KR102253939B1 - Guide RNA for editing tyrosinase gene and use thereof - Google Patents

Abstract

The present invention relates to a method for producing melanin-deficient Paralichthys olivaceus by correcting tyrosinase of Paralichthys olivaceus with gene scissors. Therefore, when the tyrosinase gene is corrected using the guide RNA of the present invention, the pigment-deficient Paralichthys olivaceus has a golden or yellow body color, thereby being usefully used for mass production of high value-added mutant body color Paralichthys olivaceus.

Description

Guide RNA for editing tyrosinase gene and use thereof {Guide RNA for editing tyrosinase gene and use thereof}

The present invention relates to a guide RNA for tyrosinase gene correction and a method for producing melanin-deficient flounder using the same.

Flounder is a major aquaculture species in Korea, and is a major aquatic export product exported to the United States, Vietnam, and Canada, including China and Japan (2018 Seafood Export Evaluation and Outlook for 2019). According to data released by the Ministry of Oceans and Fisheries, gold flounder, which is a body color mutant that has a golden color in its entire body, unlike ordinary flounder that has a dark gray color, is traded at a price more than 1.5 times higher than that of ordinary flounder, and is currently produced in Korea. Golden flounder is obtained by crossing male and female of naturally occurring mutant individuals, and it is known that the descendant generation produces golden flounder with a 20% probability. The development of a mutant flounder deficient in melanin pigment so that the offspring can produce 100% body color mutant individuals is a task to be solved for mass production of high value-added melanin-deficient flounder.

The tyrosinase gene is a melanin-pigmenting gene, and its deficiency causes a dark gray body color that is close to black to be lost, and a yellow or golden body color in fish.

Genome Editing is a technology that freely edits the genetic information of living organisms. Genetic scissors are molecular tools designed and made to accurately cut desired genetic information and play a key role in genome editing technology. Like the next generation sequencing technology that took the field of gene sequencing to the next level, gene scissors are becoming a key technology that expands the speed and range of using genetic information and creates new industrial fields.

The genetic scissors developed so far can be divided into three generations according to their order. ZFN (Zinc Finger Nuclease) for the first-generation gene scissors, TALEN (Transcription Activator-Like Effector Nuclease) for the second-generation gene scissors, the most recently studied CRISPR (Cluster regularly interspaced short palindromic repeat)-Cas (CRISPR associated) 9 Is the third generation of gene scissors. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci found in the genome of approximately 40% of gene sequenced bacteria and 90% of gene sequenced archaea.

CRISPR functions as a prokaryotic immune system in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short portions of exogenous DNA called spacers are incorporated into the genome between CRISPR repeats and serve to remember past exposures. The CRISPR spacer is then used in eukaryotic organisms to recognize and silence exogenous genetic elements in a manner similar to RNAi.

Cas9, an essential protein element in the Type II CRISPR/Cas system, forms an active endonuclease when it forms a complex with two RNAs named CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). And, by doing so, protects the host cell by ignoring foreign genetic elements from invasion of the phage or plasmid. The crRNA is transcribed from the CRISPR element of the host genome that has been occupied by foreign invaders for delivery.

Recently, Jinek et al. demonstrated that single-chain chimeric RNA produced by the fusion of essential parts of crRNA and tracrRNA can replace two RNAs in the Cas9/RNA complex, thereby forming a functional endonuclease. I did. Because the site specificity of the nucleotide-binding CRISPR-Cas protein is controlled by an RNA molecule instead of a DNA-binding protein, which can be more difficult to design and synthesize, the CRISPR/Cas system is the first generation gene (ZFN). Scissors) and transcription activator-like effector nuclease (TALEN).

The introduction of the latest gene editing technology, CRISPR/Cas9 system, is effective for the development of varieties through active genotypic change and removal.

Accordingly, the present inventors developed a method of manufacturing golden flounder deficient in melanin pigment by applying genetic scissors technology.

Korean Patent Registration No. 10-1961332

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. Science. 2012 Aug 17;337(6096):816-21.

An object to be solved of the present invention is to provide a guide RNA for tyrosinase gene correction, a recombinant vector including the guide RNA, a composition for gene correction, and a method for producing melanin-deficient flounder using the same.

In order to solve the above problems, the present invention provides a guide RNA for correcting a tyrosinase gene comprising a nucleotide sequence identical to or complementary to SEQ ID NO: 3.

The tyrosinase gene may be a tyrosinase gene of flounder.

In addition, the present invention provides a recombinant vector for tyrosinase gene editing comprising the guide RNA.

In addition, the present invention provides a composition for correcting a tyrosinase gene comprising the guide RNA or the recombinant vector.

The composition may further include a Cas9 protein.

The composition may further include a dye.

In addition, the present invention provides a method for producing a melanin-deficient flounder comprising the step of injecting the gene editing composition into a fertilized flounder egg.

When the tyrosinase gene of flounder is corrected using the guide RNA (gRNA) of the present invention, the melanin pigment-deficient flounder has a golden or yellow body color, so it can be usefully used for mass production of high-value mutant color golden flounder. .

1 is a time-dependent generation step of a flatfish fertilized egg and an image thereof.
2 is a result of RT-PCR analysis of tyrosinase gene expression according to the stage of development of fertilized flounder eggs.
FIG. 3 is a result of analyzing the expression patterns of tyrosinase genes according to development stages of flounder by in situ hybridization.
Figure 4 is a comparison of the sequence of the tyrosinase gene (tyrosinase) of the flounder with the other six kinds of sequences.
5 is a guide RNA design (A) of the target site of the genetic scissors and microinjection (B) it into a fertilized egg.
6 is a result of confirming the location of the injection material (A) after microinjection into the fertilized halibut, the gene editing status according to the injection location (B), and the nucleotide sequence of the gene editing mutation (C).
7 is a result of confirming the change of melanin pigment by gene correction by measuring the number of melanophores, photographs (A) of cells after 24 hours and 48 hours of genetically corrected fertilized eggs (B), and This is the result of analyzing the number (C) of melanophores.
Figures 8 to 10 show the nucleotide sequence of the variant (X1: SEQ ID NO: 8, X2: SEQ ID NO: 9, and X3: SEQ ID NO: 10) of the tyrosinase transcript derived from flounder.

The present invention provides a guide RNA for correcting a tyrosinase gene comprising a nucleotide sequence identical to or complementary to SEQ ID NO: 3.

In the present invention, "tyrosinase" is a gene encoding a melanin pigment synthesis protein, and the tyrosinase gene may be a tyrosinase gene of flounder, and specifically, may be composed of SEQ ID NOs: 8 to 10.

In the present invention, "gene editing" refers to an action of causing nucleotide mutation by generating double-stranded DNA cleavage at a target site in a target gene, a tyrosinase gene.

The mutation includes deletion of all or part of the nucleotides in the target gene, inversion, conversion, or insertion of an exogenous gene, as a result of which the function of the target gene is lost. The loss of the function means that all or part of a biological function or role normally performed when the target gene is not modified, for example, the protein expressed by the target gene is terminated early, or the protein It has lost its normal function.

Specifically, the gene correction is a non-coding DNA sequence that knocks out a target gene or does not produce a protein by generating a stop codon at the target site or by generating a codon encoding an amino acid different from the wild type. It may be in various forms, such as introducing a mutation to, but is not limited thereto.

The gene correction of the present invention may be performed in vitro or in vivo.

The guide RNA of the present invention is for suppressing the expression of tyrosinase by modifying the tyrosinase gene.

The guide RNA includes a nucleotide sequence identical to or complementary to SEQ ID NO: 3.

The guide RNA includes a protospacer adjacent motif (PAM) sequence, which is a site specifically recognized by the Cas9 nuclease. The PAM sequence means 5'-NGG-3' (including the complementary nucleotide sequence 5'-CCN-3'), and the nucleotide sequence N can be any nucleotide sequence of A, T, G, or C.

In the present invention, "guide RNA" refers to a polynucleotide that cuts, inserts, or links target DNA in cells through RNA editing. The guide RNA may be a single-chain guide RNA (sgRNA). The guide RNA may be crRNA (CRISPR RNA) specific for a target nucleotide sequence. The guide RNA may further include a tracrRNA (trans-activating crRNA) that interacts with the Cas9 nuclease. The tracrRNA may include a polynucleotide forming a loop structure.

The guide RNA may include RNA, DNA, PNA, or a combination thereof. The guide RNA may be chemically modified.

The guide RNA can edit some sequences of the tyrosinase gene by non-homologous end-joining (NHEJ) in the genome of the cell, and specifically can be removed or modified.

In one embodiment of the present invention, gRNA (50 ng/µl) and Cas9 protein (100 ng/µl), 0.5% rhodamine dextran (10,000 MW, neutral), 0.05% phenol red, 0.1 M KCl was mixed to prepare an injection composition for gene editing, and after injecting it into a halibut fertilized egg (2 to 8 cell phase), tyrosinase correction was confirmed. As a result, only when injected into a fertilized egg, tyrosinase gene correction occurs. Was confirmed, and it was confirmed that the number of melanin-producing cells (melanophore) decreased.

Therefore, it is possible to correct the tyrosinase gene in the flounder using the gRNA comprising SEQ ID NO: 3 of the present invention, and it can be utilized in the production of melanin pigment-deficient flounder.

In addition, the present invention provides a recombinant vector for tyrosinase gene editing comprising the guide RNA.

In the present invention, the description of tyrosinase, gene editing, and guide RNA is as described above.

The “recombinant vector” of the present invention includes a gene sequence encoding a guide RNA (gRNA) that specifically recognizes the target gene, and is functionally linked with an expression control sequence so that guide RNA (gRNA) can be expressed. Has been. For example, the gRNA expression vector includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, and can be prepared in various ways according to the purpose. I can.

The gRNA expression vector may include a promoter that enables transcription of RNA, specifically includes a promoter of RNA polymerase, and more specifically includes U3, U6, and H1 promoters.

In addition, the gRNA expression vector may contain a selectable marker, and the vector may be self-replicating or integrated into the host DNA. The vector of the present invention can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation use enzymes generally known in the art.

In addition, the present invention provides a composition for correcting a tyrosinase gene comprising the guide RNA or the recombinant vector.

The composition for gene editing of the present invention includes a guide RNA comprising a nucleotide sequence identical to or complementary to SEQ ID NO: 3 or a recombinant vector comprising the same.

When the guide RNA is injected into the Cas9 protein and cells, the target gene, tyrosinase gene, can be corrected.

In the above-described embodiment of the present invention, gRNA (50 ng/µl) and Cas9 protein (100 ng/µl), 0.5% rhodamine dextran (10,000 MW, neutral), 0.05% phenol red, 0.1M KCl was mixed to prepare an injection composition for gene correction.

Therefore, the composition may further include a Cas9 protein.

In the present invention, the Cas9 protein recognizes a specific sequence (protospacer associated motif, PAM) of the target gene and has a nucleotide cleavage activity, and is selected from among all Cas9s capable of causing insertion and/or deletion (Indel) in the target gene. It can be more than a species.

Specifically, the Cas9 protein is a Cas9 protein derived from the genus Streptococcus , specifically, Streptococcus thermophiles , Streptocuccus aureus , or Streptococcus pyogenes. ; Campylobacter genus, specifically, Campylobacter jejuni Cas9 protein derived from; Ney ceria (Neisseria), A specifically Ney ceria mening gidi tooth (Neisseria meningitidis) derived Cas9 protein; Cas9 protein derived from the genus Pasteurella , specifically, Pasteurella multocida; Fran when cellar (Francisella) in, in particular, when Francisco Cellar Novi Let (Francisella novicida), but may be one or more selected from the group consisting of Cas9 protein derived from, without being limited thereto.

The Cas9 protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI).

The Cas9 protein is used together with a target gene-specific guide RNA for guiding to a target site of genomic DNA. The Cas9 protein may form a complex with a guide RNA to act in the form of ribonucleic acid protein (RNP).

In addition, the composition may further include a dye. The dye may include fluorescein-dextran, Bodipy-FL-dextran, or Oregon Green-dextran.

In addition, the present invention provides a method for producing a melanin-deficient flounder comprising the step of injecting the gene editing composition into a fertilized flounder egg.

The description of the composition for gene editing in the present invention is as described above.

The "method for producing melanin-deficient halibut" of the present invention includes the step of injecting the gene editing composition into fertilized halibut.

In one embodiment of the present invention, after injecting a composition containing gRNA and Cas9 protein into a flatfish fertilized egg (2 to 8 cell phase), tyrosinase correction was confirmed. As a result, only when injected into a fertilized egg, tyrosinase gene correction is It was confirmed that it occurred, and it was confirmed that the number of melanin-producing cells (melanophore) decreased.

In an embodiment of the present invention, after obtaining and transferring the collected halibut eggs to sterilized seawater, the wounded eggs (normal) that rise to the surface of the seawater are first selected to exclude dead or unfertilized eggs. I did. After entering the 1st cell phase, it was accurately classified into unfertilized eggs and fertilized eggs through microscopic observation, and the selected eggs were arranged in a 1% agarose mold for microinjection and microinjected. In other words, the stage of the fertilized egg during microinjection was the 2 cell stage, and the experiment was carried out up to the 8 cell stage.

Therefore, the step of injecting may include injecting the gRNA and Cas9 protein into the fertilized eggs of the 2 to 8 cell stage.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

1. Expression patterns of tyrosinase according to the stage of development of fertilized flounder eggs

For genetic correction of fertilized flounder eggs, it is necessary to check the stages of development of fertilized flounder eggs over time. Therefore, while culturing the fertilized flounder eggs in an incubator at 19.5±0.5° C., the stages of occurrence were confirmed by time (FIG. 1 ). Based on this, 20 fertilized eggs were sampled at each stage, RNA was extracted (RNeasy mini kit, Qiagen), and cDNA was synthesized from them (PrimeScript 1st strand cDNA synthesis kit, Takara).

In order to analyze the expression pattern of the tyrosinase gene using the previously obtained cDNA, RT-PCR was performed using EF-1a as a control to confirm whether the total amount of the sample was prepared equally. (Figure 2A). Using the identified cDNA for each stage of development as a template, the expression pattern for each stage of development of the tyrosinase gene (tyrosinase) was confirmed (FIG. 2B). As a result, the tyrosinase gene was a maternal gene, and the expression level was low, but expression was observed from the 4-cell stage, and the expression level gradually increased and was modified to a high level after the 80% epiboly stage passed. It was confirmed that it was maintained continuously until 48 hours after that.

Next, in situ hybridization analysis was performed to analyze the spatial expression pattern of the tyrosinase gene.

To this end, 1003 bp inside the ORF of the tyrosinase gene using a tyrosinase-specific primer set (SEQ ID NO: 1: forward 5'-CCATGTCAGGCCGTGGTTTCT-3', SEQ ID NO: 2: reverse 5'-GAACCCTGCAGGCACCAGAT-3) A clone of was obtained, and a dig-labeled antisense RNA probe was synthesized therefrom (DIG RNA Labeling kit, Roche). Fertilized eggs for each stage of development were fixed in 4% paraformaldehyde, and each sample 48 hours before was fixed with a fixative, the egg membrane was removed 24 hours, transferred to MeOH, and stored at -20°C until the experiment. The sample was transferred to the PBS buffer in steps of concentration, reacted with the synthesized probe at 70° C. for 12 hours, and washed. A blocking step was performed using 5% sheep serum, followed by reaction with dig-Ab at room temperature for 2 hours, followed by washing with PBS buffer to remove non-specific reactions. Thereafter, the color was developed through reaction with BCIP/NBT (substrate), and a picture was taken using a microscope (FIG. 3).

As shown in the RT-PCR result, tyrosinase gene expression can be observed from the four-cell stage fertilized egg, and it was confirmed that the overall expression of the fertilized egg cells was limited as the shield stage passed. At the 80% epiboly stage, tyrosinase began to be expressed on both sides of the body truck. These cells then synthesize eumelanin to become melanophores with black pigment.

As the development progressed, the number of melanophores increased, and it was confirmed that cells having melanin were generated at the 22 somite stage 28 hours after fertilization.

2. Analysis of tyrosinase gene in flounder

Tyrosinase (tyrosinase) is well known as a gene that plays an important role in the process of forming black melanin pigment. When it is used as a target, the reduction of epidermal melanin pigment in embryos in the F0 generation after injection is performed. By checking, it is easy to predict whether gene editing has occurred. In the present invention, the tyrosinase gene of flounder was analyzed to edit the tyrosinase gene by synthesizing sgRNA targeting the tyrosinase gene of flounder and using this to introduce the CRISPR/Cas9 system.

4 is a result of comparing and analyzing the tyrosinase gene sequence of the flounder (Paralichthys olivaceus ) used to make melanin deficient flounder with six other types of tyrosinase genes.

Compared to the tyrosinase of zebrafish ( Danio rerio ), medaka ( Oryzias latipes ), true frog ( Pelophylax nigromaculatus ), red field ( Gallus gallus ), mouse ( Mus musculus ) and human ( Homo sapiens ), well-conserved residues are black. As a result, partially conserved residues appeared gray. The functional domain of tyrosinase is indicated as follows. The orange box indicates the central domain of the tyrosinase, the blue arrow indicates the signal peptide, the red arrow indicates the two Cu-binding domains, and the green arrow indicates the laminin-type-EGF-like domain. like domain).

3. Preparation and microinjection of guide RNA

The tyrosinase transcript of flounder had the same ORF sequence, but there were three variants having different length UTR regions (FIGS. 8 to 10, SEQ ID NOs: 8 to 10). Therefore, amino acid 28 was selected as a target to include the PAM sequence recognized by the Cas9 nuclease behind the start codon (ATG).

Single guide RNA (sgRNA) synthesis

Specifically, the target sequence was selected using the zifit site (http://zifit.partners.org/ZiFiT/). 5'-ACTCAGAGGGACTACGGACC-3' (SEQ ID NO: 3, 20 bp) in front of the tyrosinase domain was selected as a guide RNA (gRNA) target (Fig. 5).

To synthesize the entire sequence of gRNA including the backbone, the forward primer (sgRNA forward primer, SEQ ID NO: 4) attaches the T7 promoter sequence to the front of the selected target sequence, and the sgRNA backbone sequence to the rear. A total of 57 mer oligos were designed by attaching some parts, and 80 mer sgRNA backbone oligos were ordered and used for the reverse primer (sgRNA reverse primer, SEQ ID NO: 5).

The gRNA was synthesized and obtained using the cloning free method presented by Gaurav et al. ( Genome Res. 2015 Jul; 25(7): 1030-1042). Forward primer (100 pmol/µl) 2 µl, reverse primer (100 pmol/µl) 2 µl, HS prime star (Takara, Japan) 0.5 µl, 5 × prime star buffer 10 µl, dNTP DNA template was synthesized by 1 cycle PCR of 95°C for 2 minutes, 50°C for 10 minutes, and 72°C for 30 minutes using 4 μl, and then purified with a PCR purification kit (Bioneer, Korea). Ethanol precipitation was performed by adding 3M sodium acetate. Thereafter, it was dissolved in nuclease free water and used as a template for in vitro transcription. To 14 µl of DNA template, add 1 µl of T7 RNA polymerase, 2 µl of 10 × T7 polymerase butter, 1 µl of RNase inhibitor, and 2 µl of sense NTP (10 pmol/µl) at 37℃ for 4 hours Reacted during.

Thereafter, 1 µl of RNase free DNase I was added and reacted at 37° C. for 20 minutes to remove the DNA template, followed by ethanol precipitation using 4M LiCl. The synthesized gRNA was quantified and stored at -80°C until use.

Preparation and injection method of composition for gene editing

Injection materials are the gRNA synthesized above (50 ng/µl) and Cas9 protein (100 ng/µl), 0.5% rhodamine dextran (10,000 MW, neutral), 0.05% phenol red (phenol). red), prepared by mixing 0.1M KCl.

After obtaining and transferring the collected flounder eggs to sterile seawater, the flounder eggs (normal) floating on the surface of the seawater were first selected to exclude dead or unfertilized eggs. After entering the 1st cell phase, it was accurately classified into unfertilized eggs and fertilized eggs through microscopic observation, and the selected eggs were arranged in a 1% agarose mold for microinjection, and microinjection was performed. Therefore, the stage of the fertilized egg during microinjection was the 2-cell stage, and the experiment was carried out up to the 8-cell stage.

The halibut fertilized eggs were arranged in a 1% agarose mold, and the injection composition was microinjected at a concentration of 0.5 nl/embryo. The material was microinjected into the correct position as shown in FIG. 5B using a microinjection needle made with a sharp tip using a grinder.

Gene editing and mutation rate verification method

In order to check whether mutation occurred in the microinjected embryo, genomic DNA was extracted from the embryo at 48 hours after fertilization, and through PCR reaction and T7 endonuclease 1 (T7E1) assay. The mutation was confirmed.

For genomic DNA, microinjected embryos were placed in an e-tube, followed by 50 µl of Lysis buffer (lysis buffer, 10 mM Tris-Cl, pH 8.0, 50 mM KCl, 0.3% tween 20, 0.3% triton X-100) and pro 1 µl of Teinase K (proteinase K, 20 mg/ml) was added and incubated at 60° C. for 1 hour, followed by extraction. At this time, it was confirmed that the embryo was lysed by vortexing every 20 minutes, and after that, inactivation was performed at 95°C for 10 minutes, and then used in the experiment.

A primer for T7E1 assay (SEQ ID NO: 6, SEQ ID NO: 7) was designed to check mutations with a targeting site in between. PCR reaction was performed using genomic DNA as a template, and amplification was performed by 35 cycles of 1 cycle at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.

A hetero duplex reaction was performed on the DNA amplified through PCR using a PCR machine. After the PCR reaction was completed, the reaction was carried out by reacting at 95°C for 4 minutes to lower the temperature by 1°C per second to 85°C and 0.1°C per second from 85°C to 25°C. After that, T7 endonuclease I (New England BioLab, USA) enzyme and buffer were added to the PCR product, reacted at 37°C for 2 hours, and then electrophoresed on a 2% agarose gel to confirm.

Confirmation of halibut mutation by genetic modification

0.5% rhodamine dextran (10,000 MW, neutral: Sigma), which shows fluorescence, was injected together with gRNA and Cas9 protein to confirm that the injection material was injected at the correct location, and the location of the fluorescence 24 hours after fertilization. Was confirmed. Compared to the non-microinjected flatfish fertilized egg, the location of fluorescence is expressed in case 1 (case 1) expressed in cells above the yolk and in the perivitelline space (space between the egg membrane and the fertilized egg) outside the cells. It was observed in two cases of case 2 (Fig. 6A).

A T7 endonuclease 1 assay was performed in order to check whether gene correction occurred by collecting the fertilized eggs of each group. The T7 E1 assay is a T7 endonuclease that recognizes and cuts a hetero duplex formed at the site where gene correction has occurred in the process of amplifying the selected target site by PCR and slowly regenerating the reaction product after denaturation. This is how to check using 1.

As a result of the analysis, it was confirmed that only when injected into the fertilized egg (case 1), gene correction occurred (FIG. 6B). Next, in order to accurately confirm gene correction in the fertilized egg corresponding to Case 1, the target site was amplified by PCR and nucleotide sequence analysis was performed. As a result, it was confirmed that various gene editing patterns occurred as shown in FIG. 6C.

4. Changes in melanin pigment by gene editing

In order to confirm whether the mutation caused by gene correction of tyrosinase induces the change in melanin pigment, the head of the fertilized egg is imaged 24 hours after fertilization (Fig. 7A) and the number of melanophores is counted ( counting) (Fig. 7C). As a result, it was confirmed that the number of melanin-producing cells (melanophore) in microinjected individuals decreased by 47.5% from 39.6 in the control group to 20.8 in the control group.

It can be judged that this result is observed because F0 individuals undergo gene correction in a mosaic pattern. After an additional 24 hours (48 hpf), it was confirmed that the formation of melanin-producing cells (melanophore) was further reduced in the body trunk (FIG. 7A), and all of these individuals were sampled and genotyping for each individual by T7E1 assay. (genotyping) was performed. As a result, it was confirmed that all the microinjected individuals were 100% genetically corrected as shown in FIG. 7B.

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aggttggagt tgccctgatt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7E1 reverse primer <400> 7 accccagtaa ccaaacctgc 20 <210> 8 <211> 3227 <212> DNA <213> Paralichthys olivaceus <400> 8 gtggaaggag aggaggtctt ggggagggtt agaggactga gtggaggggt aacaaaattg 60 taatcgatta tctttggaaa catttgtgta ctggtagaca gtgttgtctt tgataaaagt 120 aaaaaactaa aacaaattca gtgggatgtg aatcctgttg ttctctgctt gcagcctaaa 180 aatctctcca gctctgaatc tattcctctg tgctgtttca ggttcatttg cgtccatcct 240 ctcatcagga gcttactttc agactgtggt taaggttgga gttgccctga tttttcatca 300 tgaggactgt gtgtttatct gcactcctgc tgcagctcct cgggacgtct ttctgtcagt 360 tcccccgccc gtgtgccaac tcagagggac tacggaccaa agagtgctgc ccggtgtggg 420 acggtgacgg ctcagtctgc ggtgccatgt caggccgtgg tttctgctcc gaggtgttgg 480 tctcagatga gccccacggg ccccagtacc ctcaccgtgg gattgatgac agagagcgct 540 ggcctttagc cttctttaac cggacgtgtc gctgtgctgg aaactatgga ggctttaact 600 gtggagaatg caggtttggt tactggggtt cgaactgtgc agagtacagg gaatctgtgc 660 gcaggaacat catgaccatg tccactgctg agcagcagaa gtttgtctct tacctgaacc 720 tggctaaaaa caccatcagc caagactacg tcatatccac agcaacaaga gcagagatgg 780 gcgagaacgg tgagaacccc atgttctctg acatcaacac ctatgacctg tttgtgtgga 840 tgcactacta cgtgtcccgg gacgccttcc tggggggggc agggaacgta tggacagaca 900 tcgactttgc ccatgaatct gcggccttcc ttccgtggca ccgagtcttc ctgcttcact 960 gggagaatga gatcaggaag ctgacgggag attttaactt caccatcccg tactgggact 1020 ggagggacgc ccagtcctgt gaggtgtgca ccgatgctct gatgggtgga cgcagctccc 1080 gcaatcccaa cctcatcagc cccggctccg tcttctcctc atggaaggtg atctgcaccc 1140 agccagagga gtacaacagt cgagaagcat tgtgtaacgc caccggggag ggcccactgt 1200 tgcgtaaccc aggcaaccat gataggaacc gcgtgcctcg actccccaca agagctgacg 1260 ttgatttcac tgtgggcctt cctgagtacg agacggggcc catggaccga ttctccaact 1320 tgagctttag aaacgtcctg gagggctttg ccagtccagt gacaggcatg gcggtgccgg 1380 gccagagcac gatgcacaac tccctgcacg tcttcatgaa cggctccatg tcctcggtgc 1440 agggttcagc caacgacccc atattcttgc tgcaccacgc tttcattgac agtatctttg 1500 aacgctggct caggacccac atgcctctcc gggccaacta ccccctcgcc aatgccccca 1560 ttggccacaa tgatggctac tacatggtgc ccttcctgcc tctctttaga aacggagact 1620 acttcttgtc caacaaggct ctgggattcg agtacgccta tttgctggac cctggacaga 1680 ggttcgtgca ggagttcctg acgccatacc tcgaacaggc ccaacagatc tggcagtggc 1740 tcctgggggc cgggatcctc ggggctctca tcgcagcggt cctcgctgcg gtgattgttg 1800 ttgcgaggag gaagtggagg cgtaaccaga ggaggaagag ggtgtcaagc tacggagaga 1860 gacaaccact actgcagagc agctcagagg aaggctcggc ctcatatcag accactctgt 1920 aacacacaca ctgtacatac tgttacatga atgtgcaaac tattaaggac acatgattcc 1980 atgacacaaa tgaataagat gaatcttagt acaaggatac gtcttttttt tattcctgag 2040 aataaatcaa tttagatttt ttaataaagg tacagatgag ttagagaaga gcctttaaag 2100 tcagttcacc cgaaatctca aaaagtaaat atttctccct catagtatct ggcagaccgc 2160 tttgtgtgtg tgtgtgtgtg ttgttgagat acctgctaat atatctgctc atctgctttt 2220 gaacaatagg ggtgaagtga attttatttg tggtggtcat agcgctcatt aggaacacaa 2280 tttattttcc aaggttttct ttcaatttct ttgtgcaaaa aaagacagag gagaacagaa 2340 gagacggttt aagacatctc cacaaatgga ttgtttaaac gttgatgagg ggagatttgg 2400 gtgaaatgac cctttacaga ttgagacaag gagcaaagtc cttcagatgt gcctgatatt 2460 ttacacattc agtacatgtc atgtcagaaa tatctctgtg tgacacaaac gtacaacgtg 2520 acagactttc agagccatca agagatcttt atcacaaagc aacacctccc tgcagtttgc 2580 aggtcaaaca ccttgaggga tatttattgt ctatttacag aggccactct gcagccaatg 2640 acagcagctg tagttagata agatcattga cttgtgcaga gacacatttc atctaattta 2700 ttgtccactt gtatgttact ggaaatcatt gagattcagt aaatagtcac ttaaagccag 2760 gtcatgtctg ctgatctgtg gggacagctc gagttgttga aagcacttta tttgttgctt 2820 catacagata aagtcccggt cacacgtacc agagtgtgtc agcaatgaac cctgttcact 2880 tccaatcagc accagctagg gaggtgagga aaacatttgt tttatgtggc gaagcaaatg 2940 gcagagtgat ttcacgtaga gtttaacttt gttgaacttt gacccatgat attctcttta 3000 cattcatgta tctgtgaccg ggcctgaaat ctgagtccca aaaaacatac ttttaatcag 3060 tttatgtctt tcagtgcaaa attctgaaaa aactgaccaa accgacaaca cactctcgtg 3120 tgttagcact ggtgctaaag gagcgcagca tactcacaat atgtatcatg ataatgaaga 3180 tgaatcagga ttttgtaaat gctgcattaa aatcagactt gatccaa 3227 <210> 9 <211> 3064 <212> DNA <213> Paralichthys olivaceus <400> 9 gagagcgagg gtggaaggag aggaggtctt ggggagggtt agaggactga gtggaggggt 60 tcatttgcgt ccatcctctc atcaggagct tactttcaga ctgtggttaa ggttggagtt 120 gccctgattt ttcatcatga ggactgtgtg tttatctgca ctcctgctgc agctcctcgg 180 gacgtctttc tgtcagttcc cccgcccgtg tgccaactca gagggactac ggaccaaaga 240 gtgctgcccg gtgtgggacg gtgacggctc agtctgcggt gccatgtcag gccgtggttt 300 ctgctccgag gtgttggtct cagatgagcc ccacgggccc cagtaccctc accgtgggat 360 tgatgacaga gagcgctggc ctttagcctt ctttaaccgg acgtgtcgct gtgctggaaa 420 ctatggaggc tttaactgtg gagaatgcag gtttggttac tggggttcga actgtgcaga 480 gtacagggaa tctgtgcgca ggaacatcat gaccatgtcc actgctgagc agcagaagtt 540 tgtctcttac ctgaacctgg ctaaaaacac catcagccaa gactacgtca tatccacagc 600 aacaagagca gagatgggcg agaacggtga gaaccccatg ttctctgaca tcaacaccta 660 tgacctgttt gtgtggatgc actactacgt gtcccgggac gccttcctgg ggggggcagg 720 gaacgtatgg acagacatcg actttgccca tgaatctgcg gccttccttc cgtggcaccg 780 agtcttcctg cttcactggg agaatgagat caggaagctg acgggagatt ttaacttcac 840 catcccgtac tgggactgga gggacgccca gtcctgtgag gtgtgcaccg atgctctgat 900 gggtggacgc agctcccgca atcccaacct catcagcccc ggctccgtct tctcctcatg 960 gaaggtgatc tgcacccagc cagaggagta caacagtcga gaagcattgt gtaacgccac 1020 cggggagggc ccactgttgc gtaacccagg caaccatgat aggaaccgcg tgcctcgact 1080 ccccacaaga gctgacgttg atttcactgt gggccttcct gagtacgaga cggggcccat 1140 ggaccgattc tccaacttga gctttagaaa cgtcctggag ggctttgcca gtccagtgac 1200 aggcatggcg gtgccgggcc agagcacgat gcacaactcc ctgcacgtct tcatgaacgg 1260 ctccatgtcc tcggtgcagg gttcagccaa cgaccccata ttcttgctgc accacgcttt 1320 cattgacagt atctttgaac gctggctcag gacccacatg cctctccggg ccaactaccc 1380 cctcgccaat gcccccattg gccacaatga tggctactac atggtgccct tcctgcctct 1440 ctttagaaac ggagactact tcttgtccaa caaggctctg ggattcgagt acgcctattt 1500 gctggaccct ggacagaggt tcgtgcagga gttcctgacg ccatacctcg aacaggccca 1560 acagatctgg cagtggctcc tgggggccgg gatcctcggg gctctcatcg cagcggtcct 1620 cgctgcggtg attgttgttg cgaggaggaa gtggaggcgt aaccagagga ggaagagggt 1680 gtcaagctac ggagagagac aaccactact gcagagcagc tcagaggaag gctcggcctc 1740 atatcagacc actctgtaac acacacactg tacatactgt tacatgaatg tgcaaactat 1800 taaggacaca tgattccatg acacaaatga ataagatgaa tcttagtaca aggatacgtc 1860 ttttttttat tcctgagaat aaatcaattt agatttttta ataaaggtac agatgagtta 1920 gagaagagcc tttaaagtca gttcacccga aatctcaaaa agtaaatatt tctccctcat 1980 agtatctggc agaccgcttt gtgtgtgtgt gtgtgtgttg ttgagatacc tgctaatata 2040 tctgctcatc tgcttttgaa caataggggt gaagtgaatt ttatttgtgg tggtcatagc 2100 gctcattagg aacacaattt attttccaag gttttctttc aatttctttg tgcaaaaaaa 2160 gacagaggag aacagaagag acggtttaag acatctccac aaatggattg tttaaacgtt 2220 gatgagggga gatttgggtg aaatgaccct ttacagattg agacaaggag caaagtcctt 2280 cagatgtgcc tgatatttta cacattcagt acatgtcatg tcagaaatat ctctgtgtga 2340 cacaaacgta caacgtgaca gactttcaga gccatcaaga gatctttatc acaaagcaac 2400 acctccctgc agtttgcagg tcaaacacct tgagggatat ttattgtcta tttacagagg 2460 ccactctgca gccaatgaca gcagctgtag ttagataaga tcattgactt gtgcagagac 2520 acatttcatc taatttattg tccacttgta tgttactgga aatcattgag attcagtaaa 2580 tagtcactta aagccaggtc atgtctgctg atctgtgggg acagctcgag ttgttgaaag 2640 cactttattt gttgcttcat acagataaag tcccggtcac acgtaccaga gtgtgtcagc 2700 aatgaaccct gttcacttcc aatcagcacc agctagggag gtgaggaaaa catttgtttt 2760 atgtggcgaa gcaaatggca gagtgatttc acgtagagtt taactttgtt gaactttgac 2820 ccatgatatt ctctttacat tcatgtatct gtgaccgggc ctgaaatctg agtcccaaaa 2880 aacatacttt taatcagttt atgtctttca gtgcaaaatt ctgaaaaaac tgaccaaacc 2940 gacaacacac tctcgtgtgt tagcactggt gctaaaggag cgcagcatac tcacaatatg 3000 tatcatgata atgaagatga atcaggattt tgtaaatgct gcattaaaat cagacttgat 3060 ccaa 3064 <210> 10 <211> 3076 <212> DNA <213> Paralichthys olivaceus <400> 10 agagagcgag ggtggaagga gaggaggtct tggggagggt tagaggactg agtggagggg 60 taacaaaatt gttcatttgc gtccatcctc tcatcaggag cttactttca gactgtggtt 120 aaggttggag ttgccctgat ttttcatcat gaggactgtg tgtttatctg cactcctgct 180 gcagctcctc gggacgtctt tctgtcagtt cccccgcccg tgtgccaact cagagggact 240 acggaccaaa gagtgctgcc cggtgtggga cggtgacggc tcagtctgcg gtgccatgtc 300 aggccgtggt ttctgctccg aggtgttggt ctcagatgag ccccacgggc cccagtaccc 360 tcaccgtggg attgatgaca gagagcgctg gcctttagcc ttctttaacc ggacgtgtcg 420 ctgtgctgga aactatggag gctttaactg tggagaatgc aggtttggtt actggggttc 480 gaactgtgca gagtacaggg aatctgtgcg caggaacatc atgaccatgt ccactgctga 540 gcagcagaag tttgtctctt acctgaacct ggctaaaaac accatcagcc aagactacgt 600 catatccaca gcaacaagag cagagatggg cgagaacggt gagaacccca tgttctctga 660 catcaacacc tatgacctgt ttgtgtggat gcactactac gtgtcccggg acgccttcct 720 ggggggggca gggaacgtat ggacagacat cgactttgcc catgaatctg cggccttcct 780 tccgtggcac cgagtcttcc tgcttcactg ggagaatgag atcaggaagc tgacgggaga 840 ttttaacttc accatcccgt actgggactg gagggacgcc cagtcctgtg aggtgtgcac 900 cgatgctctg atgggtggac gcagctcccg caatcccaac ctcatcagcc ccggctccgt 960 cttctcctca tggaaggtga tctgcaccca gccagaggag tacaacagtc gagaagcatt 1020 gtgtaacgcc accggggagg gcccactgtt gcgtaaccca ggcaaccatg ataggaaccg 1080 cgtgcctcga ctccccacaa gagctgacgt tgatttcact gtgggccttc ctgagtacga 1140 gacggggccc atggaccgat tctccaactt gagctttaga aacgtcctgg agggctttgc 1200 cagtccagtg acaggcatgg cggtgccggg ccagagcacg atgcacaact ccctgcacgt 1260 cttcatgaac ggctccatgt cctcggtgca gggttcagcc aacgacccca tattcttgct 1320 gcaccacgct ttcattgaca gtatctttga acgctggctc aggacccaca tgcctctccg 1380 ggccaactac cccctcgcca atgcccccat tggccacaat gatggctact acatggtgcc 1440 cttcctgcct ctctttagaa acggagacta cttcttgtcc aacaaggctc tgggattcga 1500 gtacgcctat ttgctggacc ctggacagag gttcgtgcag gagttcctga cgccatacct 1560 cgaacaggcc caacagatct ggcagtggct cctgggggcc gggatcctcg gggctctcat 1620 cgcagcggtc ctcgctgcgg tgattgttgt tgcgaggagg aagtggaggc gtaaccagag 1680 gaggaagagg gtgtcaagct acggagagag acaaccacta ctgcagagca gctcagagga 1740 aggctcggcc tcatatcaga ccactctgta acacacacac tgtacatact gttacatgaa 1800 tgtgcaaact attaaggaca catgattcca tgacacaaat gaataagatg aatcttagta 1860 caaggatacg tctttttttt attcctgaga ataaatcaat ttagattttt taataaaggt 1920 acagatgagt tagagaagag cctttaaagt cagttcaccc gaaatctcaa aaagtaaata 1980 tttctccctc atagtatctg gcagaccgct ttgtgtgtgt gtgtgtgtgt tgttgagata 2040 cctgctaata tatctgctca tctgcttttg aacaataggg gtgaagtgaa ttttatttgt 2100 ggtggtcata gcgctcatta ggaacacaat ttattttcca aggttttctt tcaatttctt 2160 tgtgcaaaaa aagacagagg agaacagaag agacggttta agacatctcc acaaatggat 2220 tgtttaaacg ttgatgaggg gagatttggg tgaaatgacc ctttacagat tgagacaagg 2280 agcaaagtcc ttcagatgtg cctgatattt tacacattca gtacatgtca tgtcagaaat 2340 atctctgtgt gacacaaacg tacaacgtga cagactttca gagccatcaa gagatcttta 2400 tcacaaagca acacctccct gcagtttgca ggtcaaacac cttgagggat atttattgtc 2460 tatttacaga ggccactctg cagccaatga cagcagctgt agttagataa gatcattgac 2520 ttgtgcagag acacatttca tctaatttat tgtccacttg tatgttactg gaaatcattg 2580 agattcagta aatagtcact taaagccagg tcatgtctgc tgatctgtgg ggacagctcg 2640 agttgttgaa agcactttat ttgttgcttc atacagataa agtcccggtc acacgtacca 2700 gagtgtgtca gcaatgaacc ctgttcactt ccaatcagca ccagctaggg aggtgaggaa 2760 aacatttgtt ttatgtggcg aagcaaatgg cagagtgatt tcacgtagag tttaactttg 2820 ttgaactttg acccatgata ttctctttac attcatgtat ctgtgaccgg gcctgaaatc 2880 tgagtcccaa aaaacatact tttaatcagt ttatgtcttt cagtgcaaaa ttctgaaaaa 2940 actgaccaaa ccgacaacac actctcgtgt gttagcactg gtgctaaagg agcgcagcat 3000 actcacaata tgtatcatga taatgaagat gaatcaggat tttgtaaatg ctgcattaaa 3060 atcagacttg atccaa 3076

Claims (7)

Guide RNA for correction of tyrosinase gene of flounder comprising a nucleotide sequence identical to or complementary to SEQ ID NO: 3. delete Recombinant vector for tyrosinase gene editing comprising the guide RNA of claim 1. A composition for correcting a tyrosinase gene comprising the guide RNA of claim 1 or the recombinant vector of claim 3. The method of claim 4,
The composition is a composition that further comprises a Cas9 protein. The method of claim 5,
The composition of the composition further comprises a dye. Including the step of injecting the composition for editing the gene of claim 4 in the fertilized flounder egg,
Method for producing melanin deficient flounder.

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