Patent Publication Number: US-2022214327-A1

Title: Method and composition for sorting out of cell comprising a modified gene

Description:
TECHNICAL FIELD 
     Sorting Out Genetically Modified Cells 
     The present disclosure relates to a method for sorting out genetically modified cells. Recently, research for producing genetically modified cells or animals through modification of inserting or removing a gene having a specific trait has been conducted. In the above research, in order to confirm whether the cell has been genetically modified or to use the genetically modified cell, a process for checking whether the cell is genetically modified is essential. 
     An embodiment of the present disclosure relates to a method for more efficiently sorting out genetically modified cells in the genetic modification study. 
     BACKGROUND 
     Genetic modification technology is widely used for the treatment of genetic diseases and incurable diseases and the improvement of animal and plant varieties. In this case, genetic modification means inserting, deleting, or substituting a specific DNA in the genome of an organism. 
     Recently, widely used genetic modification technologies include Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR/Cas system. The current genetic modification-related market is expected to expand from $3.62 billion (about KRW 4.86 trillion) in 2018 to $7.12 billion (about KRW 8.37 trillion) in 2023, growing at an average annual rate of 14.5%, of which CRISPR/Cas9 technology is the largest market size with $1.95 billion (53.8% occupied) in 2018 (Biotechnology Policy Research Center, Global Genome Editing Technologies Industry Outlook (Frost &amp; Sullivan Analysis), 2019.3). 
     In the above genetic modification technology, after modifying a specific DNA in the genome of an organism, it is essential to check whether the specific DNA has been modified or not. That is, the process of sorting out genetically modified cells is essential. Conventionally, PCR has been widely used as a method for checking whether genes have been modified. However, the method of confirming the insertion, deletion, or substitution of a specific DNA through PCR was inefficient in that cells were consumed in this process, or an additional analysis step and time were required. 
     RELATED ART LITERATURE 
     Patent Literature 
     
         
         U.S. Ser. No. 16/612,142 
       
    
     SUMMARY 
     Technical Problem 
     Conventionally, in order to sort out genetically modified cells, a process of analyzing the cells was required. That is, in addition to the step of transforming cells or animals according to the genetic modification method, an additional step of checking whether the genetic modification was performed or not was required. 
     In this way, in order to select genetically modified cells, an additional analysis whether genes are genetically modified is inefficient in the cell sorting out process. In addition, since cells that have undergone the analysis step cannot be utilized, there is a problem that cells are consumed. 
     Accordingly, an objective of the present disclosure is to provide a more efficient method for sorting out genetically modified cells. 
     Technical Solution 
     The present disclosure provides a method for sorting out a cell comprising a modified gene on a target locus in the genome. 
     According to an embodiment of the present disclosure, the method comprises: preparing fluorescent bovine cells (bovine cells exhibiting fluorescence); treating the fluorescent bovine cells with a composition; and selecting non-fluorescent bovine cells (bovine cells that do not exhibit fluorescence). 
     In this case, the fluorescent bovine cell includes a fluorescent protein gene on one or more positions in a genome, wherein the fluorescent protein gene is a different gene from a gene on the target locus in the genome, and the composition comprises: a guide RNA for the fluorescent protein gene or a nucleic acid encoding the same; a guide RNA for the gene on the target locus in the genome or a nucleic acid encoding the same; and a Cas protein or a nucleic acid encoding the same, wherein the non fluorescent bovine cell comprises a modified gene on the target locus in the genome. 
     In this case, in the method, the composition further comprises a transgene to be inserted into the target locus in the genome. 
     In this time, in the method, the step of preparing the fluorescent bovine cells comprises to use a cow comprising the fluorescent protein gene located on 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in a genome. 
     In this case, in the step of treating the composition on the fluorescent bovine cell in the method, the guide RNA for the fluorescent protein gene or the nucleic acid encoding the same, and the guide RNA for the gene on the target locus in a genome or the nucleic acid encoding the same are simultaneously treated. 
     In this case, in the step of treating the fluorescent bovine cells with the composition in the method, the composition is treated in a vector form. 
     In this case, in the step of treating the fluorescent bovine cells with the composition in the method, the composition is treated in a ribonucleoprotein (RNP) form. 
     The present disclosure provides a cell including a modified gene on a target locus in the genome. 
     As an embodiment of the present disclosure, the cell is a bovine cell, the bovine cell includes a modified gene on a target locus in a genome and a modified fluorescent protein gene on one or more positions in the genome, wherein the fluorescent protein gene is a different gene from the gene on the target locus in the genome. 
     In this case, the modified gene in the bovine cell is a beta-lactoglobulin (BLG) gene or a prion (PRNP) gene. 
     In this case, the bovine cell includes the modified fluorescent protein gene on three positions in the genome. 
     In this case, the one or more positions in the genome include at least one of 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6. 
     The present disclosure provides a method for producing an animal including a modified gene on a target locus in the genome. 
     As an embodiment of the present disclosure, the animal is a cow, and the method includes: preparing a cell that expresses fluorescence; treating composition to the cell that expresses fluorescence; selecting a non-fluorescent cell; and transplanting the non fluorescent cell into the uterus of a surrogate mother. 
     In this case, the cell includes a fluorescent protein gene on one or more positions in a genome, the fluorescent protein gene is a different gene from a gene on the target locus in a genome, and the composition includes a guide RNA for the fluorescent protein gene or a nucleic acid encoding the same; a guide RNA for the gene on the target locus in the genome or a nucleic acid encoding the same; and a Cas protein or a nucleic acid encoding the same, wherein the non fluorescent cell includes a modified gene on the target locus in a genome. 
     In this case, the modified gene is a beta-lactoglobulin (BLG) gene or a prion (PRNP) gene. 
     In this case, the composition further includes a transgene to be inserted on the target locus in the genome. 
     In this case, the one or more positions in the genome include at least one of 95433564-95434563 positions of chromosome 4; 113823097-113823101 positions of chromosome 4; and 20085913-20086912 positions of chromosome 6. 
     The present disclosure provides an animal including a modified gene on a target locus in the genome. 
     As an embodiment of the present disclosure, the animal is a cow, the cow includes a modified gene on a target locus in a genome, the cow includes a modified fluorescent protein gene on one or more positions in the genome, and the fluorescent protein gene is a different gene from the gene on the target locus gene in the genome, and the fluorescent protein gene present in the genome is transferred the same site in a next generation. 
     In this case, the modified gene in the cow is a beta-lactoglobulin gene or a prion (PRNP) gene. 
     In this case, the cow comprises a fluorescent protein gene modified on three positions in the genome. 
     In this case, the one or more positions in the genome comprise at least one of 95433564-95434563 positions of chromosome 4; 113823097-113823101 positions of chromosome 4; and 20085913-20086912 positions of chromosome 6. 
     The present disclosure provides a kit for sorting out cells including a modified gene on a target locus in the genome. 
     As an embodiment of the present disclosure, the kit comprises: a fluorescent bovine cell; a guide RNA for a fluorescent protein gene, or a nucleic acid encoding the same; and a Cas protein, or a nucleic acid encoding the same, wherein the fluorescent bovine cell includes a fluorescent protein gene on one or more positions in a genome. 
     In this case, the fluorescent protein in the kit is a green fluorescent protein. 
     In this case, the kit further comprises a guide RNA for a gene on a target locus in the genome or a nucleic acid encoding the same. 
     In this case, the kit further includes a transgene to be inserted on the target locus in the genome, in addition to the guide RNA for a gene on a target locus gene in the genome or a nucleic acid encoding the same. 
     In this case, the Cas protein in the kit is a Cas9 protein or a Cpf1 protein. 
     In this case, in the kit, the fluorescent bovine cell includes a fluorescent protein gene on three positions in the genome. 
     In this case, the one or more positions in the genome include at least one of 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6. 
     Advantageous Effects of Invention 
     The methods and materials for sorting out genetically modified cells disclosed herein provide a more efficient method of sorting out cells. 
     1) Since an additional analysis process for sorting out genetically modified cells is not required, it is possible to prevent cell consumption during the analysis process. 
     2) Since an additional analysis process for sorting out genetically modified cells is not required, the time and effort required for the analysis process may be saved. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a mutation test result of a prion (PRNP) gene in a single cell colony. 
       3, 4, 5, 8, 9, 10, 19, 21, 22, 24, 25 in (a); 3, 4, 5, 6, 7, 8, 9, 10, 11 in (b) represent mutant colonies, M represents a marker, NC represents a negative control group, and PC represents a positive control group. 
         FIG. 2  shows a mutation test result of the beta-lactoglobulin (BLG) gene in a single cell colony. 
       2, 7, 8, 9, 10, 11, 12 in (a); 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14 in (b) represent mutant colonies, M represents a marker, NC represents a negative control group, and PC represents a positive control group. 
         FIG. 3  is a photograph of cells. 
       a) Bright-field image and a′) fluorescence image of early passage cultured cells; b) bright-field images and b′) fluorescence images of primary cells transfected with Cas9 and sgRNA for GFP; c) bright-field images and c′) fluorescence images of primary cells transfected with Cas9 and sgRNA for GFP and PRNP. 
         FIG. 4  shows a result showing the ratio of (a) prion (PRNP) gene knock-out cell ratio and (b) beta-lactoglobulin (BLG) gene knock-out cell ratio in GFP (+) cells and GFP (−) cells. The mutant colony ratio of the prion (PRNP) gene in the GFP negative cell group was higher (90.0% vs. 58.3%) than in the GFP positive cell group. The mutant colony ratio of beta-lactoglobulin (BLG) genes in the GFP negative cell group was higher (79% vs. 58%) than in the GFP positive cell group. 
         FIG. 5  is a photomicrograph of a group of GFP-expressing bovine cells (control group), a group in which GFP-expressing bovine cells were treated with GFP sgRNA and Cas9 protein (knock-out group), and a group in which GFP-expressing bovine cells were treated with GFP sgRNA, Cas9 protein, and Donor DNA (knock-in group). 
         FIG. 6  is a PCR test result for confirming the GFP gene extracted from blastocysts of the obtained three groups and the gene inserted into the GFP gene sequence. 
     
    
    
     DETAILED DESCRIPTION 
     Term Definition 
     Definitions of key terms used in this specification are as follows. 
     Gene Modification or Gene Editing 
     Genetic modification or genetic manipulation, as used herein, refers to the creation of an insertion, deletion and/or substitution of a specific DNA sequence on a target locus in the genome of a cell. The genetic modification includes a gene mutation. The genetic modification includes a gene knock-out of a target locus in the genome and/or a knock-in of a transgene. The gene knock-out refers to a modification that reduces the function of a gene so that the expression of the gene is undetectably small or not expressed. The gene knock-in refers to a modification of inserting a gene to be expressed in a cell. In this case, in the present disclosure, the genetically modified or genetically edited cell may be referred to as an ‘engineered cell’. 
     Safe Harbor 
     Safe harbor, as used herein, means a specific position in the genome where an inserted gene can be stably expressed in the cell without interfering with the expression or regulation of the gene adjacent to the inserted position after a specific gene is inserted into the genome of a cell. 
     Target Locus (Target Region) 
     As used herein, a ‘target locus’ or a ‘target region’ refers to a region on a genome in which a gene to be edited exists. That is, it means including a region to be artificially manipulated on the genome and is a region including the protospacer sequence and the target sequence indicated below. 
     Gene on a Target Locus 
     As used herein, a gene on a target locus means a gene located at a specific target locus (target region) in a genome, and the gene is a gene in which cleavage occurs by gene editing. As an example, the gene may be knocked out through cleavage. As another example, after the gene is cleavage, a transgene may be inserted on the cleavage site. 
     Protospacer Sequence 
     The term ‘protospacer sequence’ refers to about 20 sequences adjacent to the PAM sequence in the target region of the present application. The protospacer sequence and the target sequence are complementary sequences. That is, it means the same sequence as the guide sequence that complementarily binds to the target sequence. However, the guide sequence may have the same sequence in which T (thymine) of the protospacer sequence is substituted with U (uracil). 
     Target Sequence 
     The term ‘target sequence’ of the present application is a sequence included in the target region of the present application, and is a sequence complementary binding to a protospacer sequence. The target sequence may bind complementary to the guide sequence. 
     Meaning of A, T, C, G, and U 
     As used herein, the symbols A, T, C, G, and U are interpreted as meanings understood by those of ordinary skilled in the art. It may be properly interpreted as a base, a nucleoside, or a nucleotide on DNA or RNA according to context and technology. For example, when it means a base, it can be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U) itself, respectively. When it means a nucleoside, it can be interpreted as adenosine (A), thymine (T), cytidine (C), guanosine (G), or uridine (U), respectively, and when it means a nucleotide in the sequence, it should be interpreted to mean a nucleotide including each of the nucleosides. 
     CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas System 
     The CRISPR-Cas system means that it is derived from an acquired immune system that stores genetic information of pathogens that have invaded from the outside in bacteria and cuts them when re-invading later. 
     The CRISPR Cas system used herein is composed of a guide RNA capable of recognizing a specific DNA sequence and a Cas protein capable of cutting DNA. The guide RNA may interact with the Cas protein. The guide RNA may form a guide RNA-Cas protein complex through this interaction with the Cas protein. The guide RNA-Cas protein complex guides the Cas protein to a specific region of DNA, allowing DNA cleavage to occur in that region. 
     DNA cleavage caused by the CRISPR-Cas system is repaired by homology directed repair (HDR) or non-homologous end joining in cells. In homologous recombination, when template DNA of a homologous base sequence exists, repair occurs based on the homologous template DNA. On the other hand, in non-homologous end joining, DNA repair occurs when several bases are inserted or deleted (indels, insertions, or deletions) in the process of joining the cut ends. 
     The CRISPR-Cas system may be used for gene editing through DNA cleavage. 
     Guide RNA 
     Guide RNA, as used herein, refers to an RNA that recognizes a partial nucleotide sequence of DNA in a cell and interacts with a Cas protein. 
     The guide RNA includes crRNA and/or tracrRNA. 
     As an example, the guide RNA may be composed of only crRNA, and in another example, the guide RNA may be composed of crRNA and tracrRNA. 
     The guide RNA may be a single guide RNA in which the crRNA and the tracrRNA are composed of a single strand, or a dual guide RNA in which the crRNA and the tracrRNA are composed of two strand separated from each other. 
     crRNA 
     The crRNA comprises a guide sequence and may further comprise a first complementary sequence complementary binding to the tracrRNA. 
     The guide sequence is a sequence in which a sequence complementary to a target sequence has the same identity as a protospacer sequence and is an RNA sequence consisting of U (uracil) instead of T (thymine) among the corresponding protospacer sequences. In this case, the guide sequence has complete complementarity with respect to the protospacer sequence or has at least 60, 70, 80, 90%, or more complementarity. As an example, the guide sequence may be 5 to 30 base sequences. As an example, the guide sequence may be 10 to 25 base sequences. 
     The first complementary sequence may be derived from a first complementary sequence derived from a natural origin, or may include a sequence having sequence identity therewith. AS an example, the first complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the first complementary sequence may include 5′-GUUUUAGAGCUA-3′ (SEQ ID NO: 270), or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the first complementary sequence may include 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′ (SEQ ID NO: 271) or 5′-GUUUUAGUCCCUU-3′ (SEQ ID NO: 272), or may include a sequence having at least 50% sequence identity therewith. 
     tracrRNA 
     The tracrRNA includes a second complementary sequence for complementary binding to the crRNA. 
     The second complementary sequence may be derived from a naturally occurring second complementary sequence, or may include a sequence having sequence identity therewith. As an example, the second complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the second complementary sequence may include 5′-UAGCAAGUUAAAAU-3′ (SEQ ID NO: 273), or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the second complementary sequence may include 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′ (SEQ ID NO: 274) or 5′-AAGGGACUAAAAU-3′ (SEQ ID NO: 275), or may include a sequence having at least 50% sequence identity therewith. 
     The tracrRNA may further comprise a tail sequence. 
     The tail sequence may be derived from a naturally occurring tail sequence, or may include a sequence having sequence identity therewith. AS an example, the tail sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the tail sequence may include 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′ (SEQ ID NO: 276), or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the tail sequence may include 5′-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3′ (SEQ ID NO: 277), or may include a sequence having at least 50% sequence identity therewith. 
     Cas Protein 
     Cas protein, as used herein, refers to a protein capable of cleaving DNA in a cell. 
     As an example, the Cas protein may be at least one selected from the group consisting of  Streptococcus pyogenes -derived Cas9 protein,  Campylobacter jejuni -derived Cas9 protein,  Streptococcus  thermophiles-derived Cas9 protein,  Streptococcus aureus -derived Cas9 protein,  Neisseria meningitidis -derived Cas9 protein, and Cpf1. The Cas protein includes an artificially modified protein in addition to the wild-type protein. 
     The Cas protein may comprise a domain capable of cleaving DNA and a domain recognizing a PAM sequence. 
     The domain capable of cleaving DNA can cleave both strands of DNA. Alternatively, only the strand that interacts with the guide sequence may be cleaved. Alternatively, only the complementary strand of the strand interacting with the guide sequence may be cleaved. Also, the DNA cleavage method may be different depending on the type of Cas protein. As an example, the Cas9 protein can cut two DNA strands side by side. As another example, the Cpf1 protein may not cut two DNA strands side by side. 
     A domain recognizing a PAM sequence may have a different recognized PAM sequence depending on the type of Cas protein. As an example, the Cas9 protein derived from  Streptococcus pyogenes  may recognize the PAM sequence of 5′-NGG-3′ (N is A, T, C, or G). 
     The Cas protein may interact with a guide RNA. The Cas protein may interact with the guide RNA to form a guide RNA-Cas protein complex. As an example, the Cas9 protein may interact with a guide RNA including both crRNA and tracrRNA. As another example, the Cpf1 protein may interact with a guide RNA that does not include tracrRNA. 
     PAM Sequence 
     As used herein, a PAM sequence is a base sequence present in DNA, and the PAM sequence may be recognized by a Cas protein. Recognizing the PAM sequence of the Cas protein may affect the DNA cleavage function of the Cas protein. 
     The PAM sequence exists in a strand in which a protospacer sequence exists in the target region. 
     The PAM sequence may have a different sequence depending on the origin of the Cas protein. 
     As an example, the PAM sequence may be one of 5′-NGG-3′ (N is A, T, C, or G), 5′-NNGRR(N)-3′ (N is each independently A, T, C, or G, and R is A or G), 5′-TTN-3′ (N is A, T, C, or G), 5′-NNNNGATT-3′ (N is A, T, C, or G), 5′-NNAGAA-3′ (N is A, T, C, or G), and the like. 
     Transgene 
     As used herein, a transgene may be a gene encoding a protein of interest. In the present disclosure, the transgene may be inserted into a genome of a cell to replace an existing protein or express a novel protein. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skilled in the art to which this invention belongs. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. 
     Hereinafter, specific details of the present invention will be disclosed. 
     I. Genetically Modified Cell Selection Method 
     An example disclosed in the present disclosure is a method for sorting out a cell including a modified gene at a target locus in the genome. The method uses a fluorescent cell to sorting out a genetically modified cell without an additional analysis process. The fluorescent cell refers to a cell expressing a fluorescent protein gene by including the fluorescent protein gene in the genome of the cell. 
     In one embodiment, the method comprises the following steps. 
     (a) preparing a fluorescent cell; 
     (b) treating the fluorescent cell with a composition; and 
     (c) selecting a non fluorescent cell. 
     Each step will be described in detail below. 
     1. Preparing a Fluorescent Cell 
     In the present disclosure, in order to efficiently sort out the genetically modified cells without an additional analysis method, a cell expressing fluorescence (fluorescent cell) is used. 
     1) Fluorescent Cell that Expresses Fluorescence 
     The fluorescent cell disclosed in the present disclosure refers to a cell expressing a fluorescent protein gene by including the fluorescent protein gene in the genome of the cell. 
     i) Cells 
     As an example, the cell may be a non-human mammalian cell. As an example, the cell may be a cell of a cow, a pig, a mouse, a rat, and the like. As a specific example, the cell is a bovine cell. 
     As another example, the cell may be a somatic cell or a germ cell. As a specific example, the cell may be a fertilized egg obtained by the fertilization of a germ cell. As another specific example, the cell may be a blastocyst in which a fertilized egg is cell-divided. 
     ii) Fluorescent Protein Gene 
     The fluorescent protein gene is contained in the genome of the cell. As an example, the fluorescent protein gene is contained in a safe harbor in the genome of the cell. The safe harbor may include AAVS1, CCR5, ROSA26, ACTB, and the like. As another example, the fluorescent protein gene is included in an intron in the genome of a cell. As a specific example, when the cell is a bovine cell, the fluorescent protein gene may be included in one or more positions of 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 positions of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome of the bovine cell. As an example, the fluorescent protein gene may be included in one or more positions of 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6. 
     The fluorescent protein gene is present on one or more positions in the cell genome. As an example, the fluorescent protein gene is present on two positions in the cell genome. As an example, the fluorescent protein gene is present on three or more positions in the cell genome. 
     As an example, the fluorescent protein gene may be one or more among a green fluorescent protein gene (GFP), a blue fluorescent protein gene (BFP), a cyan fluorescent protein gene (CFP), a yellow fluorescent protein gene (YFP), a red fluorescent protein gene (RFP), and the like. As a specific example, the fluorescent protein gene is a green fluorescent protein gene. 
     iii) Relationship Between Fluorescent Protein Gene and Target Locus Gene 
     The fluorescent protein gene and an interest gene on the target locus in the cell genome are different genes. The fluorescent protein gene and the gene on the target locus in the cell genome exist on different positions. 
     In this case, the fluorescent cell may be prepared as a method of using a wild-type cell or an animal expressing a fluorescent protein gene. 
     2) Method of Using Wild-Type Cell 
     i) Wild-Type Cell 
     In one aspect of the method of preparing the cell expressing fluorescence, a method of inserting a fluorescent protein gene into a wild-type cell may be used. 
     The wild-type cell may be a somatic cell or a germ cell. As an example, a fluorescent cell may be prepared by inserting a fluorescent protein gene into a somatic cell. As another example, a fluorescent cell may be prepared by inserting a fluorescent protein gene into a fertilized egg using germ cells. As another example, a fluorescent cell may be prepared by inserting a fluorescent protein gene into a blastocyst in which a fertilized egg is cell-divided. 
     ii) Method of Inserting a Fluorescent Protein Gene into a Wild-Type Cell 
     As a method of inserting the fluorescent protein gene, a method of using plasmid DNA or a virus, a method of using a transposon or a method of using gene scissors may be used. The method of using the transposon may be at least one of a using piggyBac transposon system and a sleeping beauty transposon system. The method of using the gene scissors may be a method of using one or more of specific target nucleases. As an example, the method of using the gene scissors may be one or more of methods of using zinc finger nuclease (ZFN), TALEN, and CRISPR-Cas. 
     In the method of inserting the fluorescent protein gene, the fluorescent protein gene may be inserted on one or more positions in the genome of a cell. As an example, the fluorescent protein gene may be inserted on two positions in the genome of a cell. As another example, the fluorescent protein gene may be inserted on three or more positions in the genome of a cell. 
     At this time, the fluorescent protein gene is inserted into the safe harbor in the genome of the cell. The safe harbor may include AAVS1, CCR5, ROSA26, ACTB, and the like. As an example, the fluorescent protein gene is inserted into an intron position in the genome of a cell. As a specific example, the fluorescent protein gene may be inserted on one or more positions in the genome of a bovine cell. As a specific example, the fluorescent protein gene may be inserted on at least one position of 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome of a bovine cell. As an example, the fluorescent protein gene may be inserted into at least one position of 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6. 
     3) Method of Using an Animal Expressing a Fluorescent Protein Gene 
     i) An Animal Expressing a Fluorescent Protein Gene 
     As another aspect of the method for preparing the cell expressing a fluorescence, a method using a transgenic animal expressing a fluorescent protein gene may be used. As an example, the animal expressing the fluorescent protein gene may be one or more of a cow, a pig, a mouse, a rat, and the like. As an example, a cow expressing the fluorescent protein gene may be used. 
     The animal expressing the fluorescent protein gene may be an animal expressing one or more genes among green fluorescent protein gene (GFP), blue fluorescent protein gene (BFP), cyan fluorescent protein gene (CFP), yellow fluorescent protein gene (YFP), red fluorescent protein gene (RFP), and the like. As an example, the animal expressing the green fluorescent protein gene may be used. 
     In the animal expressing the fluorescent protein gene, the fluorescent protein gene is inserted on one or more positions in the genome. As an example, an animal in which a fluorescent protein gene is inserted into a safe harbor in the genome may be used. The safe harbor may include AAVS1, CCR5, ROSA26, ACTB, and the like. As an example, an animal in which a fluorescent protein gene is inserted into an intron in the genome may be used. As a specific example, a cow in which the fluorescent protein gene is inserted into one or more positions among 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome may be used. As an example, a cow in which the fluorescent protein gene is inserted into one or more positions among 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6 may be used. As a specific example, a cow including a fluorescent protein gene described in the Yum S Y et al. literature (Long-term health and germ line transmission in transgenic cattle following transposon-mediated gene transfer. BMC Genomics 2018; 19:387) may be used. 
     ii) Fluorescent Cell Prepared Using Animal Expressing Fluorescent Protein Gene 
     The fluorescent cell may be a somatic cell or a germ cell. As an example, a fluorescent cell may be prepared by separating a somatic cell from an animal expressing a fluorescent protein gene. As another example, a fluorescent cell may be prepared by separating a germ cell from an animal expressing a fluorescent protein gene. As a specific example, the fluorescent cell may be prepared by separating germ cells from an animal expressing a fluorescent protein gene and then using them to make fertilized eggs or blastocysts. As another specific example, the fluorescent cell may be prepared by separating germ cells from a cow including fluorescent protein gene described in the Yum S Y et al. literature (Long-term health and germ line transmission in transgenic cattle following transposon-mediated gene transfer. BMC Genomics 2018; 19:387) and then using them to make fertilized eggs or blastocysts. 
     2. Treating the Fluorescent Cell with a Composition 
     In the present disclosure, the cell are treated with a composition. 
     1) Construction of Composition 
     The composition comprises 
     i) a guide RNA for a gene on a target locus in the genome or a nucleic acid sequence encoding the same; 
     ii) a guide RNA for a fluorescent protein gene or a nucleic acid sequence encoding the same; and 
     iii) Cas protein or a nucleic acid sequence encoding the same. 
     In this case, the composition may further comprise a transgene to be inserted into the target locus in the genome. 
     i) A Guide RNA (First Guide RNA) for the Gene on the Target Locus in the Genome 
     The composition includes a guide RNA for a gene on a target locus in a genome. The first guide RNA is a component for modifying the gene on the target locus in the genome. 
     The first guide RNA is a single guide RNA or a dual guide RNA. The first guide RNA may recognize the gene on the target locus in the genome of the cell and interact with a Cas protein. 
     The first guide RNA includes crRNA and/or tracrRNA. 
     The crRNA may include a first guide sequence and additionally include a first complementary sequence that binds complementary to tracrRNA. 
     The first guide sequence is a sequence having the same identity with a protospacer sequence, which is a sequence complementary to a target sequence of DNA in the cell, and is an RNA sequence composed of U (uracil) instead of T (thymine) among the corresponding protospacer sequences. In this case, the guide sequence has complete complementarity to the protospacer sequence or has at least 60, 70, 80, 90%, or more complementarity. As an example, the guide sequence may be 5 to 30 base sequences. As an example, the guide sequence may be 10 to 25 base sequences. 
     As an example, the first guide RNA may have a first guide sequence having the same identity with a protospacer sequence, which is a sequence complementary to a target sequence of a gene DNA to suppress expression. As another example, the first guide RNA may have a first guide sequence capable of interacting with a protospacer sequence, which is a sequence complementary to a target sequence of gene DNA present at a position into which a transgene is to be inserted. 
     As an example, the first guide RNA may have one or more guide sequences from SEQ ID NO: 1 to 150. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Guide sequence of guide RNA for 
               
               
                 beta-lactoglobulin (BLG) gene 
               
            
           
           
               
               
               
            
               
                   
                 Gudie Sequence(5′ to 3′) 
                 SEQ ID NO 
               
               
                   
                   
               
               
                   
                 GGAGAUGUCGCUGGCCGCCA 
                 1 
               
               
                   
                   
               
               
                   
                 GUACUCCUUGGCCAUGGCGGCCA 
                 2 
               
               
                   
                   
               
               
                   
                 GCCAUGGCGGCCAGCGACAUCUC 
                 3 
               
               
                   
                   
               
               
                   
                 AGCUCCUCCACAUACACUCUCAG 
                 4 
               
               
                   
                   
               
               
                   
                 UGCAGCAGGAUCUCCAGGUCGCC 
                 5 
               
               
                   
                   
               
               
                   
                 CUGCAGCAGGAUCUCCAGGUCGC 
                 6 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Guide sequence of guide RNA for 
               
               
                 prion (PRNP) gene 
               
            
           
           
               
               
               
            
               
                   
                 Gudie Sequence(5′ to 3′) 
                 SEQ ID NO 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 AUCAUGGUGAAAAGCCACAU 
                 7 
               
               
                   
                   
               
               
                   
                 UGAAAAGCCACAUAGGCAGU 
                 8 
               
               
                   
                   
               
               
                   
                 CCACAUAGGCAGUUGGAUCC 
                 9 
               
               
                   
                   
               
               
                   
                 CCAGGAUCCAACUGCCUAUG 
                 10 
               
               
                   
                   
               
               
                   
                 UUGGAUCCUGGUUCUCUUUG 
                 11 
               
               
                   
                   
               
               
                   
                 ACAUGGCCACAAAGAGAACC 
                 12 
               
               
                   
                   
               
               
                   
                 UGGUUCUCUUUGUGGCCAUG 
                 13 
               
               
                   
                   
               
               
                   
                 UGUGGCCAUGUGGAGUGACG 
                 14 
               
               
                   
                   
               
               
                   
                 GUGGCCAUGUGGAGUGACGU 
                 15 
               
               
                   
                   
               
               
                   
                 GAGGCCCACGUCACUCCACA 
                 16 
               
               
                   
                   
               
               
                   
                 GUUUUGGUCGCUUCUUGCAG 
                 17 
               
               
                   
                   
               
               
                   
                 UGCAAGAAGCGACCAAAACC 
                 18 
               
               
                   
                   
               
               
                   
                 AAGAAGCGACCAAAACCUGG 
                 19 
               
               
                   
                   
               
               
                   
                 AAGCGACCAAAACCUGGAGG 
                 20 
               
               
                   
                   
               
               
                   
                 GACCAAAACCUGGAGGAGGA 
                 21 
               
               
                   
                   
               
               
                   
                 UUCCAUCCUCCUCCAGGUUU 
                 22 
               
               
                   
                   
               
               
                   
                 CCUGGAGGAGGAUGGAACAC 
                 23 
               
               
                   
                   
               
               
                   
                 CCAGUGUUCCAUCCUCCUCC 
                 24 
               
               
                   
                   
               
               
                   
                 CUGGAGGAGGAUGGAACACU 
                 25 
               
               
                   
                   
               
               
                   
                 UGGAGGAGGAUGGAACACUG 
                 26 
               
               
                   
                   
               
               
                   
                 GGAGGAGGAUGGAACACUGG 
                 27 
               
               
                   
                   
               
               
                   
                 GAGGAGGAUGGAACACUGGG 
                 28 
               
               
                   
                   
               
               
                   
                 ACUGGGGGGAGCCGAUACCC 
                 29 
               
               
                   
                   
               
               
                   
                 GGGGAGCCGAUACCCAGGAC 
                 30 
               
               
                   
                   
               
               
                   
                 GGGAGCCGAUACCCAGGACA 
                 31 
               
               
                   
                   
               
               
                   
                 GACUGCCCUGUCCUGGGUAU 
                 32 
               
               
                   
                   
               
               
                   
                 UACCCAGGACAGGGCAGUCC 
                 33 
               
               
                   
                   
               
               
                   
                 CUCCAGGACUGCCCUGUCCU 
                 34 
               
               
                   
                   
               
               
                   
                 CCAGGACAGGGCAGUCCUGG 
                 35 
               
               
                   
                   
               
               
                   
                 CCUCCAGGACUGCCCUGUCC 
                 36 
               
               
                   
                   
               
               
                   
                 GGUGGAUAACGGUUGCCUCC 
                 37 
               
               
                   
                   
               
               
                   
                 AGGCAACCGUUAUCCACCUC 
                 38 
               
               
                   
                   
               
               
                   
                 GGCAACCGUUAUCCACCUCA 
                 39 
               
               
                   
                   
               
               
                   
                 AACCGUUAUCCACCUCAGGG 
                 40 
               
               
                   
                   
               
               
                   
                 ACCGUUAUCCACCUCAGGGA 
                 41 
               
               
                   
                   
               
               
                   
                 CCGUUAUCCACCUCAGGGAG 
                 42 
               
               
                   
                   
               
               
                   
                 CCCCUCCCUGAGGUGGAUAA 
                 43 
               
               
                   
                   
               
               
                   
                 CGUUAUCCACCUCAGGGAGG 
                 44 
               
               
                   
                   
               
               
                   
                 UAUCCACCUCAGGGAGGGGG 
                 45 
               
               
                   
                   
               
               
                   
                 CAGCCACCCCCUCCCUGAGG 
                 46 
               
               
                   
                   
               
               
                   
                 CACCUCAGGGAGGGGGUGGC 
                 47 
               
               
                   
                   
               
               
                   
                 ACCUCAGGGAGGGGGUGGCU 
                 48 
               
               
                   
                   
               
               
                   
                 CCUCAGGGAGGGGGUGGCUG 
                 49 
               
               
                   
                   
               
               
                   
                 CCCCAGCCACCCCCUCCCUG 
                 50 
               
               
                   
                   
               
               
                   
                 GGUGGCUGGGGUCAGCCCCA 
                 51 
               
               
                   
                   
               
               
                   
                 GGCUGGGGUCAGCCCCAUGG 
                 52 
               
               
                   
                   
               
               
                   
                 UGGGGUCAGCCCCAUGGAGG 
                 53 
               
               
                   
                   
               
               
                   
                 GUCAGCCCCAUGGAGGUGGC 
                 54 
               
               
                   
                   
               
               
                   
                 UCAGCCCCAUGGAGGUGGCU 
                 55 
               
               
                   
                   
               
               
                   
                 CAGCCCCAUGGAGGUGGCUG 
                 56 
               
               
                   
                   
               
               
                   
                 UGGCCCCAGCCACCUCCAUG 
                 57 
               
               
                   
                   
               
               
                   
                 CUGGCCCCAGCCACCUCCAU 
                 58 
               
               
                   
                   
               
               
                   
                 GCUGGCCCCAGCCACCUCCA 
                 59 
               
               
                   
                   
               
               
                   
                 GGUGGCUGGGGCCAGCCUCA 
                 60 
               
               
                   
                   
               
               
                   
                 GGCUGGGGCCAGCCUCAUGG 
                 61 
               
               
                   
                   
               
               
                   
                 UGGGGCCAGCCUCAUGGAGG 
                 62 
               
               
                   
                   
               
               
                   
                 GCCAGCCUCAUGGAGGUGGC 
                 63 
               
               
                   
                   
               
               
                   
                 CCAGCCUCAUGGAGGUGGCU 
                 64 
               
               
                   
                   
               
               
                   
                 CCCAGCCACCUCCAUGAGGC 
                 65 
               
               
                   
                   
               
               
                   
                 CAGCCUCAUGGAGGUGGCUG 
                 66 
               
               
                   
                   
               
               
                   
                 UGGCCCCAGCCACCUCCAUG 
                 67 
               
               
                   
                   
               
               
                   
                 GGUGGCUGGGGCCAGCCUCA 
                 68 
               
               
                   
                   
               
               
                   
                 GGCUGGGGCCAGCCUCAUGG 
                 69 
               
               
                   
                   
               
               
                   
                 UGGGGCCAGCCUCAUGGAGG 
                 70 
               
               
                   
                   
               
               
                   
                 GCCAGCCUCAUGGAGGUGGC 
                 71 
               
               
                   
                   
               
               
                   
                 CCAGCCUCAUGGAGGUGGCU 
                 72 
               
               
                   
                   
               
               
                   
                 CCCAGCCACCUCCAUGAGGC 
                 73 
               
               
                   
                   
               
               
                   
                 CAGCCUCAUGGAGGUGGCUG 
                 74 
               
               
                   
                   
               
               
                   
                 UGACCCCAGCCACCUCCAUG 
                 75 
               
               
                   
                   
               
               
                   
                 GGUGGCUGGGGUCAGCCCCA 
                 76 
               
               
                   
                   
               
               
                   
                 GGCUGGGGUCAGCCCCAUGG 
                 77 
               
               
                   
                   
               
               
                   
                 UGGGGUCAGCCCCAUGGUGG 
                 78 
               
               
                   
                   
               
               
                   
                 GUCAGCCCCAUGGUGGUGGC 
                 79 
               
               
                   
                   
               
               
                   
                 UCAGCCCCAUGGUGGUGGCU 
                 80 
               
               
                   
                   
               
               
                   
                 CAGCCCCAUGGUGGUGGCUG 
                 81 
               
               
                   
                   
               
               
                   
                 UGUCCCCAGCCACCACCAUG 
                 82 
               
               
                   
                   
               
               
                   
                 CUGUCCCCAGCCACCACCAU 
                 83 
               
               
                   
                   
               
               
                   
                 GCUGUCCCCAGCCACCACCA 
                 84 
               
               
                   
                   
               
               
                   
                 GGUGGCUGGGGACAGCCACA 
                 85 
               
               
                   
                   
               
               
                   
                 GGCUGGGGACAGCCACAUGG 
                 86 
               
               
                   
                   
               
               
                   
                 UGGGGACAGCCACAUGGUGG 
                 87 
               
               
                   
                   
               
               
                   
                 GGACAGCCACAUGGUGGUGG 
                 88 
               
               
                   
                   
               
               
                   
                 AGCCACAUGGUGGUGGAGGC 
                 89 
               
               
                   
                   
               
               
                   
                 GCCACAUGGUGGUGGAGGCU 
                 90 
               
               
                   
                   
               
               
                   
                 CCACAUGGUGGUGGAGGCUG 
                 91 
               
               
                   
                   
               
               
                   
                 CCCCAGCCUCCACCACCAUG 
                 92 
               
               
                   
                   
               
               
                   
                 GGUGGUGGAGGCUGGGGUCA 
                 93 
               
               
                   
                   
               
               
                   
                 GGUGGAGGCUGGGGUCAAGG 
                 94 
               
               
                   
                   
               
               
                   
                 UGGGGUCAAGGUGGUACCCA 
                 95 
               
               
                   
                   
               
               
                   
                 AAGGUGGUACCCACGGUCAA 
                 96 
               
               
                   
                   
               
               
                   
                 GGGUUUGUUCCAUUGACCGU 
                 97 
               
               
                   
                   
               
               
                   
                 UGGGUUUGUUCCAUUGACCG 
                 98 
               
               
                   
                   
               
               
                   
                 AUGUUGGUUUUUGGCUUACU 
                 99 
               
               
                   
                   
               
               
                   
                 CAUGUUGGUUUUUGGCUUAC 
                 100 
               
               
                   
                   
               
               
                   
                 ACAUGCUUCAUGUUGGUUUU 
                 101 
               
               
                   
                   
               
               
                   
                 AAAAACCAACAUGAAGCAUG 
                 102 
               
               
                   
                   
               
               
                   
                 ACCAACAUGAAGCAUGUGGC 
                 103 
               
               
                   
                   
               
               
                   
                 UCCUGCCACAUGCUUCAUGU 
                 104 
               
               
                   
                   
               
               
                   
                 GUGGCAGGAGCUGCUGCAGC 
                 105 
               
               
                   
                   
               
               
                   
                 AGCUGCUGCAGCUGGAGCAG 
                 106 
               
               
                   
                   
               
               
                   
                 GCUGCAGCUGGAGCAGUGGU 
                 107 
               
               
                   
                   
               
               
                   
                 CUGCAGCUGGAGCAGUGGUA 
                 108 
               
               
                   
                   
               
               
                   
                 UGCAGCUGGAGCAGUGGUAG 
                 109 
               
               
                   
                   
               
               
                   
                 GCAGCUGGAGCAGUGGUAGG 
                 110 
               
               
                   
                   
               
               
                   
                 GGAGCAGUGGUAGGGGGCCU 
                 111 
               
               
                   
                   
               
               
                   
                 GCAGUGGUAGGGGGCCUUGG 
                 112 
               
               
                   
                   
               
               
                   
                 GGGCCUUGGUGGCUACAUGC 
                 113 
               
               
                   
                   
               
               
                   
                 GGCCUUGGUGGCUACAUGCU 
                 114 
               
               
                   
                   
               
               
                   
                 UUCCCAGCAUGUAGCCACCA 
                 115 
               
               
                   
                   
               
               
                   
                 UGCUGGGAAGUGCCAUGAGC 
                 116 
               
               
                   
                   
               
               
                   
                 AUGUAUAAGAGGCCUGCUCA 
                 117 
               
               
                   
                   
               
               
                   
                 AGCAGGCCUCUUAUACAUUU 
                 118 
               
               
                   
                   
               
               
                   
                 UCACUGCCAAAAUGUAUAAG 
                 119 
               
               
                   
                   
               
               
                   
                 ACAUUUUGGCAGUGACUAUG 
                 120 
               
               
                   
                   
               
               
                   
                 GCAUGUUUUCACGAUAGUAA 
                 121 
               
               
                   
                   
               
               
                   
                 AGUACACUUGGUUGGGGUAA 
                 122 
               
               
                   
                   
               
               
                   
                 ACCCCAACCAAGUGUACUAC 
                 123 
               
               
                   
                   
               
               
                   
                 GCCUGUAGUACACUUGGUUG 
                 124 
               
               
                   
                   
               
               
                   
                 GGCCUGUAGUACACUUGGUU 
                 125 
               
               
                   
                   
               
               
                   
                 UGGCCUGUAGUACACUUGGU 
                 126 
               
               
                   
                   
               
               
                   
                 CCAAGUGUACUACAGGCCAG 
                 127 
               
               
                   
                   
               
               
                   
                 CCACUGGCCUGUAGUACACU 
                 128 
               
               
                   
                   
               
               
                   
                 UGGUUACUAUACUGAUCCAC 
                 129 
               
               
                   
                   
               
               
                   
                 AGUCAUGCACAAAGUUGUUC 
                 130 
               
               
                   
                   
               
               
                   
                 CUGUGUCAACAUCACAGUCA 
                 131 
               
               
                   
                   
               
               
                   
                 CACAGUCACCACCACCACCA 
                 132 
               
               
                   
                   
               
               
                   
                 ACAGUCACCACCACCACCAA 
                 133 
               
               
                   
                   
               
               
                   
                 CAGUCACCACCACCACCAAG 
                 134 
               
               
                   
                   
               
               
                   
                 AGUCACCACCACCACCAAGG 
                 135 
               
               
                   
                   
               
               
                   
                 GUUCUCCCCCUUGGUGGUGG 
                 136 
               
               
                   
                   
               
               
                   
                 GAAGUUCUCCCCCUUGGUGG 
                 137 
               
               
                   
                   
               
               
                   
                 GGUGAAGUUCUCCCCCUUGG 
                 138 
               
               
                   
                   
               
               
                   
                 UUCGGUGAAGUUCUCCCCCU 
                 139 
               
               
                   
                   
               
               
                   
                 CAUCAUCUUGAUGUCAGUUU 
                 140 
               
               
                   
                   
               
               
                   
                 CGAAACUGACAUCAAGAUGA 
                 141 
               
               
                   
                   
               
               
                   
                 CAUCAAGAUGAUGGAGCGAG 
                 142 
               
               
                   
                   
               
               
                   
                 CAAGAUGAUGGAGCGAGUGG 
                 143 
               
               
                   
                   
               
               
                   
                 CUGGGAUUCUCUCUGGUACU 
                 144 
               
               
                   
                   
               
               
                   
                 CCAGUACCAGAGAGAAUCCC 
                 145 
               
               
                   
                   
               
               
                   
                 CCUGGGAUUCUCUCUGGUAC 
                 146 
               
               
                   
                   
               
               
                   
                 AAUAAGCCUGGGAUUCUCUC 
                 147 
               
               
                   
                   
               
               
                   
                 UCCCAGGCUUAUUACCAACG 
                 148 
               
               
                   
                   
               
               
                   
                 CCCAGGCUUAUUACCAACGA 
                 149 
               
               
                   
                   
               
               
                   
                 CCCUCGUUGGUAAUAAGCCU 
                 150 
               
               
                   
                   
               
            
           
         
       
     
     As a specific example, the first guide RNA has a first guide sequence of 5′-GGAGAUGUCGCUGGCCGCCA-3′ (SEQ ID NO: 1). In another specific example, the first guide RNA has a first guide sequence of 5′-AAAAACCAACAUGAAGCAUG-3′ (SEQ ID NO: 102). 
     The first complementary sequence included in the first guide RNA of the present disclosure may be derived from a naturally occurring first complementary sequence or may include a sequence having sequence identity therewith. As an example, the first complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the first complementary sequence may include 5′-GUUUUAGAGCUA-3′ (SEQ ID NO: 270) or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the first complementary sequence may include 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′ (SEQ ID NO: 271) or 5′-GUUUUAGUCCCUU-3′ (SEQ ID NO: 272), or may include a sequence having at least 50% sequence identity therewith. 
     The tracrRNA included in the first guide RNA of the present disclosure comprises a second complementary sequence that complementarily binds to the crRNA. 
     The second complementary sequence may be derived from a naturally occurring second complementary sequence or may include a sequence having sequence identity therewith. AS an example, the second complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the second complementary sequence may include 5′-UAGCAAGUUAAAAU-3′ (SEQ ID NO: 273) or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the second complementary sequence may include 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′ (SEQ ID NO: 274) or 5′-AAGGGACUAAAAU-3′ (SEQ ID NO: 275), or may include a sequence having at least 50% sequence identity therewith. 
     The tracrRNA included in the first guide RNA of the present disclosure may further comprise a tail sequence. 
     The tail sequence may be derived from a naturally occurring tail sequence or may include a sequence having sequence identity therewith. AS an example, the tail sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the tail sequence may include 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′ (SEQ ID NO: 276) or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the tail sequence may include 5′-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3′ (SEQ ID NO: 277) or may include a sequence having at least 50% sequence identity therewith. 
     Meanwhile, in another embodiment of the present disclosure, a DNA encoding the first guide RNA may be provided. 
     In this case, the DNA sequence encoding the first guide RNA is a sequence comprising a sequence encoding the first guide sequence, the DNA sequence may include at least one of the same DNA sequences (sequences in which U is changed to T in each sequence) as the RNA sequence of SEQ ID NOs: 1 to 150. 
     ii) A Guide RNA for Fluorescent Protein Gene (Second Guide RNA) 
     The composition comprises a guide RNA (second guide RNA) for a fluorescent protein gene. The second guide RNA is a component for modifying the fluorescent protein gene. 
     The second guide RNA may be in the form of a single guide RNA or a dual guide RNA. 
     The second guide RNA may recognize a fluorescent protein gene in a cell genome and interact with a Cas protein. 
     The second guide RNA comprises crRNA and/or tracrRNA. 
     The crRNA included in the second guide RNA of the present disclosure may comprise a second guide sequence and may further comprise a first complementary sequence that complementarily binds to the tracrRNA. 
     The second guide sequence is a sequence having the same identity as a protospacer sequence which is a sequence complementary to a target sequence of DNA in a cell, and is an RNA sequence composed of U (uracil) instead of T (thymine) among the corresponding protospacer sequences. In this case, the guide sequence has complete complementarity to the protospacer nucleotide sequence or has at least 60, 70, 80, 90%, or more complementarity. As an example, the guide sequence may be 5 to 30 base sequences. As an example, the guide sequence may be 10 to 25 base sequences. 
     As an example, the second guide RNA has a second guide sequence, and the second guide sequence may interact with a partial nucleotide sequence of a fluorescent protein gene in a cell genome. The second guide sequence is a sequence having the same identity as a protospacer sequence which is a sequence complementary to a target sequence of the fluorescent protein gene DNA in the cell genome, and is an RNA sequence composed of U (uracil) instead of T (thymine) among the corresponding protospacer sequences. In this case, the guide sequence has complete complementarity or at least 60, 70, 80, 90%, or more complementarity. The second guide sequence may be 10 to 25 base sequences. 
     As an example, the fluorescent protein gene may be one or more genes among a green fluorescent protein gene (GFP), a blue fluorescent protein gene (BFP), a cyan fluorescent protein gene (CFP), a yellow fluorescent protein gene (YFP), a red fluorescent protein gene (RFP), etc. 
     As an example, the second guide RNA may have one or more guide sequences among SEQ ID NOs 151 to 269. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Guide sequence of guide RNA for green 
               
               
                 fluorescent protein (GFP) gene. 
               
            
           
           
               
               
               
            
               
                   
                 Gudie Sequence(5′ to 3′) 
                 SEQ ID NO 
               
               
                   
                   
               
               
                   
                 AAGGGCGAGGAGCUGUUCAC 
                 151 
               
               
                   
                   
               
               
                   
                 AGGGCGAGGAGCUGUUCACC 
                 152 
               
               
                   
                   
               
               
                   
                 GGGCGAGGAGCUGUUCACCG 
                 153 
               
               
                   
                   
               
               
                   
                 CGAGGAGCUGUUCACCGGGG 
                 154 
               
               
                   
                   
               
               
                   
                 CACCGGGGUGGUGCCCAUCC 
                 155 
               
               
                   
                   
               
               
                   
                 GACCAGGAUGGGCACCACCC 
                 156 
               
               
                   
                   
               
               
                   
                 GGUGCCCAUCCUGGUCGAGC 
                 157 
               
               
                   
                   
               
               
                   
                 CCCAUCCUGGUCGAGCUGGA 
                 158 
               
               
                   
                   
               
               
                   
                 CCGUCCAGCUCGACCAGGAU 
                 159 
               
               
                   
                   
               
               
                   
                 GCCGUCCAGCUCGACCAGGA 
                 160 
               
               
                   
                   
               
               
                   
                 CGUCGCCGUCCAGCUCGACC 
                 161 
               
               
                   
                   
               
               
                   
                 GAGCUGGACGGCGACGUGAA 
                 162 
               
               
                   
                   
               
               
                   
                 GGCCACAAGUUCAGCGUGUC 
                 163 
               
               
                   
                   
               
               
                   
                 CGCCGGACACGCUGAACUUG 
                 164 
               
               
                   
                   
               
               
                   
                 CAAGUUCAGCGUGUCCGGCG 
                 165 
               
               
                   
                   
               
               
                   
                 AAGUUCAGCGUGUCCGGCGA 
                 166 
               
               
                   
                   
               
               
                   
                 CAGCGUGUCCGGCGAGGGCG 
                 167 
               
               
                   
                   
               
               
                   
                 AGCGUGUCCGGCGAGGGCGA 
                 168 
               
               
                   
                   
               
               
                   
                 GGCAUCGCCCUCGCCCUCGC 
                 169 
               
               
                   
                   
               
               
                   
                 GGCGAGGGCGAUGCCACCUA 
                 170 
               
               
                   
                   
               
               
                   
                 CAGGGUCAGCUUGCCGUAGG 
                 171 
               
               
                   
                   
               
               
                   
                 CUUCAGGGUCAGCUUGCCGU 
                 172 
               
               
                   
                   
               
               
                   
                 GGUGGUGCAGAUGAACUUCA 
                 173 
               
               
                   
                   
               
               
                   
                 CGGUGGUGCAGAUGAACUUC 
                 174 
               
               
                   
                   
               
               
                   
                 CUGAAGUUCAUCUGCACCAC 
                 175 
               
               
                   
                   
               
               
                   
                 GGGCACGGGCAGCUUGCCGG 
                 176 
               
               
                   
                   
               
               
                   
                 CCGGCAAGCUGCCCGUGCCC 
                 177 
               
               
                   
                   
               
               
                   
                 CCAGGGCACGGGCAGCUUGC 
                 178 
               
               
                   
                   
               
               
                   
                 ACGAGGGUGGGCCAGGGCAC 
                 179 
               
               
                   
                   
               
               
                   
                 CACGAGGGUGGGCCAGGGCA 
                 180 
               
               
                   
                   
               
               
                   
                 GUGGUCACGAGGGUGGGCCA 
                 181 
               
               
                   
                   
               
               
                   
                 GGUGGUCACGAGGGUGGGCC 
                 182 
               
               
                   
                   
               
               
                   
                 GUCAGGGUGGUCACGAGGGU 
                 183 
               
               
                   
                   
               
               
                   
                 GGUCAGGGUGGUCACGAGGG 
                 184 
               
               
                   
                   
               
               
                   
                 GUAGGUCAGGGUGGUCACGA 
                 185 
               
               
                   
                   
               
               
                   
                 CGUAGGUCAGGGUGGUCACG 
                 186 
               
               
                   
                   
               
               
                   
                 CUCGUGACCACCCUGACCUA 
                 187 
               
               
                   
                   
               
               
                   
                 CUGCACGCCGUAGGUCAGGG 
                 188 
               
               
                   
                   
               
               
                   
                 GCACUGCACGCCGUAGGUCA 
                 189 
               
               
                   
                   
               
               
                   
                 AGCACUGCACGCCGUAGGUC 
                 190 
               
               
                   
                   
               
               
                   
                 GCUGAAGCACUGCACGCCGU 
                 191 
               
               
                   
                   
               
               
                   
                 GCUUCAUGUGGUCGGGGUAG 
                 192 
               
               
                   
                   
               
               
                   
                 CGUGCUGCUUCAUGUGGUCG 
                 193 
               
               
                   
                   
               
               
                   
                 UCGUGCUGCUUCAUGUGGUC 
                 194 
               
               
                   
                   
               
               
                   
                 GUCGUGCUGCUUCAUGUGGU 
                 195 
               
               
                   
                   
               
               
                   
                 AGAAGUCGUGCUGCUUCAUG 
                 196 
               
               
                   
                   
               
               
                   
                 UUCAAGUCCGCCAUGCCCGA 
                 197 
               
               
                   
                   
               
               
                   
                 GACGUAGCCUUCGGGCAUGG 
                 198 
               
               
                   
                   
               
               
                   
                 CUGGACGUAGCCUUCGGGCA 
                 199 
               
               
                   
                   
               
               
                   
                 CAUGCCCGAAGGCUACGUCC 
                 200 
               
               
                   
                   
               
               
                   
                 CGCUCCUGGACGUAGCCUUC 
                 201 
               
               
                   
                   
               
               
                   
                 GCGCUCCUGGACGUAGCCUU 
                 202 
               
               
                   
                   
               
               
                   
                 UGAAGAAGAUGGUGCGCUCC 
                 203 
               
               
                   
                   
               
               
                   
                 GGAGCGCACCAUCUUCUUCA 
                 204 
               
               
                   
                   
               
               
                   
                 ACCAUCUUCUUCAAGGACGA 
                 205 
               
               
                   
                   
               
               
                   
                 GCCGUCGUCCUUGAAGAAGA 
                 206 
               
               
                   
                   
               
               
                   
                 CAACUACAAGACCCGCGCCG 
                 207 
               
               
                   
                   
               
               
                   
                 CUCGAACUUCACCUCGGCGC 
                 208 
               
               
                   
                   
               
               
                   
                 CCGCGCCGAGGUGAAGUUCG 
                 209 
               
               
                   
                   
               
               
                   
                 CCUCGAACUUCACCUCGGCG 
                 210 
               
               
                   
                   
               
               
                   
                 CGCGCCGAGGUGAAGUUCGA 
                 211 
               
               
                   
                   
               
               
                   
                 GUCGCCCUCGAACUUCACCU 
                 212 
               
               
                   
                   
               
               
                   
                 GAAGUUCGAGGGCGACACCC 
                 213 
               
               
                   
                   
               
               
                   
                 CAGCUCGAUGCGGUUCACCA 
                 214 
               
               
                   
                   
               
               
                   
                 UCAGCUCGAUGCGGUUCACC 
                 215 
               
               
                   
                   
               
               
                   
                 GGUGAACCGCAUCGAGCUGA 
                 216 
               
               
                   
                   
               
               
                   
                 GUGAACCGCAUCGAGCUGAA 
                 217 
               
               
                   
                   
               
               
                   
                 CGAUGCCCUUCAGCUCGAUG 
                 218 
               
               
                   
                   
               
               
                   
                 GCUGAAGGGCAUCGACUUCA 
                 219 
               
               
                   
                   
               
               
                   
                 GAAGGGCAUCGACUUCAAGG 
                 220 
               
               
                   
                   
               
               
                   
                 GGCAUCGACUUCAAGGAGGA 
                 221 
               
               
                   
                   
               
               
                   
                 CAAGGAGGACGGCAACAUCC 
                 222 
               
               
                   
                   
               
               
                   
                 AAGGAGGACGGCAACAUCCU 
                 223 
               
               
                   
                   
               
               
                   
                 AGGAGGACGGCAACAUCCUG 
                 224 
               
               
                   
                   
               
               
                   
                 CAACAUCCUGGGGCACAAGC 
                 225 
               
               
                   
                   
               
               
                   
                 UGUACUCCAGCUUGUGCCCC 
                 226 
               
               
                   
                   
               
               
                   
                 CAGCCACAACGUCUAUAUCA 
                 227 
               
               
                   
                   
               
               
                   
                 CGGCCAUGAUAUAGACGUUG 
                 228 
               
               
                   
                   
               
               
                   
                 AUGGCCGACAAGCAGAAGAA 
                 229 
               
               
                   
                   
               
               
                   
                 GAUGCCGUUCUUCUGCUUGU 
                 230 
               
               
                   
                   
               
               
                   
                 CAAGCAGAAGAACGGCAUCA 
                 231 
               
               
                   
                   
               
               
                   
                 CAAGAUCCGCCACAACAUCG 
                 232 
               
               
                   
                   
               
               
                   
                 AUCCGCCACAACAUCGAGGA 
                 233 
               
               
                   
                   
               
               
                   
                 UGCCGUCCUCGAUGUUGUGG 
                 234 
               
               
                   
                   
               
               
                   
                 CGCUGCCGUCCUCGAUGUUG 
                 235 
               
               
                   
                   
               
               
                   
                 GGUGUUCUGCUGGUAGUGGU 
                 236 
               
               
                   
                   
               
               
                   
                 UGGGGGUGUUCUGCUGGUAG 
                 237 
               
               
                   
                   
               
               
                   
                 UACCAGCAGAACACCCCCAU 
                 238 
               
               
                   
                   
               
               
                   
                 CGCCGAUGGGGGUGUUCUGC 
                 239 
               
               
                   
                   
               
               
                   
                 CAGAACACCCCCAUCGGCGA 
                 240 
               
               
                   
                   
               
               
                   
                 CACGGGGCCGUCGCCGAUGG 
                 241 
               
               
                   
                   
               
               
                   
                 GCACGGGGCCGUCGCCGAUG 
                 242 
               
               
                   
                   
               
               
                   
                 AGCACGGGGCCGUCGCCGAU 
                 243 
               
               
                   
                   
               
               
                   
                 CAGCACGGGGCCGUCGCCGA 
                 244 
               
               
                   
                   
               
               
                   
                 GGUUGUCGGGCAGCAGCACG 
                 245 
               
               
                   
                   
               
               
                   
                 UGGUUGUCGGGCAGCAGCAC 
                 246 
               
               
                   
                   
               
               
                   
                 GUGGUUGUCGGGCAGCAGCA 
                 247 
               
               
                   
                   
               
               
                   
                 GUGCUCAGGUAGUGGUUGUC 
                 248 
               
               
                   
                   
               
               
                   
                 GGUGCUCAGGUAGUGGUUGU 
                 249 
               
               
                   
                   
               
               
                   
                 CGGACUGGGUGCUCAGGUAG 
                 250 
               
               
                   
                   
               
               
                   
                 UCAGGGCGGACUGGGUGCUC 
                 251 
               
               
                   
                   
               
               
                   
                 GUCUUUGCUCAGGGCGGACU 
                 252 
               
               
                   
                   
               
               
                   
                 GGUCUUUGCUCAGGGCGGAC 
                 253 
               
               
                   
                   
               
               
                   
                 GUUGGGGUCUUUGCUCAGGG 
                 254 
               
               
                   
                   
               
               
                   
                 CUCGUUGGGGUCUUUGCUCA 
                 255 
               
               
                   
                   
               
               
                   
                 UCUCGUUGGGGUCUUUGCUC 
                 256 
               
               
                   
                   
               
               
                   
                 UGUGAUCGCGCUUCUCGUUG 
                 257 
               
               
                   
                   
               
               
                   
                 AUGUGAUCGCGCUUCUCGUU 
                 258 
               
               
                   
                   
               
               
                   
                 CAUGUGAUCGCGCUUCUCGU 
                 259 
               
               
                   
                   
               
               
                   
                 CAACGAGAAGCGCGAUCACA 
                 260 
               
               
                   
                   
               
               
                   
                 GCGCGAUCACAUGGUCCUGC 
                 261 
               
               
                   
                   
               
               
                   
                 CGGCGGUCACGAACUCCAGC 
                 262 
               
               
                   
                   
               
               
                   
                 CUGGAGUUCGUGACCGCCGC 
                 263 
               
               
                   
                   
               
               
                   
                 UGGAGUUCGUGACCGCCGCC 
                 264 
               
               
                   
                   
               
               
                   
                 ACCGCCGCCGGGAUCACUCA 
                 265 
               
               
                   
                   
               
               
                   
                 GCCGUGAGUGAUCCCGGCGG 
                 266 
               
               
                   
                   
               
               
                   
                 CAUGCCGUGAGUGAUCCCGG 
                 267 
               
               
                   
                   
               
               
                   
                 CGCCGGGAUCACUCACGGCA 
                 268 
               
               
                   
                   
               
               
                   
                 GUCCAUGCCGUGAGUGAUCC 
                 269 
               
               
                   
                   
               
            
           
         
       
     
     As a specific example, the second guide RNA has a second guide sequence of 5′-CGUCGCCGUCCAGCUCGACC-3′ (SEQ ID NO: 161). 
     The first complementary sequence included in the second guide RNA of the present disclosure may be derived from a naturally occurring first complementary sequence or may include a sequence having sequence identity therewith. As an example, the first complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the first complementary sequence may include 5′-GUUUUAGAGCUA-3′ (SEQ ID NO: 270) or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the first complementary sequence may include 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′ (SEQ ID NO: 271) or 5′-GUUUUAGUCCCUU-3′ (SEQ ID NO: 272), or may include a sequence having at least 50% sequence identity therewith. 
     The tracrRNA included in the second guide RNA of the present disclosure comprises a second complementary sequence that complementarily binds to the crRNA. 
     The second complementary sequence may be derived from a naturally occurring second complementary sequence or may include a sequence having sequence identity therewith. As an example, the second complementary sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus , or  Neisseria meningitidis  or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the second complementary sequence may include 5′-UAGCAAGUUAAAAU-3′ or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the second complementary sequence may include 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′ (SEQ ID NO: 274) or 5′-AAGGGACUAAAAU-3′ (SEQ ID NO: 275), or may include a sequence having at least 50% sequence identity therewith. 
     The tracrRNA included in the second guide RNA of the present disclosure may further comprise a tail sequence. 
     The tail sequence may be derived from a naturally occurring tail sequence or may include a sequence having sequence identity therewith. AS an example, the tail sequence may include a sequence derived from  Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophiles, Staphylococcus aureus  or  Neisseria meningitides , or the like, and may include a sequence having at least 50% sequence identity therewith. As a specific example, when derived from  Streptococcus pyogenes , the tail sequence may include 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′ (SEQ ID NO: 276) or may include a sequence having at least 50% sequence identity therewith. As another specific example, when derived from  Campylobacter jejuni , the tail sequence may include 5′-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3′ (SEQ ID NO: 277) or may include a sequence having at least 50% sequence identity therewith. 
     Meanwhile, in another embodiment of the present application, a DNA encoding the second guide RNA may be provided. 
     In this case, the DNA sequence encoding the second guide RNA is a sequence comprising a sequence encoding the second guide sequence, and may include at least one of the same DNA sequence (a sequence in which U is replaced by T in each sequence) as the RNA sequence of SEQ ID NO: 151 to 269. 
     iii) Cas Protein 
     The composition comprises a Cas protein. 
     The Cas protein is a protein that functions to cut DNA by being induced to a specific position in the genome by a guide RNA. 
     As an example, the Cas protein may be at least one selected from the group consisting of  Streptococcus pyogenes -derived Cas9 protein,  Campylobacter jejuni -derived Cas9 protein,  Streptococcus  thermophiles-derived Cas9 protein,  Staphylococcus aureus -derived Cas9 protein,  Neisseria meningitidis -derived Cas9 protein, and Cpf1. 
     The Cas protein may combine with a first guide RNA. The Cas protein combines with a first guide RNA and is induced to the gene position of the target locus in the genome. The Cas protein cuts a gene on a target locus in the genome. 
     As an example, when a gene on a target locus in the genome is cut, the cut is repaired by non-homologous end joining. Non-homologous end joining occurs when several bases are inserted or deleted (indels) in the process of joining the cleaved ends. Therefore, the gene on the target locus in the genome may be knocked out by indels in the process of non-homologous end joining. 
     As another example, when a template DNA of a homologous base sequence exists, repair occurs based on the homologous template DNA. Accordingly, when the composition includes a transgene to be inserted into a target locus in the genome, the transgene may be knocked-in into the cleaved position by homologous recombination. 
     The Cas protein may combine with the second guide RNA. The Cas protein combines with the second guide RNA and is induced to the fluorescent protein gene position. The Cas protein cuts the fluorescent protein gene. When the fluorescent protein gene is cut, the fluorescent protein gene may be knocked out by indel in the process of non-homologous end joining. 
     iv) A Transgene to be Inserted into the Target Locus in the Genome 
     The composition may further comprise a transgene to be inserted into a target locus in the genome. 
     The composition is used for a genetically modified cell selection method, wherein the genetic modification includes knock-in of a foreign gene on the genome in addition to knock-out of a gene on the genome in the cell. When the genetic modification means knock-in, the composition may further include a transgene to be inserted into a target locus in the genome. As an example, when a gene on a target locus in the genome is cut by a Cas protein, a transgene may be inserted at the position. As an example, after the gene is cut, repair occurs based on the template DNA of the homologous base sequence by homologous recombination. The transgene can be inserted into a target locus in the genome through this homologous recombination process. As an example, the transgene may include homology arms having homology with the gene sequence of the target locus in the genome to be inserted into the target locus in the genome by homogeneous recombination at both ends of the transgene. 
     2) Method of Treatment of the Composition 
     i) Form of Composition 
     The first guide RNA, the second guide RNA, the Cas protein, and/or the transgene may be treated into cells separately or in combination. 
     In one embodiment, the composition of the present disclosure may include a ribonucleoprotein (RNP) form. 
     As an example, the first guide RNA, the second guide RNA, and the Cas protein may be treated into a cell in the form of a guide RNA-Cas protein complex. As a specific example, the guide RNA and the Cas protein may be treated into cells in the form of ribonucleoprotein (RNP). In this case, only the first guide RNA may be treated into the cell in the form of a guide RNA-Cas protein complex. In this case, only the second guide RNA may be treated into the cell in the form of a guide RNA-Cas protein complex. In this case, both the first guide RNA and the second guide RNA may be treated into the cell in the form of a guide RNA-Cas protein complex. 
     In another embodiment, the composition of the present disclosure may include a vector form. 
     The first guide RNA, the second guide RNA, the Cas protein and/or the transgene may be treated into cells in the form of DNA encoding the same, RNA, or a mixture thereof. 
     The vector may be a plasmid or a viral vector. In this case, the virus may be a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes virus, or the like. 
     In this case, the nucleic acid sequences encoding the first guide RNA, the second guide RNA, the Cas protein and/or the transgene may be included in one vector or included in multiple vectors. As an example, the nucleic acid sequences encoding the first guide RNA, the second guide RNA, the Cas protein, and/or the transgene may be included in separate vectors, respectively. As an example, the nucleic acid sequences encoding the first guide RNA, the second guide RNA, the Cas protein, and/or the transgene may all be included in one vector. 
     In another embodiment, the composition of the present disclosure may include a mixed form of a vector and a non-vector. 
     The first guide RNA, the second guide RNA, the Cas protein, and/or the transgene may be treated into a cell in a mixed form of its RNA, protein, or DNA encoding them. 
     In this case, a mixed form thereof may be treated into cells in the form of a combination of vector and non-vector. 
     ii) Method of Treatment of the Composition 
     The composition may be treated to cells by a method using a vector or a method using a non-vector. When the composition is treated by a method using a vector, the vector may be a viral vector or a non-viral vector. 
     In the case of treating the composition in the form of the non-viral vector and the non-vector, the composition may be treated into cells by one or more of methods using a microinjection method, an electroporation method, and LNP (lipid nanoparticles). As a specific example, in order to treat the composition, the composition may be treated through electroporation of guide RNA and Cas protein in the form of ribonucleoprotein (RNP). 
     3) The Order of Processing the Composition 
     The composition may be treated to the cells simultaneously or in a series sequence. As an example, the first guide RNA, the second guide RNA, and the Cas protein of the composition may be simultaneously treated into cells. 
     As another example, the guide RNA and the Cas protein may be separately treated. In this case, the first guide RNA and the second guide RNA are simultaneously treated. As an example, after the first guide RNA and the second guide RNA are treated, the Cas protein may be treated. As another example, after the Cas protein is treated, the first guide RNA and the second guide RNA may be treated. 
     When the composition additionally includes a transgene, the transgene may be treated into the cell simultaneously with the guide RNA and the Cas protein or may be treated in a series sequence. In this case, the first guide RNA and the second guide RNA are simultaneously treated. As an example, the transgene may be simultaneously treated with guide RNA and Cas protein. As another example, after the transgene is treated, the guide RNA and the Cas protein may be treated. As another example, the transgene may be treated between the treatment steps of the guide RNA and the Cas protein. As another example, after the guide RNA is treated, the Cas protein and the transgene may be simultaneously treated. As another example, after the Cas protein is treated, the guide RNA and the transgene may be simultaneously treated. However, the treatment order is not limited thereto. 
     4) Effect after Composition Treatment_Genetic Modification 
     i) Knock-Out of the Gene on the Target Locus 
     When the composition does not include a transgene, the gene on the target locus in the genome of the fluorescent cell is cut by the first guide RNA and Cas protein, and the cleavage is repaired by non-homologous end joining. At this time, non-homologous end joining occurs when several bases are inserted or deleted (indels) in the process of connecting the cut ends. Accordingly, the gene on the target locus in the genome includes indels of some bases. The gene on the target locus in the genome may be knocked out by the indels of some bases. 
     In addition, the fluorescent protein gene in the genome of the fluorescent cell is cut by the second guide RNA and the Cas protein, and the cleavage is repaired by non-homologous end joining. At this time, non-homologous end joining occurs when several bases are inserted or deleted (indels) in the process of connecting the cut ends. Accordingly, the fluorescent protein gene in the genome includes indels of some bases. The fluorescent protein gene in the genome may be knocked out by the indels of some bases. 
     As a specific example, when the composition comprises a first guide RNA for the bovine beta-lactoglobulin (BLG) gene, a second guide RNA for a green fluorescent protein gene, and a Cas protein, fluorescent bovine cells are treated with the composition. Then, the beta-lactoglobulin (BLG) gene and the green fluorescent protein gene of the fluorescent bovine cell may include indels of some bases and may be knocked out by indels of some bases. 
     ii) Knock-In of the Transgene 
     When the composition includes a transgene, the gene on the target locus in the genome of the fluorescent cell is cut by the first guide RNA and the Cas protein, and when template DNA with a homologous nucleotide sequence exists, repair occurs at the basis of the homologous template DNA. Accordingly, when the composition includes a transgene to be inserted into a target locus in the genome, the transgene may be inserted into the cleaved position by homologous recombination. 
     In addition, the fluorescent protein gene in the genome of the fluorescent cell is cleaved by the second guide RNA and the Cas protein, and the cleavage is repaired by non-homologous end joining. At this time, non-homologous end joining occurs when several bases are inserted or deleted (indels) in the process of connecting the cut ends. Accordingly, the fluorescent protein gene in the genome includes indels of some bases. The fluorescent protein gene in the genome may be knocked out by the indels of some bases. 
     As a specific example, when the composition includes a first guide RNA for a gene on a bovine target locus, a second guide RNA for a green fluorescent protein gene, a Cas protein, and a transgene to be inserted into the target locus, a fluorescent bovine cell is treated with the composition, then a transgene is inserted into a target locus of a fluorescent bovine cell, and then a green fluorescent protein gene may be knocked out. 
     3. Selecting Non-Fluorescent Cells 
     In the non fluorescent cells after the composition treatment, the gene on the target locus and the fluorescent protein gene has been modified by the first guide RNA, the second guide RNA, the Cas protein and/or the transgene. Therefore, by selecting the non fluorescent cells, it is possible to select the genetically modified cells. 
     That is, in the present disclosure, non fluorescent cells (cells that do not express fluorescence) are selected after the composition treatment step to select genetically modified cells. 
     1) Characteristics of Non-Fluorescent Cells (Cells that do not Express Fluorescence) 
     The non fluorescent cell is a cell in which a gene on a target locus and a fluorescent protein gene has been modified. 
     The modified gene on the target locus in the genome may be one or more of the genes in the cell genome except for the fluorescent protein gene. The fluorescent protein gene is a gene different from the gene on the target locus in the genome. The fluorescent protein gene and the gene on the target locus in the genome are present at different positions. 
     The modified gene may be, for example, one or more of the genes whose expression is to be suppressed. As another example, the modified gene may be one or more of the genes present at a position into which a transgene is to be inserted. 
     The non-fluorescent cell includes a modified gene on a target locus in the genome. 
     As an example, the gene on the target locus in the genome of the non-fluorescent cell includes indels of some bases. That is, in the non-fluorescent cell, the gene on the target locus in the genome is knocked out by the indels of some bases. As a specific example, when the cell is a bovine cell, the beta-lactoglobulin (BLG) gene in the non fluorescent cell includes indels of some bases. That is, in the non fluorescent cell, the beta-lactoglobulin (BLG) gene is knocked out by the indels of some bases. In another example, in the non-fluorescent cell, the prion (PRNP) gene includes indels of some bases. That is, in the non-fluorescent cell, the prion (PRNP) gene is knocked out by the indels of some of the bases. 
     As another example, in the non-fluorescent cell, a transgene may be inserted at a target locus in the genome. As a specific example, when the cell is a bovine cell, the non-fluorescent cell has a transgene inserted (knocked-in) into the beta-lactoglobulin (BLG) gene. As another example, the non-fluorescent cell has a transgene inserted (knocked-in) into the prion (PRNP) gene position. 
     The non fluorescent cell includes a modified fluorescent protein gene in the genome. The modified fluorescent protein gene means that the fluorescent protein gene is knocked out. In the non-fluorescent cell, all fluorescent protein genes present in the genome of the cell are knocked out. 
     2) Method for Selecting Non-Fluorescent Cells 
     Various methods of analyzing fluorescence to select for non-fluorescent cells may be used. As an example, as a method for selecting non-fluorescent cells, a method using FACS or a method using a microscope may be used. As a specific example, by analyzing the light emitted by the fluorescent material stimulated by a laser beam, the non-fluorescent cell and the fluorescent cell may be classified, and the non-fluorescent cell may be selected. As another specific example, non fluorescent cells may be selected using fluorescence observation through a microscope. 
     4. Specific Example_Beta-Lactoglobulin (BLG) Genetically Modified Cell Selection Method 
     1) Preparation of Fluorescent Cells 
     Prepare cells including a green fluorescent protein (GFP) gene in the genome of a bovine cell. 
     2) Composition Treatment 
     The fluorescent bovine cells are treated with guide RNA having at least one guide sequence of SEQ ID Nos: 1 to 6 (guide RNA for BLG gene), guide RNA having at least one guide sequence of SEQ ID Nos: 151 to 269 (guide RNA for GFP gene) and Cas9 protein. 
     3) Non-Fluorescent Cell Selection 
     Cells that do not express fluorescence after treatment with the composition are selected. The cells that do not express the fluorescence (non-fluorescent cells) are cells in which the beta-lactoglobulin gene and the green fluorescent protein gene contain indels of some bases and are knocked out by the indels of some bases. 
     Through the above method, genetically modified cells may be efficiently selected. 
     II. Characteristics of the Genetically Modified Cell Selection Method 
     As an example, the present specification discloses a method for sorting out genetically modified cells. The method for sorting out genetically modified cells disclosed herein has the following characteristics. 
     First, the method uses a cell expressing fluorescence, a first guide RNA for a gene on a target locus, a second guide RNA for a fluorescent protein gene, and a Cas protein. 
     In this case, the method has the characteristic of treating material for modifying a gene on a target locus and a material for modifying a fluorescent protein gene in a cell expressing fluorescence through one single experimental process. Due to these characteristics, by using the method, two results of genetic modification of a target locus of a cell and genetic modification of a fluorescent protein may be obtained at the same time. That is, by using the method, cells in which the gene on the target locus is modified may be selected through the selection of non-fluorescent cells. Therefore, an additional analysis process for cell selection is not required. Conventionally, in order to check whether the cell is genetically modified, the gene on the target locus is PCR amplified and sequenced. However, the method for sorting out genetically modified cells disclosed herein does not require an additional sequencing step, so it is possible to avoid consuming time and effort for sequencing. 
     Second, in the method, cells are not consumed in the process of selecting genetically modified cells. 
     The conventional method of PCR amplification and sequencing of genes on the target locus is a method in which cells are consumed, and there is a problem in that cells that have undergone the sequencing step are not utilized and consumed thereafter. However, the method for sorting out genetically modified cells disclosed in the present disclosure is a method using fluorescence expression of cells and does not cause a problem in that cells are consumed in order to check and select whether cell is genetically modified or not. 
     III. Genetically Modified Cells 
     Disclosed herein are genetically modified cells. 
     As an example, the genetically modified cell disclosed herein is a cell selected through the method for sorting out genetically modified cells. 
     As an example, the genetically modified cell disclosed herein is a cell in which a gene on a target locus and a fluorescent protein gene in the genome has been modified. 
     The genetically modified cells may be interchanged with non fluorescent cells. 
     1. Cell 
     The genetically modified cells disclosed herein may be one or more of the non-human mammalian cells. As a specific example, the cell may be one or more of bovine, pig, mouse, and rat cells. As a specific example, the cell is a bovine cell. 
     As another example, the cell may be a somatic cell or a germ cell. As a specific example, the cell is a fertilized egg or blastocyst generated through the fertilization of a germ cell. As another specific example, the cell may be a cell in which a nucleus of a somatic cell is transplanted into an enucleated egg. 
     2. Cells Including the Modified Gene on the Target Locus 
     The genetically modified cell disclosed herein is a cell in which a gene on a target locus in the genome has been modified. 
     In this case, the gene on the target locus may be one or more of the genes whose expression is to be suppressed. Alternatively, the gene on the target locus may be one or more of the genes present at a position into which a transgene is to be inserted. 
     As an example, in the cell, a gene on a target locus in the genome is knocked out. As a specific example, when the cell is a bovine cell, the non-fluorescent cell has a beta-lactoglobulin (BLG) gene knocked out. As another example, the non-fluorescent cell has a prion (PRNP) gene knocked out. The knock-out gene has a reduced function of the gene so that the expression of the gene in the cell is not detectably small or is not expressed. 
     As another example, in the non-fluorescent cell, a transgene is inserted at a target locus in the genome. As a specific example, the cell may be a cell in which a transgene is knocked-in at a target locus in the genome to replace an existing protein or to express a new protein. As an example, when the cell is a bovine cell, the non-fluorescent cell has a transgene inserted (knock-in) into the beta-lactoglobulin (BLG) gene position. As another example, the non-fluorescent cell has a transgene inserted (knock-in) into the prion (PRNP) gene position. 
     3. Cells in which the Fluorescent Protein Gene has been Modified 
     The genetically modified cell disclosed herein is a cell in which a fluorescent protein gene in the genome has been modified. In this case, the fluorescent protein gene is not located at the target locus in the genome. 
     The cell is a cell in which the fluorescent protein gene in the cell genome is knocked out. The cell is a cell in which the function of the fluorescent protein gene is reduced such that the expression of the fluorescent protein gene in the cell is not detectably small or is not expressed. 
     As an example, the modified fluorescent protein gene is present at one or more positions in the genome. As an example, the modified fluorescent protein gene is present at two positions in the genome. As an example, the modified fluorescent protein gene is present at three or more positions in the genome. As a specific example when the cell is a bovine cell, the modified fluorescent protein gene includes at least one of the 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome. As an example, the modified fluorescent protein gene includes at least one of 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6. 
     The cell is a cell in which all fluorescent protein genes present in the cell genome are knocked out. As an example, if the cell includes one fluorescent protein gene, one fluorescent protein gene is knocked out. As another example, if the cell includes two fluorescent protein genes, the two fluorescent protein genes are knocked out. As another example, if the cell includes three or more fluorescent protein genes, all three or more fluorescent protein genes are knocked out. 
     As an example, the fluorescent protein gene may be one or more among a green fluorescent protein gene (GFP), a blue fluorescent protein gene (BFP), a cyan fluorescent protein gene (CFP), a yellow fluorescent protein gene (YFP), a red fluorescent protein gene (RFP), etc. As a specific example, the fluorescent protein gene is a green fluorescent protein gene. 
     IV. Methods for Producing Genetically Modified Animals 
     Disclosed herein is a method for producing a genetically modified animal including a modified gene on a target locus in a genome. 
     The method for producing a genetically modified animal disclosed herein includes 
     a) preparing a cells expressing fluorescence; 
     b) treating the cell expressing the fluorescence with a composition; 
     c) selecting a non fluorescent cell; and 
     d) implanting the non-fluorescent cell into the uterus of a surrogate mother. 
     Hereinafter, each step will be described in detail. 
     1. Preparing a Cells Expressing Fluorescence 
     In the present disclosure, in order to efficiently produce a genetically modified animal in which a gene is modified, a cell expressing fluorescence is used. The cell expressing the fluorescence is a cell expressing the fluorescent protein gene by including the fluorescent protein gene in the genome. 
     The description of preparing the cell expressing fluorescence disclosed in 1. Preparing a fluorescent cell of I. Genetically modified cell selection method) above are applied. 
     2. Treating the Cell Expressing the Fluorescence with the Composition 
     In the present disclosure, the cell is treated with a composition. 
     The description of the step of treating the composition disclosed in 2. Treating the fluorescent cell with a composition of I. Genetically modified cell selection method above is applied. 
     3. Selecting a Non-Fluorescent Cell (a Cell that does not Display Fluorescence) after Treatment with the Composition 
     In the present disclosure, a non-fluorescent cell is selected after the cell is treated with the composition. 
     The description of the step of selecting the non fluorescent cell disclosed in 3. Selecting non-fluorescent cells after treatment with the composition of I. Genetically modified cell selection method above is applied. 
     4. Transplanting the Non-Fluorescent Cell into the Uterus of a Surrogate Mother 
     In the present disclosure, after selecting the non-fluorescent cell to produce a genetically modified animal, the non fluorescent cell is transplanted into the uterus of a surrogate mother. 
     In the step of transplanting the non-fluorescent cell into the uterus of the surrogate mother, the process of culturing the non-fluorescent cell in a transplantable state into the surrogate mother may be included. 
     1) Preparation of Transplantable Cells into Surrogate Mothers 
     When the non fluorescent cell is a fertilized egg, the non-fluorescent cell can be cultured in a state capable of being transplanted into the surrogate mother and then transplanted into the uterus of the surrogate mother. As an example, after culturing the fertilized egg to a blastocyst stage, the fertilized egg may be transplanted into the uterus of a surrogate mother. 
     When the non fluorescent cell is a somatic cell, a somatic cell nuclear transfer (SCNT) fertilized egg may be prepared and implanted in the uterus of a surrogate mother. 
     i) Method for Generating a Nuclear-Transferred Fertilized Egg 
     The nuclear-transferred fertilized egg includes the nucleus of the non-fluorescent somatic cell. The method for generating the nuclear-transferred fertilized egg may include nuclear removal of the egg, nuclear transfer of non-fluorescent somatic cells, and induction of cell fusion after transplantation. Hereinafter, a method for generating a nuclear-transferred fertilized egg using a known technique will be briefly described. 
     Egg type—The method may use an egg obtained from a wild-type animal or an egg obtained from an animal expressing a fluorescent protein gene. 
     Egg preparation—The method may use mature eggs that have undergone in vitro maturation after obtaining immature eggs from animals. 
     Nucleus Removal—The method may utilize a micropipette to remove the nucleus from a mature egg. As an example, a portion of the cytoplasm and nucleus of an egg may be removed using a micropipette. 
     Nuclear transfer—The method may include injecting donor cells of nuclear transfer into an egg. As an example, the method may use the non-fluorescent cells as donor cells of nuclear transfer. 
     Cell fusion—The method may include a process of applying a stimulus to a nuclear-transferred oocyte to achieve cell fusion between the cytoplasm of the enucleated oocyte and the nucleus of a donor cell. As an example, the stimulation may include electrical stimulation. 
     2) Transplantation 
     The cells described above are transplanted into a surrogate mother. As an example, the cells are transplanted into an animal of the same species as the cells. As a specific example, when the cells are bovine cells, the cells are transplanted into a cow. 
     The cells are transplanted into the uterus of a surrogate mother animal. As an example, the cells are transplanted into the uterine horn. As a specific example, cells are transplanted into the uterine horn without damaging the cervix and the uterus. 
     The step of transplanting the non-fluorescent cell into the uterus of the surrogate mother may further include determining pregnancy after transplantation. As an example, rectal examination or ultrasonography may be used to determine embryo survival and pregnancy. As a specific example, on the 45th day after estrus, it is possible to determine the pregnancy of a cow by rectal examination and/or ultrasonography. 
     5. Production of Animal 
     An animal born after being transplanted into a surrogate mother animal through the above process is an animal in which the gene on the target locus and the fluorescent protein gene in the genome have been modified. As an example, the gene on the target locus and the fluorescent protein gene in the genome of the animal born through the above process are knocked out. As another example, in animals born through the above process, a transgene is knocked in on a target locus in the genome, and a fluorescent protein gene is knocked out. As a specific example, in a cow born through using the above method, the bovine prion (PRNP) gene and fluorescent protein gene are mutated. As another specific example, in a cow born through using the above process, the bovine beta-lactoglobulin (BLG) gene and fluorescent protein gene are knocked out. 
     V. Genetically Modified Animals 
     Disclosed herein are genetically modified animals. 
     The genetically modified animal is an animal including a modified gene on a target locus in the genome. 
     1. Animal 
     The genetically modified animal disclosed herein may be one or more of the non-human mammals. As a specific example, the cell may be one or more cells of a cow, a pig, a mouse, and a rat. As a specific example, the animal is a cow. 
     2. Animal Including the Modified Gene on the Target Locus 
     The genetically modified animal disclosed herein has a cell in which a gene on a target locus in a genome has been modified. 
     In this case, the gene on the target locus may be one or more of the genes whose expression is to be suppressed. Alternatively, the gene on the target locus may be one or more of the genes present at a position into which a transgene is to be inserted. 
     As an example, the animal may be an animal in which a gene on a target locus in the genome is cut and knocked out. As a specific example, when the animal is a cow, a beta-lactoglobulin (BLG) gene or a prion (PRNP) protein gene is mutated in the animal. The knock-out gene has a reduced function of the gene so that the expression of the gene in the cell is not detectably small or is not expressed. 
     As another example, the animal may be an animal in which a gene on a target locus in the genome is cut, and a transgene is inserted into the cleaved site. As a specific example, the cell may be an animal in which a transgene is knocked in on a target locus in the genome to replace an existing protein or to express a new protein. As an example, when the animal is a cow, the animal may have a transgene inserted (knock-in) on the beta-lactoglobulin (BLG) gene position. As another example, the animal may have a transgene inserted (knock-in) on the prion (PRNP) gene position. 
     3. Cells in which the Fluorescent Protein Gene has been Modified 
     The genetically modified animal disclosed herein is a cell in which a fluorescent protein gene in the genome has been modified. In this case, the fluorescent protein gene is a gene different from the gene on the target locus in the genome. 
     The animal is an animal in which a fluorescent protein gene in the genome is knocked out. The animal is an animal in which the function of the fluorescent protein gene is reduced such that the expression of the fluorescent protein gene is not detectably small or is not expressed in the cell. 
     As an example, the modified fluorescent protein gene is present at one or more positions in the genome. As an example, the modified fluorescent protein gene is present at two positions in the genome. As an example, the modified fluorescent protein gene is present at three or more positions in the genome. As a specific example, when the animal is a cow, the modified fluorescent protein gene includes at least one of the 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome. As an example, the modified fluorescent protein gene includes at least one of 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6. 
     The animal is an animal in which all fluorescent protein genes present in the genome are knocked out. As an example, if one fluorescent protein gene is included in the genome of the animal, one fluorescent protein gene is knocked out. As another example, if two fluorescent protein genes are included in the genome of the animal, both fluorescent protein genes are knocked out. As another example, if three or more fluorescent protein genes are included in the genome of the animal, all three or more fluorescent protein genes are knocked out. 
     As an example, the fluorescent protein gene may be one or more among a green fluorescent protein gene (GFP), a blue fluorescent protein gene (BFP), a cyan fluorescent protein gene (CFP), a yellow fluorescent protein gene (YFP), a red fluorescent protein gene (RFP), etc. As a specific example, the fluorescent protein gene is a green fluorescent protein gene. 
     Fluorescent protein genes in the animal may be transferred to the same site in the next generation. As an example, when the animal is a cow, the fluorescent protein gene in the cow&#39;s genome may be transferred to the same site in the next generation. 
     VI. Kit for Selection of Genetically Modified Cells 
     Another example disclosed by the present disclosure is a kit for sorting out a cell including a modified gene on a target locus in the genome. Each component of the kit has the same meaning as the component used in the method for selecting genetically modified cells disclosed herein. 
     The kit disclosed in the present disclosure includes 
     i) a cell that express fluorescence; 
     ii) a guide RNA for a fluorescent protein gene or a nucleic acid encoding the same; and 
     iii) Cas protein or a nucleic acid encoding the same. 
     The kit may further include a guide RNA for a gene on a target locus in the genome or a nucleic acid encoding the same. 
     In addition, the kit may further include a transgene to be inserted into the target locus in the genome. 
     1. Cells that Express Fluorescence 
     A kit for sorting out a cell in which a gene on a target locus in a genome has been modified as disclosed in the present disclosure includes a cell expressing fluorescence. 
     1) Cells 
     As an example, the cell may be a non-human mammalian cell. As an example, the cell may be a cell of a cow, a pig, a mouse, or a rat. As a specific example, the cell is a bovine cell. 
     As another example, the cell may be a somatic cell or a germ cell. As a specific example, the cell is a blastocyst generated through the fertilization of germ cells. As another specific example, the cell is a cell in which a nucleus of a somatic cell is transplanted into an enucleated egg. 
     2) Fluorescent Protein 
     The cell is a cell that expresses fluorescence. The cell includes a fluorescent protein gene in the genome. As an example, the fluorescent protein gene may be one or more genes among a green fluorescent protein gene (GFP), a blue fluorescent protein gene (BFP), a cyan fluorescent protein gene (CFP), a yellow fluorescent protein gene (YFP), a red fluorescent protein gene (RFP), etc. As a specific example, the fluorescent protein gene is a green fluorescent protein gene. 
     The fluorescent protein gene is present in the cell genome. As an example, the fluorescent protein gene is present in a safe harbor in the genome of the cell. The safe harbor may include AAVS1, CCR5, ROSA26, ACTB, and the like. As an example, the fluorescent protein gene is present in an intron in the genome of a cell. As a specific example, when the cell is a bovine cell, the fluorescent protein gene is present on one or more positions among 105665894 position of chromosome 1; 79750136 position of chromosome 3; 71122343 position of chromosome 4; 85854536 position of chromosome 10; 51221667 position of chromosome 12; 80581377 position of chromosome X; 95433564-95434563 position of chromosome 4; 113823097-113823101 position of chromosome 4; and 20085913-20086912 position of chromosome 6 in the genome of the bovine cell. As an example, the fluorescent protein gene is present on one or more positions among 95433564 position of chromosome 4; 113823097 position of chromosome 4; and 20085913 position of chromosome 6. 
     The fluorescent protein gene is present on one or more positions in the cell genome. As an example, the fluorescent protein gene is present on three positions in the cell genome. 
     The fluorescent protein gene is not present on a target locus in the cell genome. The fluorescent protein gene and the gene on the target locus in the cell genome exist in different positions. 
     3) Storage 
     The Cell expressing fluorescence, which is a component of the kit, may be stored in an appropriate environment for the storage of cells. As an example, the cell may be stored frozen in an appropriate medium according to the cell type, and the medium may contain a cryopreservative agent. As a specific example, the cell may be stored in a medium including a cryopreservative agent and stored at about −60° C. to −80° C. in a deep freezer. As another specific example, the cell may be stored in a medium including a cryopreservative agent and stored at about −180° C. to −200° C. in a liquid nitrogen tank. 
     2. Guide RNA for Fluorescent Protein Gene or Nucleic Acid Encoding the Same 
     The kit disclosed in the present disclosure includes a guide RNA (second guide RNA) for a fluorescent protein gene or a nucleic acid encoding the same. 
     1) Guide RNA 
     The second guide RNA is a single guide RNA or a dual guide RNA. The second guide RNA may recognize a fluorescent protein gene in a cell genome and may interact with a Cas protein. The second guide RNA includes a region capable of combining with a Cas protein. The guide RNA combines to the Cas protein and induces the Cas protein to the fluorescent protein gene position so that the fluorescent protein gene can be cut. 
     2) Storage 
     The second guide RNA, which is a component of the kit, may be stored in an appropriate environment for RNA storage. As an example, the second guide RNA may be stored in a dried state or in a buffer solution. As a specific example, the second guide RNA may be stored at −20° C. in a dried state or in a buffer solution. 
     3. Cas Protein or Nucleic Acid Encoding the Same 
     The kit disclosed in the present disclosure comprises a Cas protein or a nucleic acid encoding the same. 
     1) Cas Protein 
     The Cas protein may combine with a guide RNA. The Cas protein may be induced to the gene on the target locus in the genome by the guide RNA, thereby allowing the gene on the target locus in the genome to be cut. As an example, the Cas protein is a Cas9 protein or a Cpf1 protein. However, the present disclosure is not limited thereto. 
     2) Storage 
     The Cas protein, which is a component of the kit, may be stored in an appropriate environment to maintain nuclease activity. As an example, the Cas protein may be stored in a dried state or in a buffer solution. As a specific example, the Cas protein may be stored at −20° C. in a dried state or in a buffer solution. 
     4. Guide RNA for the Gene on the Target Locus in the Genome or Nucleic Acid Encoding the Same 
     The kit disclosed in the present disclosure may further include a guide RNA (first guide RNA) for a gene on a target locus in the genome or a nucleic acid encoding the same. 
     1) First Guide RNA 
     The first guide RNA is a single guide RNA or a dual guide RNA. The first guide RNA may recognize a gene on a target locus in a cell genome and interact with a Cas protein. The first guide RNA includes a region capable of combining with a Cas protein. The guide RNA combines with the Cas protein and induces the Cas protein to the gene location of the target locus in the cell genome, thereby allowing the gene on the target locus to be cut. 
     2) Storage 
     The first guide RNA, which is a component of the kit, may be stored in an appropriate environment for RNA storage. As an example, the first guide RNA may be stored in a dried state or in a buffer solution. As a specific example, the first guide RNA may be stored at −20° C. in a dried state or in a buffer solution. 
     5. Transgene to be Inserted into the Target Locus in the Genome 
     The kit disclosed in the present disclosure may further include a transgene to be inserted into a target locus in the genome. 
     1) Transgene 
     In the genetically modified cell selection method disclosed in the present disclosure, genetic modification may include knock-in in addition to knock-out. When the genetic modification means knock-in, the kit for selecting cells in which the gene on the target locus in the genome is modified may further include a transgene to be inserted into the target locus in the genome. As an example, the transgene may be inserted into a target locus in the genome by homologous recombination. 
     2) Storage 
     The transgene, which is a component of the kit, may be stored in an appropriate environment for storing the transgene. As an example, the transgene may be stored in a dried state or in a buffer solution. As a specific example, the transgene may be stored at −20° C. in a dried state or in a buffer solution. 
     EXAMPLE 
     Example 1. Fluorescent Cow Production 
     For a fluorescent cow production method, the full text of Yum S Y et al. literature (Long-term health and germline transmission in transmission following transposon-mediated gene transfer. BMC Genomics 2018; 19:387) is referenced. 
     Example 1-1. DNA Vector 
     GFP was amplified by gateway PCR cloning (MultiSite Gateway Pro Plus, Invitrogen, 12537100, Life Technologies, Carlsbad, Calif., USA) and inserted into a final expression vector, PB-CAG (http://www.addgene.org/, #20960). 
     Example 1-2. Egg Collection and In Vitro Maturation (IVM) 
     Ovaries were collected in saline at 35° C. in the slaughterhouse and transported to the laboratory within 2 hours. The cumulus-oocyte complex (COC) from follicles with a diameter of 2 to 8 mm was aspirated by using an 18 gauge needle attached to a 10 ml disposable syringe. COCs with evenly granulated cytoplasm and surrounded by three or more layers of compact cumulus cells were selected and washed three times in HEPES buffered tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, Calif., USA) supplemented with 10% FBS, 2 mM NaHCO 3 (Sigma-Aldrich Corp., St. Louis, Mo., USA), and 1% penicillin-streptomycin (v/v). For IVM, COC was incubated in a 4-well dish (30-40 oocytes per well; Falcon, Becton-Dickinson Ltd., Plymouth, UK) for 22 hours in a 38.5° C. and 5% CO2 environment in 450 μL TCM-199 tissue culture medium supplemented with 10% FBS, 0.005 AU/ml FSH (Antrin, Teikoku, Japan), 100 μM Cysteamine (Sigma-Aldrich), 1 μg/ml 17β-estradiol (Sigma-Aldrich). 
     Example 1-3. Sperm Preparation, In Vitro Fertilization (IVF) and Embryo In Vitro Culture (IVC) 
     Motile sperm were purified and selected using the Percoll gradient method. Briefly, sperm were selected from thawed semen straws by centrifugation on a Percoll discontinuous gradient (45 to 90%) at 1500 rpm for 15 minutes. A 45% Percoll solution was prepared with 1 mL of 90% Percoll (Nutricell, Campinas, SP, Brazil) and 1 mL of capacitation-TALP (Nutricell). The sperm pellet was centrifuged at 1500 rpm for 5 minutes and washed twice with capacitation-TALP. The active motile sperms of the pellet were used for fertilization of mature oocytes (at 24 hours IVM). Oocytes were fertilized with 1 to 2×10 6  sperm/mL for 18 hours in 30 μL microdrops of IVF-TALP medium (Nutricell) coated with mineral oil in an environment of 38.5° C. and 5% CO2 (day 0). Putative zygotes were removed and cultured in a chemically defined two-step culture medium covered with mineral oil (Sigma-Aldrich). All cultures were performed in an environment of 38.5° C., 5% 02, 5% CO2, and 90% N2. On day 2, division rates were recorded and embryonic development was monitored according to the stages of the International Society for Embryo Transplantation (IETS). 
     Example 1-4. Microinjection 
     Transposon DNA was microinjected into the cytoplasm by a microinjection machine (Femtojet Eppendorf, Germany) after removing the cumulus cells of the fertilized oocytes. The amount of injected DNA was 100 ng/mL (1:1 ratio of transposon and transposase). After 7 days, preimplantation stage embryos expressing GFP were selected and transplanted into surrogate mothers. 
     Example 1-5. Embryo Transfer and Pregnancy Diagnosis 
     GFP-expressing blastocysts in PBS supplemented with 20% FBS were transferred to the uterine horn of each surrogate mother by a cervical method at day 7 (estrus=0=day of fusion) by a non-surgical approach. The surrogate mothers were examined by rectal examination and ultrasonography on day 45 after estrus to determine embryo survival and pregnancy. Pregnant surrogates were subsequently monitored regularly by rectal examination and ultrasonography. 
     Fluorescent Cow Production 
     The cow born by transplantation into the surrogate mother is a cow showing fluorescence, and the fluorescent cow was produced through the above process. 
     Example 2. Fluorescent Bovine Cell Preparation 
     2-1. Fluorescent Bovine Cell Primary Culture and Single-Cell Colony Culture 
     The primary cells derived from the ear skin of cow (SNU-F1-2) born by transplanting blastocysts obtained by fertilization of frozen semen of SNU-PB-1 described in Yum S Y et al. Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer. BMC Genomics 2018; 19:387 and wild-type cow eggs into a surrogate mother were cultured in DMEM supplemented with 10% bovine fetal serum, 1% penicillin/streptomycin (P/S) (Gibco), 1% non-essential amino acids (NEAA) (Gibco), and 100 mM β(2-ME) (Sigma-Aldrich) in an environment of 38.5° C. and 5% CO 2  humidified air. For single-cell colony culture, 100 cells were cultured with cell culture medium in 100 mm cell culture dish (Falcon). On day 10, single-cell colonies were picked up and transported to 12-well plates. When the single-cell colonies were fully grown in 12-well plates, the single-cell colonies were trypsinized for gDNA extraction and stored in Eppendorf tubes. 
     2-2. sgRNA Synthesis and Transfection 
     The single guide RNAs (sgRNA) for GFP (Green Fluorescent Protein), PRNP (Prion), and BLG (beta-lactoglobulin) genes were designed by CHOPCOHP software (https://chopchop.cbu.uib.no/) to select sgRNA candidates for target sites. And these sgRNAs were synthesized by GeneArt™ Precision gRNA Synthesis Kit (Invitrogen). Cas9 protein (TrueCut™ Cas9 Protein v2, Invitrogen) and sgRNA were transfected into bovine fibroblasts using an electroporation device (program #16, Neon Invitrogen). 
     guide sequence of sgRNA 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene 
                 Guide sequence(5′ to 3′) 
                 SEQ ID NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 GFP 
                 CGUCGCCGUCCAGCUCGACC 
                 161 
               
               
                   
               
               
                 PRNP 
                 AAAAACCAACAUGAAGCAUG 
                 102 
               
               
                   
               
               
                 BLG 
                 GGAGAUGUCGCUGGCCGCCA 
                 1 
               
               
                   
               
            
           
         
       
     
     Example 3. Analysis of the Incidence of Mutations Between the GFP-Expressing Group and the Non-Expressing Group 
     Cas9 protein (Thermo Fisher), GFP guide RNA, and PRNP or BLG guide RNA were delivered to GFP-expressing fibroblasts through transfection. After 5 days, cells in which GFP expression disappeared were identified, and single-cell culture was performed to analyze the mutation rate between the GFP-expressing group and the non-expressing group. The single-cell culture was performed by culturing 150 cells in a 100 mm culture dish. On the 10th day of culture, after checking whether single-cell colonies expressed GFP through a fluorescence microscope, cultured cells were divided into a GFP expression group and a non-expressing group, and each single cell colony was subcultured in a 6-well culture dish. When the culture dish was full of cells, genomic DNA was extracted from each single cell colony. Thereafter, the mutation of PRNP or BLG was confirmed through the T7E1 analysis. 
     Example 4. T7E1 Analysis 
     After transfection, genomic DNA was extracted from the cells using a DNA extraction kit (DNeasy Blood &amp; Tissue kit, Qiagen, Limburg, Netherlands). Target locus gene primers were designed by PRIMER3 software (PRNP; Forward: GAGGTGTTCGTTCGTTTTTC(SEQ ID NO: 278), Reverse: CTACCAGTTTCCTGTGCTTA(SEQ ID NO: 279), BLG; Forward: CTTGTCTAAGAGGCTGACCC(SEQ ID NO: 280), Reverse: GAGAAGATGGCTGTCTGCTC (SEQ ID NO: 281)). The PCR reaction was performed under the same conditions. (94° C. 5 minutes, 94° C. 20 seconds/57° C. 30 seconds/72° C. 35 seconds, 72° C. 5 minutes). Mutations in the target locus gene were detected in the T7E1 assay. The T7E1 assay was performed by thawing the amplicon, denaturing, and annealing to make a DNA heteroduplex, which was then subjected to the addition of 5 units of T7 endonuclease 1 (New England Biolabs, Massachusetts, USA) for 15 minutes at 37° C., and then the T7E1 assay was analyzed by 1% agarose gel electrophoresis. 
       FIG. 4  shows a result showing (a) prion (PRNP) gene knock-out cell ratio and (b) beta-lactoglobulin (BLG) gene knock-out cell ratio in GFP (+) cells and GFP (−) cells. The mutation colony ratio of the prion (PRNP) gene in the GFP negative cell group was higher (90.0% vs. 58.3%) than in the GFP positive cell group. The mutation colony ratio of beta-lactoglobulin (BLG) genes in the GFP negative cell group was higher (79% vs. 58%) than in the GFP positive cell group. 
     Example 5. Genetically Modified Cow Production Using Bovine Cells Including the GFP Gene 
     5-1. Egg Collection of Wild-Type Cow and In Vitro Maturation (IVM) 
     Ovaries are collected in saline at 35° C. in the slaughterhouse and transported to the laboratory within 2 hours. The cumulus-oocyte complex (COC) from follicles with a diameter of 2 to 8 mm is aspirated by using an 18 gauge needle attached to a 10 ml disposable syringe. COCs with evenly granulated cytoplasm and surrounded by three or more layers of compact cumulus cells are selected and washed three times in HEPES buffered tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, Calif., USA) supplemented with 10% FBS, 2 mM NaHCO 3 (Sigma-Aldrich Corp., St. Louis, Mo., USA), and 1% penicillin-streptomycin (v/v). For IVM, COC is incubated in a 4-well dish (30-40 oocytes per well; Falcon, Becton-Dickinson Ltd., Plymouth, UK) for 22 hours in a 38.5° C. and 5% CO2 environment in 450 μL TCM-199 tissue culture medium supplemented with 10% FBS, 0.005 AU/ml FSH (Antrin, Teikoku, Japan), 100 μM Cysteamine (Sigma-Aldrich), 1 μg/ml 17β-estradiol (Sigma-Aldrich). 
     5-2. Sperm Preparation in GFP-Expressing Bovines, In Vitro Fertilization (IVF) and In Vitro Embryo Culture (IVC) 
     Motile sperm obtained from cows expressing GFP are purified and selected using the Percoll gradient method. Briefly, sperm are selected from thawed semen straws by centrifugation on a Percoll discontinuous gradient (45-90%) at 1500 rpm for 15 minutes. A 45% Percoll solution is prepared with 1 mL of 90% Percoll (Nutricell, Campinas, SP, Brazil) and 1 mL of capacitation-TALP (Nutricell). The sperm pellet is centrifuged at 1500 rpm for 5 minutes and washed twice with capacitation-TALP. The active motile sperms of the pellet are used for fertilization of mature oocytes (at 24 h IVM). Oocytes are fertilized with 1 to 2×10 6  sperm/mL for 18 hours in 30 μL micro drops of IVF-TALP medium (Nutricell) coated with mineral oil in an environment of 39° C. and 5% CO2 (day 0). Putative zygotes are removed and cultured in a chemically defined two-step culture medium covered with mineral oil (Sigma-Aldrich). All cultures are performed in an environment of 38.5° C., 5% 02, 5% CO2, and 90% N2. On day 2, division rates are recorded, and embryonic development is monitored according to the stages of the International Society for Embryo Transplantation (IETS). 
     5-3. Transformation and Transplantation 
     5-3-1. Prion (PRNP) Gene Knock-Out Cow Production 
     A single guide RNAs (sgRNA) for GFP (green fluorescent protein) and PRNP (prion) genes are designed by CHOPCOHP software (https://chopchop.cbu.uib.no/) to select sgRNA candidates for target sites. And these sgRNAs are synthesized by GeneArt™ Precision gRNA Synthesis Kit (Invitrogen). Cas9 protein (TrueCut™ Cas9 Protein v2, Invitrogen) and sgRNA transform fertilized eggs by using an electroporation device (program #16, Neon Invitrogen). Embryos that do not express GFP are selected and transferred to a surrogate mother. The cow born by transplantation into the surrogate mother is a cow in which the prion (PRNP) gene is knocked out, and the prion (PRNP) gene knock-out cow is produced through the above process. 
     5-3-2. Beta-Lactoglobulin(BLG) Gene Knock-Out Cow Production 
     A single guide RNAs (sgRNA) for GFP (Green Fluorescent Protein) and BLG (beta-lactoglobulin) genes are designed by CHOPCOHP software (https://chopchop.cbu.uib.no/) to select sgRNA candidates for target sites. And these sgRNAs are synthesized by GeneArt™ Precision gRNA Synthesis Kit (Invitrogen). Cas9 protein (TrueCut™ Cas9 Protein v2, Invitrogen) and sgRNA transform fertilized eggs by using an electroporation device (program #16, Neon Invitrogen). Embryos that do not express GFP are selected and transferred to a surrogate mother. The cow born by transplantation to the surrogate mother is a cow in which the beta-lactoglobulin (BLG) gene is knocked out, and the beta-lactoglobulin (BLG) gene knock-out cow is produced through the above process. 
     5-3-3. Loxp-Loxp2272 Knock-In Cow Production 
     A single guide RNA (sgRNA) for the GFP (green fluorescent protein) gene was designed by CHOPCHOP software (https://chopchop.cbu.uib.no/) to select sgRNA candidates for the target site. The GFP sgRNA sequence was obtained as a RNA sequence using 5′-cctcgagctggacggcgacg-3′ (SEQ ID NO.: 282). 
     In order to insert the desired gene, loxp-loxp2272, into the position where the GFP gene was edited, ssODN was synthesized in Integrated DNA Technologies (US) company. 
     The length of the sequence of the gene to be inserted is 79 bp, and the sequence is 5′-ATAACTTCGTATAATGTATGCTATACGAAGTTATCaCGatCGaCGATAACTTCGTATA GGATACTTTATACGAAGTTAT-3′ (SEQ ID NO: 283). 
     For the GFP gene knock-in experiment, the experiment was divided into three groups: a control group; a group treated with GFP sgRNA and Cas9 protein (knock-out); and a group treated with GFP sgRNA, Cas9 protein, and donor DNA (knock-in). 
     In the production of fertilized bovine eggs, in vitro fertilization was performed by thawing the frozen semen including the GFP gene. After 18 hours of in vitro fertilization, GFP sgRNA, Cas9 protein (TrueCut™ Cas9 Protein v2, Invitrogen), and donor DNA were transferred to the fertilized egg using an electroporation machine (BEX, GEB 15, Japan). 
     Thereafter, blastocysts of the cow were produced through culture for 7 days. The presence or absence of expression of the GFP gene was confirmed through a fluorescence microscope ( FIG. 5 ). It was confirmed that the target position of GFP was knocked out (indel generation), or a desired sequence was inserted into the target position, thereby confirming that GFP existing in the genome of a bovine cell was no longer expressed. 
     By sampling each of these blastocysts, the presence or absence of insertion into the GFP gene was confirmed through PCR. At this time, the GFP primer sequence used for PCR is forward: 5′-GCTCTAGAGCCTCTGCTAA-3′ (SEQ ID NO: 284), reverse: 5′-CACATGAAGCAGCACGACTTC-3′ (SEQ ID NO: 285). The results are shown in  FIG. 6  (3,4,5 are knock-out cells, 6,7,8,9 are knock-in cells). 
     From these results, it can be seen that the engineered bovine cell in which the desired gene-editing, for example, knock-out or knock-in, has occurred, can be easily selected using the bovine cell having the GFP gene of the present disclosure. Without additional complicated screening processes, such as existing antibiotic resistance markers, cells with additional artificial engineering can be easily selected using bovine cells with GFP genes of this disclosure, thereby being used for various research activities using manipulated cells.