Genetically modified zebrafish

Abstract

To solve the problem in which a useful disease model has not sufficiently been established for symptoms caused by trap genome mutation (for example, facies and mental retardation).SOLUTION: A genetically modified zebrafish expresses mutant Trrap protein. The genetically modified zebrafish has a nucleic acid sequence encoding the mutant Trrap protein, incorporated into the genome of the genetically modified zebrafish. The mutant Trrap protein lacks in the functions of FAT domain, PIKK-TRRAP domain, and FATC domain existing in wild-type Trrap protein.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to genetically modified zebrafish, and in particular to genetically modified zebrafish that express a mutant Trrap protein. [Background technology]

[0002] Human TRRAP (transformation / transcription domain-associated protein) is a component of the HAT (histone acetyltransferase) complex and is thought to regulate the transcription of target genes and DNA replication. Recently, various genomic mutations have been discovered in the human TRRAP gene, and it has been reported that affected individuals exhibit similar symptoms, including characteristic facial features and mental retardation (Non-Patent Document 1). Non-Patent Document 1 reported that analysis of human genetic mutations in Europe and the United States identified missense mutations in the human TRRAP gene in 17 affected individuals, and that these individuals exhibited characteristic facial features and mental retardation. However, the relationship between missense mutations in the TRRAP gene and characteristic facial features and mental retardation remains unknown.

[0003] Patent Document 1 shows genes associated with heart failure, and discloses a zebrafish heart failure model as a means for diagnosing and controlling heart failure, using the genes and proteins encoded by the genes.

[0004] Non-patent document 2 found that knockdown and knockout embryos of zebrafish trrap genes exhibited suppressed responses to sound and showed abnormalities in the development of the lateral line (an organ unique to fish that is functionally similar to the organ that receives hearing), and stated that the function of the human TRRAP gene is to regulate hearing.

[0005] In Non-Patent Document 3, phenotypic analysis was performed on conditional knockout mice in which the mouse Trrap gene was disrupted only in the central nervous system, and it was reported that differentiation of nerve cells was arrested. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2009-242388 [Non-patent literature]

[0007] [Non-Patent Document 1] The American Journal of Human Genetics 104, 530-541, March 7, 2019 [Non-patent document 2] Clinical Genetics. 2019;1-9. [Non-patent document 3] Cell Stem Cell 14, 632-643, May 1, 2014 Summary of the Invention [Problem to be solved by the invention]

[0008] Although the establishment of disease model organisms is essential for elucidating the pathogenesis of human diseases and developing therapeutic drugs, there have been few useful disease models established for symptoms caused by TRRAP genomic mutations (e.g., facial features and mental retardation). Furthermore, since pathological analysis using a human model is not possible, it was necessary to create disease model organisms in which mutations in the trrap gene were introduced into zebrafish and to analyze the pathology of these diseases. [Means for solving the problem]

[0009] Through extensive research, the present inventors have succeeded in producing zebrafish in which a mutation has been introduced into the zebrafish trrap gene, which closely resembles the pathology of human TRRAP genetic disorders.

[0010] The object of the present invention is to A genetically modified zebrafish that expresses a mutant Trrap protein, The genetically modified zebrafish has a nucleic acid sequence encoding the mutant Trrap protein integrated into the genome of the genetically modified zebrafish; The mutant Trrap protein is a Trrap protein lacking the functions of the FAT domain, the PIKK-TRRAP domain, and the FATC domain present in the wild-type Trrap protein. The purpose is to provide

[0011] The genetically modified zebrafish of the present invention can serve as a model for human diseases and can be used to elucidate the pathogenesis of the above-mentioned human diseases and to develop therapeutic drugs by in vivo screening of small molecule compounds using the suppression of mutant phenotypes as an indicator.

[0012] Furthermore, the only known symptoms of human TRRAP genetic disorders are characteristic facial features and mental retardation (Non-Patent Document 1) and hearing abnormalities (Non-Patent Document 2). Analysis of morphogenetic abnormalities during the development of trrap gene-modified zebrafish may lead to the discovery of new pathologies and symptoms associated with human TRRAP genetic disorders.

[0013] The mutant Trrap protein is A part or all of the amino acid sequence encoding the FAT domain A part or all of the amino acid sequence encoding the PIKK-TRRAP domain and A part or all of the amino acid sequence encoding the amino acid sequence encoding the FATC domain may be deleted.

[0014] The FAT domain may have the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent thereto. The PIKK-TRRAP domain may have the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence equivalent thereto. The FATC domain may have the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence equivalent thereto.

[0015] The nucleic acid sequence encoding the mutant Trrap protein may be a nucleic acid sequence encoding the amino acid sequence from position 1 to position n (1002≦n≦2826) in the wild-type Trrap protein.

[0016] The nucleic acid sequence encoding the mutant Trrap protein may be a nucleic acid sequence encoding the amino acid sequence consisting of SEQ ID NO:4.

[0017] The genetically modified zebrafish may not express the wild-type Trrap protein.

[0018] Another object of the present invention is to utilize trrap gene-modified zebrafish as a system for evaluating the activity of the human TRRAP gene and mutant human TRRAP genes. By crossbreeding heterozygous trrap gene-modified zebrafish, fertilized eggs lacking the function of the Trrap molecule can be obtained. By injecting mRNA for a mutant TRRAP gene identified in a human TRRAP genetic disorder into these fertilized eggs, the function of the human TRRAP gene product can be evaluated using as indicators the functional recovery of microphthalmia, mandibular hypoplasia, and tooth hypoplasia observed during embryonic development in trrap zebrafish mutants (- / -).

[0019] Another object of the present invention is to provide A base sequence consisting of SEQ ID NO: 5, a base sequence having 95% or more sequence homology with the base sequence of SEQ ID NO: 5; A base sequence in which one or several bases are deleted, substituted or added to the base sequence of SEQ ID NO: 5; Or, a nucleotide sequence capable of hybridizing under stringent conditions with a sequence complementary to the nucleotide sequence of SEQ ID NO: 5; a first nucleic acid molecule for producing the genetically modified zebrafish, the first nucleic acid molecule comprising: The purpose is to provide

[0020] Another object of the present invention is to provide A base sequence consisting of SEQ ID NO: 6, a base sequence having 95% or more sequence homology with the base sequence of SEQ ID NO: 6; A base sequence in which one or several bases are deleted, substituted or added to the base sequence of SEQ ID NO: 6; Or, a nucleotide sequence capable of hybridizing under stringent conditions with a sequence complementary to the nucleotide sequence of SEQ ID NO: 6; a second nucleic acid molecule for producing the genetically modified zebrafish, the second nucleic acid molecule comprising: The purpose is to provide

[0021] The genetically modified zebrafish can be produced by using the first nucleic acid molecule and the second nucleic acid molecule.

[0022] Another object of the present invention is to provide the first nucleic acid molecule; the second nucleic acid molecule; A kit for producing the genetically modified zebrafish, comprising: The purpose is to provide

[0023] By using the kit, the genetically modified zebrafish can be produced.

[0024] Another object of the present invention is to provide A method for screening a candidate substance for preventing and / or treating a disease caused by a mutation in a Trrap protein, comprising: The above method is an administration step of administering a test sample to the genetically modified zebrafish; an evaluation step of evaluating whether the test sample is a candidate substance for preventing and / or treating the disease using a phenotype in the genetically modified zebrafish as an indicator; A screening method comprising: The purpose is to provide

[0025] By using the above screening method, it is possible to screen for candidate substances that prevent and / or treat diseases caused by mutations in the Trrap protein.

[0026] The phenotype may be at least one selected from the group consisting of microphthalmia, mandibular hypoplasia, and dental hypoplasia. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows the molecular structure of the wild-type Trrap protein (Trrap-WT), which consists of 3841 amino acids. [Figure 2] Figure 2 shows the expression of the trrap gene during zebrafish embryonic development. The expression of trrap mRNA in zebrafish embryos is visualized by the purple signal, which indicates the localization of digoxigenin (DIG)-labeled antisense trrap RNA. No purple signal is observed for sense trrap RNA in control experiments. The scale bar is 200 μm. 2-cell stage: 0.75 hours post-fertilization, shield stage: 6 hours post-fertilization, bud stage: 10 hours post-fertilization, 15-somite stage: 12 hours post-fertilization, 20-somite stage: 19 hours post-fertilization, and 24 hpf stage (15-hour embryo): 24 hours post-fertilization. [Figure 3] Figure 3 shows the molecular structures of the wild-type Trrap protein (Trrap-WT) and the mutant Trrap protein (Trrap-Mut). The wild-type Trrap protein consists of 3,841 amino acids, while the mutant Trrap protein consists of 1,002 amino acids. [Figure 4] Figure 4 shows the positions of the deleted bases (-: 5-base deletion) and the stop codon (TAG) in the base sequence encoding the mutant Trrap protein. [Figure 5]Figure 5 shows a comparison of the morphology and diameter of wild-type (+ / +) and trrap mutant (- / -) eyes and ocular tissue stained with toluidine blue. Scale bars are 200 μm (panels a and b) and 50 μm (panels d and e). Error bars indicate standard deviation. Asterisks indicate statistical significance between wild-type and mutant. ****P<0.0001. RGL: retinal ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. [Figure 6] Figure 6 shows photographs of wild-type (+ / +) and trrap mutant (- / -) cartilage formation stained with Alcian blue and a graph of the anterior-posterior distance of the ventral pharyngeal arch. The scale bar is 200 μm. Asterisks indicate statistical significance between wild-type and mutant. ****P<0.0001. eth: ethmoid plate; m: Meckel's cartilage; pq: quadrate palatine cartilage; ch: hyoid angular cartilage; h: hyomandibular bone; cb: ceratobranchial bone. [Figure 7] Figure 7 shows photographs of bone formation in wild-type (+ / +) and trrap mutants (- / -) stained with Alizarin Red. Scale bar: 200 μm. ot: otolith, vb: vertebra, n: notochord, cb5: ceratobranchial bone 5, c: pseudoclavicular bone, p: parasphenoid, br: ray bone, op: opercular bone. [Figure 8] Figure 8 shows the expression of genes in the pharyngeal arches of wild-type (+ / +), heterozygous (+ / -), and trrap mutant (- / -) embryos. The dlx2a, dlx3, and nkx2.3 genes were expressed in the pharyngeal arches in 72-hour-old embryos. The scale bar is 200 μm. [Figure 9] Figure 9 shows the expression of dental genes in wild-type (+ / +), heterozygous (+ / -), and trrap mutant (- / -) embryos. The dlx2b and pitx2 genes are expressed in the molars of 72-hour-old embryos. The scale bar is 200 μm. [Figure 10]Figure 10 shows the expression of neural genes in wild-type (+ / +) and trrap mutants (- / -). In 54-hour embryos, elavl3 expression was observed in neurons, gfap expression in radial glial cells and neural stem cells, olig2 expression in primary motor neurons and oligodendrocytes, and gli2 expression in the central nervous system and pharyngeal arches. The scale bar is 200 μm. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition For convenience, certain terms used in this application are collected here. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0029] Although the numerical ranges and parameters set forth in the present invention are approximate, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each test measurement. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error as considered by one of ordinary skill in the art.

[0030] In this specification, values ​​indicating identity and homology can be calculated using known programs such as BLAST. A nucleotide sequence having sequence homology to a nucleotide sequence set forth in a SEQ ID NO: may have a sequence homology of 95% or more, for example, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more.

[0031] An amino acid sequence having sequence homology to the amino acid sequence shown in a sequence number may have sequence homology of 95% or more, for example, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more.

[0032] As used herein, the "several bases" in the "base sequence in which one or several bases are deleted, substituted or added" refers to, for example, 2 to 10 bases, 2 to 9 bases, 2 to 8 bases, 2 to 7 bases, 2 to 6 bases, 2 to 5 bases, 2 to 4 bases, 2 to 3 bases, or 2 bases. Generally, the fewer the number of deleted, substituted or added bases, the better. Two or more types of deletion, substitution or addition of bases may occur simultaneously. Note that known techniques can be used for base deletion, substitution or addition.

[0033] As used herein, "stringent conditions" include, for example, the following (1) and (2). (1) Low ionic strength and high washing temperature conditions include, for example, 0.015 M NaCl / 0.0015 M sodium citrate (citrate) / 0.1% SDS at 50°C; or (2) During hybridization, a denaturing agent such as formamide is used. For example, 50% (vol / vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 750 mM NaCl, and 75 mM sodium citrate are used at 42°C. Another example is incubation in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 5 mM EDTA, 0.1% Tween-20, 1 mg / ml torula RNA, and 1 μg / ml heparin at 65°C. The embryos are washed twice for 30 minutes each in 50% formamide, 2x SSC, and 0.1% Tween-20. Then, they are washed twice for 15 minutes each in 2x SSC and 0.1% Tween-20. Finally, the mixture is washed twice for 30 minutes with 0.2×SSC, 0.1% Tween 20. It is clear that those skilled in the art can appropriately adjust the stringency conditions to obtain a clear and detectable hybridization signal.

[0034] Hereinafter, embodiments of the present invention will be described. The following embodiments are merely examples, and the scope of the present invention is not limited to the following embodiments. Note that similar content will not be described again to avoid repetition.

[0035] Genetically modified zebrafish The genetically modified zebrafish according to this embodiment expresses a mutant Trrap protein. The mutant Trrap protein lacks the functions of the FAT (FRAP, ATM, TRRAP) domain, the PIKK-TRRAP domain (TRRAP pseudokinase domain), and the FATC (FRAP, ATM, TRRAP C-terminus) domain present in the wild-type Trrap protein encoded by the trrap gene. The genetically modified zebrafish has a nucleic acid sequence encoding the mutant Trrap protein integrated into its genome.

[0036] Figure 1 shows the molecular structure of the wild-type Trrap protein (Trrap-WT). Trrap-WT consists of 3,841 amino acids. Trrap-WT contains the FAT domain, the PIKK-TRRAP domain, and the FATC domain. Mutant Trrap proteins lack the functions of these domains. The function of a domain in a protein can be eliminated by deleting, substituting, or adding amino acids that make up the domain, or by a combination of these.

[0037] The mutant Trrap protein may (1) lack part or all of the amino acid sequence encoding the FAT domain. The mutant Trrap protein may (2) lack part or all of the amino acid sequence encoding the PIKK-TRRAP domain. The mutant Trrap protein may (3) lack part or all of the amino acid sequence encoding the FATC domain.

[0038] The FAT domain may have the amino acid sequence shown in SEQ ID NO: 1 or an equivalent amino acid sequence thereof. The PIKK-TRRAP domain may have the amino acid sequence shown in SEQ ID NO: 2 or an equivalent amino acid sequence thereof. The FATC domain may have the amino acid sequence shown in SEQ ID NO: 3 or an equivalent amino acid sequence thereof. "Equivalent amino acid sequence" means an amino acid sequence that has the function of a domain and has sequence homology with the amino acid sequence shown in a SEQ ID NO.

[0039] The nucleic acid sequence encoding the mutant Trrap protein may be a nucleic acid sequence encoding the amino acid sequence from position 1 to position n (1002≦n≦2826) of the wild-type Trrap protein.

[0040] The nucleic acid sequence encoding the mutant Trrap protein may be a nucleic acid sequence encoding the amino acid sequence consisting of SEQ ID NO: 4 or an equivalent amino acid sequence thereof.

[0041] The genotype of the gene encoding the Trrap protein in genetically modified zebrafish may be, for example, a homozygous type (homozygous type) of the gene encoding the mutant Trrap protein, or a heterozygous type (heterozygous type) of the gene encoding the mutant Trrap protein and the gene encoding the wild-type Trrap protein.

[0042] In one embodiment, the genetically modified zebrafish is a homozygous zebrafish (i.e., homozygous for the gene encoding the mutant Trrap protein). Homozygous zebrafish can be used to elucidate the pathology of human diseases and to develop therapeutic agents by in vivo screening of small molecule compounds that suppress mutant phenotypes. Herein, homozygous zebrafish are also referred to as mutant trrap zebrafish.

[0043] In another embodiment, the genetically modified zebrafish is a heterozygous zebrafish (i.e., heterozygous for a gene encoding a mutant Trrap protein and a gene encoding a wild-type Trrap protein). Heterozygous zebrafish can be crossed to produce homozygous zebrafish.

[0044] Heterozygous zebrafish always have a wild-type allele (chromosome), so not only the mutant Trrap protein but also the normal Trrap protein is produced from the normal chromosome, resulting in heterozygous zebrafish with the same normal phenotype as wild-type zebrafish.

[0045] In yet another embodiment of this invention, the genetically modified zebrafish are homozygous zebrafish and heterozygous zebrafish.

[0046] In one embodiment, the genetically modified zebrafish does not express the wild-type Trrap protein.

[0047] Nucleic acid molecules for generating genetically modified zebrafish The first nucleic acid molecule for producing a genetically modified zebrafish according to this embodiment has a base sequence consisting of SEQ ID NO: 5, a base sequence having 95% or more sequence identity with the base sequence consisting of SEQ ID NO: 5, a base sequence in which one or more bases have been deleted, substituted, or added to the base sequence consisting of SEQ ID NO: 5, or a base sequence that can hybridize under stringent conditions with a sequence complementary to the base sequence consisting of SEQ ID NO: 5.

[0048] The second nucleic acid molecule for producing a genetically modified zebrafish according to this embodiment has a base sequence consisting of SEQ ID NO: 6, a base sequence having 95% or more sequence identity with the base sequence consisting of SEQ ID NO: 6, a base sequence in which one or more bases have been deleted, substituted, or added to the base sequence consisting of SEQ ID NO: 6, or a base sequence that can hybridize under stringent conditions with a sequence complementary to the base sequence consisting of SEQ ID NO: 6.

[0049] Kit for generating genetically modified zebrafish The kit for producing genetically modified zebrafish according to this embodiment includes a first nucleic acid molecule and a second nucleic acid molecule.

[0050] Method for producing genetically modified zebrafish The method for producing homozygous zebrafish includes a mating step of crossing heterozygous zebrafish carrying a nucleic acid sequence encoding a mutant Trrap protein with each other, and may further include a recovery step of recovering fertilized eggs of the homozygous zebrafish.

[0051] The method for producing heterozygous zebrafish includes a genetic engineering step of modifying a nucleic acid sequence encoding a wild-type Trrap protein in a one-cell stage zebrafish embryo to a nucleic acid sequence encoding a mutant Trrap protein. Genetic engineering techniques include genome editing techniques such as ZFN, TALEN, and the CRISPR / Cas9 system. The CRISPR / Cas9 system uses CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and recombinant Cas9 protein. In this embodiment, the CRISPR RNA (crRNA) may be the first nucleic acid molecule and the second nucleic acid molecule.

[0052] The method for producing heterozygous zebrafish may further include the step of obtaining heterozygous zebrafish embryos having a nucleic acid sequence encoding a mutant Trrap protein.

[0053] Screening method for candidate substances for preventing and / or treating diseases caused by mutations in Trrap protein The screening method according to this embodiment is a method for screening for candidate substances for preventing and / or treating diseases caused by mutations in the Trrap protein. This screening method includes an administration step of administering a test sample to genetically modified zebrafish, and an evaluation step of evaluating whether the test sample is a candidate substance for preventing and / or treating the disease using a phenotype in the genetically modified zebrafish as an indicator.

[0054] The test sample is preferably a water-soluble substance that can be dissolved in the water in which zebrafish are kept, and more preferably a water-soluble low-molecular-weight organic compound.

[0055] The test sample can be administered, for example, by adding it to rearing water, by mixing it with feed, etc. One type of test sample may be used, or two or more types of test samples may be used in combination.

[0056] There are no particular restrictions on the administration period of the test sample and it can be selected appropriately depending on the purpose. However, as shown in the test example described below, it is preferable to administer the test sample during the rearing period from 2 to 10 days of age (between 8 days after hatching), when the phenotype caused by the mutant Trrap protein can be observed.

[0057] The phenotype may be at least one selected from the group consisting of microphthalmia, mandibular hypoplasia, and dental hypoplasia.

[0058] The disease may be a disease associated with at least one selected from the group consisting of microphthalmia, mandibular hypoplasia, and dental hypoplasia.

[0059] The amount of test sample to be administered is not particularly limited and can be appropriately selected depending on the purpose.

[0060] A test sample may be determined to be a candidate substance if administration of the test sample suppresses the phenotype caused by the mutant Trrap protein (in other words, approaches or becomes the same as the phenotype caused by the wild-type Trrap protein). [Example]

[0061] Whole-mount In Situ Hybridization (WISH) The expression of the trrap gene was examined by whole-mount in situ hybridization (WISH). Embryos were incubated with a digoxigenin (DIG)-labeled antisense RNA probe (SEQ ID NO: 12) to detect target gene mRNA. After washing away unbound probe, the embryos were incubated with an alkaline phosphatase-conjugated anti-DIG antibody. Expression of the antisense trrap DIG probe binding to the target gene mRNA was determined by incubation with BM Purple (Roche). After washing three times with phosphate-buffered saline (PBST) containing 0.1% Tween 20 and 0.2% BSA, the embryos were incubated with 4% paraformaldehyde to stop the enzyme reaction.

[0062] The results are shown in Figure 2. Panel a shows a photograph of the 2-cell stage, panel b shows a photograph of the shield stage, panel c shows a photograph of the bud stage, panel d shows a photograph of the 15-somite stage, and panel e shows a photograph of the 20-somite stage. Panels f and g show photographs of embryos 24 hours after fertilization. All photographs are lateral views. In panel b, the dorsal side is the right, and in panels c to g, the anterior side is the left. An antisense trrap DIG probe was used in panels a to f. A sense trrap DIG probe was used in panel g. 2-cell stage: 0.75 hours after fertilization, shield stage: 6 hours after fertilization, bud stage: 10 hours after fertilization, 15-somite stage: 12 hours after fertilization, 20-somite stage: 19 hours after fertilization, and 24 hpf stage (24-hour embryo): 24 hours after fertilization.

[0063] The trrap gene is maternally supplied at the 2-cell stage and expressed throughout the embryo from the shield stage to the 20-somite stage (Fig. 1, panels a to e). Strong trrap expression was detected in the head at 24 hours post-fertilization (hpf), but no such signal was detected in embryos incubated with a sense trrap DIG RNA probe (Fig. 1, panels f and g). This indicates that trrap expression is strongly induced in the head during head formation.

[0064] Genome editing of the trrap locus To investigate the function of the trrap gene, we disrupted the zebrafish trrap gene using CRISPR / Cas9 genome editing technology and established trrap gene-modified zebrafish.

[0065] To disrupt the trrap gene in zebrafish, we used a ready-to-use CRISPR / Cas9 system containing CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), and recombinant Cas9 protein. crRNA (trrap-crRNA1; SEQ ID NO: 5, trrap-crRNA2; SEQ ID NO: 6), tracrRNA (SEQ ID NO: 7), and recombinant Cas9 protein were obtained from Integrated Device Technology, Inc. (IDT).

[0066] [Table 1]

[0067] [Table 2]

[0068] Trrap-crRNA1 (25 pg), trrap-crRNA2 (25 pg), and tracrRNA (100 pg) were injected into one-cell stage zebrafish fertilized eggs together with recombinant Cas9 protein (1 ng).

[0069] Zebrafish maintenance and ethics statement Heterozygous trrap mutant zebrafish (adult) were reared in a controlled aquatic facility where artificial seawater was added to purified water using a reverse osmosis system to maintain a constant salinity under the following conditions: 14 / 10 h light / dark photoperiod, 28.5°C (±1°C), pH 7.0 (±1), and conductivity 450 mS / cm.

[0070] Homozygous trrap mutant zebrafish embryos were obtained by mating heterozygous trrap mutant zebrafish (adult fish). The collected embryos were washed with E3 water and selected for fertilization under a light microscope (Olympus SZ61).

[0071] All animal experiments were conducted in accordance with the institutional and national guidelines and regulations. This study was approved by the Institutional Animal Care and Use Committee of Yamanashi University (approval identification number: A30 - 25).

[0072] Genotyping of trrap mutant zebrafish Zebrafish embryos were transferred to a 1.5 ml tube, 108 μl of 50 mM NaOH was added, and they were cultured at 98 °C for 10 minutes. Then, 12 μl of 1 M Tris - HCl (pH 8.0) was added to the above solution to obtain a genomic extract. The target genomic fragment was amplified by PCR using the genomic extract (1 μl), locus - specific primers shown in Table 3, dNTP (deoxynucleotide) mixture, and PrimeTaq (Primetech).

[0073]

Table 3

[0074] The conditions for PCR were as follows: 98 °C × 10 seconds, 55 °C × 30 seconds, and 72 °C × 30 seconds for 40 cycles. For heteroduplex mobility analysis (HMA), the obtained PCR amplicon was electrophoresed on a 12.5% polyacrylamide gel (not shown). By this, the introduction of mutations was confirmed.

[0075] Heteroduplex mobility analysis was performed according to the following procedure. The samples subjected to the following PCR reaction were electrophoresed on a 12.5% polyacrylamide gel (12.5% acrylamide / bis solution, 1×tris glycine buffer, 10% APS, 1% TEMED) (10 mA, 100 minutes per gel). <PCR reaction solution> · 10×buffer 1 μL · 10 mM dNTP mixture 0.8 μL · trrap - HMA - F(10 μM) 0.2 μL · trrap-HMA-R (10 μM) 0.2 μL · Genomic DNA 1 μL · Prime Taq 0.05 μL · DDW 6.75 μL Total 10 μL <PCR reaction conditions> After 94°C for 2 minutes, the following cycle was repeated 40 times. 98°C for 10 seconds → 55°C for 30 seconds → 72°C for 30 seconds

[0076] Also, after extracting genomic DNA, the target site was amplified under the above PCR reaction conditions. The obtained PCR fragment was subcloned into the pGEM-T Easy vector (Promega), and the genomic sequence was determined by sequence analysis.

[0077] Figure 3 shows the molecular structures of wild-type Trrap protein (Trrap-WT) and mutant Trrap protein (Trrap-Mut). The mutant Trrap protein consists of the amino acid sequence up to the 1002nd amino acid of the wild-type Trrap protein (Trrap-WT).

[0078] Figure 4 shows the position of the stop codon (TAG) in the nucleotide sequence encoding the mutant Trrap protein. The nucleotide sequence indicated by "1002" shows the nucleotide sequence (codon) encoding the 1002nd amino acid of the wild-type Trrap protein (Trrap-WT).

[0079] The mutant Trrap protein lacks the functions of the FAT (FRAP, ATM, TRRAP), PIKK-TRRAP (the pseudokinase domain of TRRAP), and FATC (FRAP, ATM, TRRAP C-terminal) domains, and thus was found to be functionally defective.

[0080] Morphological abnormalities in the head of mutant trrap zebrafish We examined morphological abnormalities in the head of mutant trrap zebrafish (Figure 5). Asterisks indicate statistical significance between wild-type and mutant (homozygous) zebrafish. At 3 days post-fertilization (dpf), the eye diameter of homozygous zebrafish (trrap- / -: n = 8) was slightly reduced compared to wild-type zebrafish (trrap+ / +: n = 2) and heterozygous zebrafish (trrap+ / -: n = 6) (Figure 5, panels a to c).

[0081] Histological analysis of the mutant trrap zebrafish using toluidine blue staining (Fig. 5) confirmed that the retina, consisting of three nuclear layers (retinal ganglion cell layer, inner nuclear layer, and outer nuclear layer) and two plexiform layers (inner plexiform layer and outer plexiform layer), was normally formed in wild-type zebrafish (trrap+ / +: n = 2, Panel d of Fig. 5), heterozygous zebrafish (trrap+ / -: n = 6, not shown), and homozygous zebrafish (trrap- / -: n = 8, Panel e of Fig. 5).

[0082] Alcian blue staining The morphology of the pharyngeal arches was examined by staining sulfated and carboxylated acidic mucopolysaccharides with Alcian blue.

[0083] Fixed 5-day-old (5 dpf) embryos were cultured overnight in 4% paraformaldehyde. After washing three times with PBST, the embryos were incubated in bleaching buffer (1% H2O2 and 1% KOH) for 30 minutes. The embryos were then cultured overnight at room temperature in a mixture of 30% acetic acid and 70% ethanol containing Alcian blue (0.1%) (acid alcohol buffer). After washing three times with the acid alcohol buffer, the embryos were incubated with trypsin (0.5%).

[0084] The results are shown in Figure 6. At 5 dpf (5 days old), homozygous trrap zebrafish (trrap- / -: n = 8) exhibited disrupted ventral pharyngeal arch morphology compared with wild-type zebrafish (trrap+ / +: n = 6) (Figure 6, panels a to e). In homozygous zebrafish, the anterior-posterior distance of the ventral pharyngeal arch, including the Meckel's cartilage and hyoid angular cartilage (double arrows), was shorter than in wild-type and heterozygous zebrafish (trrap+ / -: n = 2, not shown). However, the morphology of the ethmoid plate and ceratobranchial bones was normal in homozygous zebrafish.

[0085] Alizarin red staining We examined bone formation in the head of mutant trrap zebrafish using alizarin red, which stains calcium deposited in tissues.

[0086] Alizarin red staining was performed according to the same procedure as described above. 10 dpf (10-day-old) embryos were incubated in PBS containing 4% paraformaldehyde and washed twice with PBST. 10-day-old embryos were incubated in bleaching buffer for 30 minutes and washed twice with PBST. Embryos were incubated in 1 mg / ml alizarin red in 0.5% KOH at room temperature for 1 hour and then washed with 0.5% KOH.

[0087] The results are shown in Figure 7. Compared with wild-type zebrafish (trrap- / -: n = 6, panels f and g), wild-type zebrafish (trrap+ / +: n = 3, panels h and i), and heterozygous zebrafish (trrap+ / -: n = 5, not shown), defective ossification was observed in the head but not in the pseudoclavicle. Hypoplasia of the fifth ceratobranchial tooth was also observed.

[0088] These experiments demonstrated that wild-type and heterozygous zebrafish had mineralized ceratobranchial teeth, whereas homozygous zebrafish had hypomorphic teeth, demonstrating that trrap is required for the development of craniofacial structures, including the eyes, ventral pharyngeal arches, and teeth.

[0089] Expression of pharyngeal arch and tooth marker genes in the trrap mutant zebrafish Because homozygous zebrafish showed craniofacial abnormalities, including hypoplasia of the pharyngeal arches and teeth, we examined the expression of pharyngeal arch marker genes (dlx2a, dlx3, nkx2.3) and tooth marker genes (dlx2b, pitx2) by WISH.

[0090] The results are shown in Figure 8. In the ventral pharyngeal arch of homozygous zebrafish, the expression patterns of dlx2a (triangle), dlx3 (triangle), and nkx2.3 (double arrow) were narrower and reduced compared to those of wild-type and heterozygous zebrafish.

[0091] Expression of dlx2b (arrow) and pitx2 (arrow) was reduced in the developing teeth of homozygous zebrafish (Figure 9). On the other hand, expression levels of neural genes in the central nervous system (CNS), elavl3 / huC (neurons), gfap (radial glia and neural stem cells), olig2 (primary motor neurons and oligodendrocytes), and gli2a (CNS and pharyngeal arches) were comparable between wild-type and heterozygous zebrafish (Figure 10). These results revealed that expression of pharyngeal arch and tooth marker genes was significantly reduced in homozygous zebrafish.

Claims

1. A genetically modified zebrafish that expresses a mutant Trrap protein, the genetically modified zebrafish has a nucleic acid sequence encoding the mutant Trrap protein integrated into the genome of the genetically modified zebrafish; The mutant Trrap protein is a Trrap protein lacking the functions of the FAT domain, the PIKK-TRRAP domain, and the FATC domain present in the wild-type Trrap protein; The nucleic acid sequence encoding the mutant Trrap protein is a nucleic acid sequence encoding the amino acid sequence from 1 to n (1002≦n≦2826) in the wild-type Trrap protein, A genetically modified zebrafish that does not express the wild-type Trrap protein.

2. The genetically modified zebrafish of claim 1, wherein the nucleic acid sequence encoding the mutant Trrap protein is a nucleic acid sequence encoding the amino acid sequence consisting of SEQ ID NO:

4.

3. A base sequence consisting of SEQ ID NO: 5, a base sequence having 95% or more sequence homology with the base sequence of SEQ ID NO: 5; A base sequence in which one or several bases are deleted, substituted or added to the base sequence of SEQ ID NO: 5; Or, a base sequence capable of hybridizing under stringent conditions with a sequence complementary to the base sequence of SEQ ID NO: 5; A first nucleic acid molecule for producing a genetically modified zebrafish according to claim 1 or 2, comprising:

4. A base sequence consisting of SEQ ID NO: 6, A base sequence having 95% or more sequence homology with the base sequence of SEQ ID NO: 6, A base sequence in which one or several bases are deleted, substituted or added to the base sequence of SEQ ID NO: 6; Or, a base sequence capable of hybridizing under stringent conditions with a sequence complementary to the base sequence of SEQ ID NO: 6; A second nucleic acid molecule for producing a genetically modified zebrafish according to claim 1 or 2, comprising:

5. A first nucleic acid molecule according to claim 3; A second nucleic acid molecule according to claim 4; 3. A kit for producing the genetically modified zebrafish of claim 1 or 2, comprising:

6. A method for screening a candidate substance for preventing and / or treating a disease caused by a mutation in a Trrap protein, comprising: The method comprises: an administration step of administering a test sample to the genetically modified zebrafish according to claim 1 or 2; an evaluation step of evaluating whether the test sample is a candidate substance for preventing and / or treating the disease using a phenotype in the genetically modified zebrafish as an indicator; A screening method comprising:

7. The screening method according to claim 6, wherein the phenotype is at least one selected from the group consisting of microphthalmia, mandibular hypoplasia, and dental hypoplasia.

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