encoding the guide nucleic acid into the cell. In particular, the polynucleotide encoding the guide nucleic acid can be introduced into the cell in the form of RNA or a polynucleotide encoding the guide nucleic acid, or can be incorporated into a vector, which can then be introduced into the cell.

[0907] Additionally, delivering the donor polynucleotide to the cell can include introducing a vector containing the donor polynucleotide into the cell.

[0908] When the guide nucleic acid is delivered to a cell, the guide nucleic acid can bind to a target site.

[0909] Additionally, the RNA-guided endonuclease can be expressed in the cell from the polynucleotide inserted in the toolbox by an expression system of the cell, and the RNA-guided endonuclease can cl...

Claims

1. A method for producing a transgenic bovine animal that expresses a protein of interest that is not naturally expressed in a wild-type bovine animal, said method comprising: preparing a bovine fertilized egg or a bovine embryo, the genome of which comprises a polynucleotide comprising a sequence encoding a target gene and a Cas9 protein; Introducing a donor containing a guide RNA or a nucleic acid encoding the guide RNA and a nucleic acid encoding the protein of interest into the prepared fertilized egg or embryo to insert the nucleic acid encoding the protein of interest into the target gene; obtaining a transgenic bovine fertilized egg or a transgenic bovine embryo, the genome of which comprises said nucleic acid encoding said protein of interest; and transferring the transgenic bovine fertilized egg or transgenic bovine embryo into the uterus of a surrogate mother. the guide RNA is capable of binding to a portion of the target gene and of forming a complex with the Cas9 protein expressed in the conditioned bovine fertilized egg or the bovine embryo, the complex being capable of inducing cleavage in the target gene; The transgenic bovine animal produced has the nucleic acid encoding the protein of interest inserted into the target gene.

2. The method described in claim 1, wherein the conditioned embryo expresses the Cas9 protein.

3. The method described in claim 1, wherein the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes (SpCas9 protein) or a mutant thereof.

4. The method of claim 1, wherein the target gene is located in a safe harbor site.

5. The method of claim 1, wherein the target gene is selected from the group consisting of a PRNP gene, a beta-lactoglobulin (BLG) gene, a retinoblastoma 1 (Rb1) gene, a Nanog gene, a TP53 gene, the polynucleotide comprising the sequence encoding the Cas9 protein, an IFNT gene, an albumin gene, and a beta-casein (BCN) gene.

6. The method of claim 1, wherein the target protein is albumin, interleukin, insulin, erythropoietin, an antibody, or a fragment of said antibody.

7. The method of claim 6, wherein the fragment of the antibody is a gamma chain, delta chain, alpha chain, mu chain, epsilon chain, kappa chain, or lambda chain.

8. The method of claim 1, wherein the conditioned bovine fertilized egg or bovine embryo is produced by in vitro fertilization between an egg and a sperm, and the egg or sperm has a genome comprising the polynucleotide that includes the sequence encoding the target gene and the Cas9 protein.

9. The method of claim 1, wherein the introducing is performed by microinjection or electroporation.

10. The method described in claim 1, wherein the nucleic acid encoding the guide RNA or the donor is introduced into the bovine fertilized egg or bovine embryo by incorporating it into a plasmid vector or a viral vector.

11. A transgenic bovine animal expressing a protein of interest that is not naturally expressed in a wild-type bovine animal, wherein the genome of the transgenic bovine animal has a polynucleotide comprising a sequence encoding a Cas9 protein and a nucleic acid encoding the protein of interest.

12. The transgenic bovine animal described in claim 11, wherein the transgenic bovine animal expresses the Cas9 protein.

13. The transgenic bovine animal described in claim 11, wherein the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes (spCas9 protein) or a mutant thereof.

14. The transgenic bovine animal of claim 11, wherein the target protein is albumin, interleukin, insulin, erythropoietin, an antibody, or a fragment of said antibody.

15. The transgenic bovine animal of claim 14, wherein the fragment of the antibody is a gamma chain, a delta chain, an alpha chain, a mu chain, an epsilon chain, a kappa chain, or a lambda chain.

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