JP7699355B2 - TRα gene modified eel - Google Patents

Description

The present invention relates to eels with modified thyroid hormone receptor α genes and methods for producing the same.

The larvae (leptocephali) of fish of the order Anguilliformes have a longer larval period than other fish, and are characterized by growing to large larvae before metamorphosing into fry. The current standard method for rearing Japanese eels requires a longer larval period (160-450 days after hatching) than in the natural environment (110-170 days after hatching). In addition, the mortality rate is higher during the larval stage than after the fry stage, and even when a small number of Japanese eel larvae are carefully reared, the survival rate of the larvae (glass eels) is often less than 10%. For this reason, rearing eel larvae requires a great deal of labor and time, and the low survival rate (survival rate) of the larvae is an obstacle to efficient mass production, and there is a need to shorten the larval period or improve the survival rate by improving rearing methods.

On the other hand, it has been reported that the length of the larval period of captive Japanese eels is a hereditary trait controlled by multiple genes (Non-Patent Document 1).
It has also been reported that in amphibians, larval growth is promoted and metamorphosis into adults begins earlier in individuals in which the function of the thyroid hormone receptor α (TRα) gene has been partially inhibited using genome editing technology (Non-Patent Document 2).
Furthermore, in the Japanese eel, high expression of the TRα gene is observed throughout the entire larval stage, suggesting that it is involved in larval growth and metamorphosis (Non-Patent Document 3), but the details have not been clarified.

Nomura, K. et al. Genetic parameters and quantitative trait loci analysis associated with body size and timing at metamorphosis into glass eels in captive-bred Japanese eels (Anguilla japonica). PLoS One 13, e0201784, 2018. Wen, L. et al. Regulation of growth rate and developmental timing by Xenopus thyroid hormone receptor α. Dev. Growth Differ. 58, 106-115, 2016. Kawakami, Y. et al. Characterization of thyroid hormone receptors during early development of the Japanese eel (Anguilla japonica). Gen. Comp. Endocrinol. 194, 300-310, 2013.

Until now, there has been no technology to shorten the larval period of eels through genetic improvement. The present invention is concerned with providing eels with a shortened larval period.

As a result of intensive research to solve the above problems, the inventors discovered that by introducing mutations into the eel thyroid hormone receptor αA gene and thyroid hormone receptor αB gene and inhibiting their functions, the eel larval period can be shortened and, quite unexpectedly, the survival rate of the larval period can be improved, thus completing the present invention.

That is, the present invention provides the following [1] to [9].
[1] An eel having reduced or lost function of the thyroid hormone receptor αA (TRαA) gene and the thyroid hormone receptor αB (TRαB) gene.
[2] The eel according to [1], which has loss-of-function mutations in the TRαA gene and the TRαB gene.
[3] The eel according to [1] or [2], wherein the TRαA gene is any one selected from the following (a) to (c), and the TRαB gene is any one selected from the following (d) to (f):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO:1;
(b) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein that functions as TRα;
(c) a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides have been deleted, inserted, substituted or added relative to the nucleotide sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα;
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO:3;
(e) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein that functions as TRα;
(f) A polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:3, and encoding a protein that functions as TRα.
[4] The eel according to any one of [1] to [3], which lacks a part or all of the TRαA gene and a part or all of the TRαB gene.
[5] A method for producing eels, comprising the step of introducing loss-of-function mutations into the TRαA gene and the TRαB gene.
[6] A method for improving the survival rate of eels during the larval stage, comprising the step of introducing a loss-of-function mutation into the TRαA gene and the TRαB gene.
[7] The method according to [5] or [6], wherein the TRαA gene is any one selected from the following (a) to (c), and the TRαB gene is any one selected from the following (d) to (f):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO:1;
(b) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein that functions as TRα;
(c) a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides have been deleted, inserted, substituted or added relative to the nucleotide sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα;
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO:3;
(e) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein that functions as TRα;
(f) A polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:3, and encoding a protein that functions as TRα.
[8] The method according to any one of [5] to [7], wherein the introduction of a loss-of-function mutation results in a deletion of a part or the whole of the TRαA gene and a part or the whole of the TRαB gene.
[9] The method according to any one of [5] to [8], wherein the introduction of a loss-of-function mutation is carried out by genome editing.

The present invention provides eels with a shortened larval period and improved survival rate during the larval period, a method for producing the eels, and a method for improving the survival rate of eels. Such eels have genetic characteristics suitable for efficient seed production. Furthermore, the eels of the present invention can achieve the effects of shortening the larval period and improving the survival rate during the larval period from F0 generation individuals into which a mutation has been introduced, and it is possible to instill these genetic characteristics in large-scale farmed populations through the raising and breeding of these F0 individuals.

FIG. 1 shows the relationship between the age at the onset of metamorphosis and the rate of onset of metamorphosis in the control group and the groups with TRαA gene and TRαB gene mutations introduced. FIG. 1 shows the transition of survival rates in a control group and groups with TRαA gene and TRαB gene mutations introduced. A diagram showing the relationship between the TRαA gene editing rate (%) per individual and the number of days until the onset of metamorphosis (A), and the relationship between the TRαB gene editing rate (%) per individual and the number of days until the onset of metamorphosis (B).

In this specification, the abbreviations for base sequences (nucleotide sequences), nucleic acids, etc. are written using symbols commonly used in the relevant field, such as the IUPAC-IUB standard (IUPAC-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138:9-37, 1984) and the "Guidelines for the preparation of specifications, etc., including base sequences or amino acid sequences" (edited by the Japan Patent Office). In this specification, "deoxyribonucleic acid (DNA)" includes not only double-stranded DNA, but also the single-stranded DNAs that constitute it, that is, the sense strand and the antisense strand.

In this specification, "nucleotide," "oligonucleotide," and "polynucleotide" are synonymous with nucleic acid and include both DNA and RNA. The DNA includes cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, rRNA, and synthetic RNA. Furthermore, "nucleotide," "oligonucleotide," and "polynucleotide" may be double-stranded or single-stranded, and when a "nucleotide" (or "oligonucleotide," "polynucleotide") having a certain sequence is mentioned, it is understood to comprehensively mean a "nucleotide" (or "oligonucleotide," "polynucleotide") having a complementary sequence, unless otherwise specified.

In this specification, "gene" includes double-stranded DNA including genomic DNA, single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments of these, and refers to DNA in which some biological information is contained in the sequence information of the bases that make up the DNA. Unless otherwise specified, "gene" refers to control regions, coding regions, exons, and introns without distinction.

In this specification, the identity of a base sequence or an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis program of the genetic information processing software GENETYX with the unit size to compare (ktup) set to 2.

In this specification, "a base sequence in which one or several bases have been deleted, inserted, substituted or added" refers to a base sequence in which preferably 1 to 30 bases, more preferably 1 to 15 bases, and even more preferably 1 to 9 bases have been deleted, inserted, substituted or added. In addition, in this specification, "an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added" refers to an amino acid sequence in which preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, and even more preferably 1 to 3 amino acids have been deleted, inserted, substituted or added. In this specification, "addition" of a base or amino acid includes addition of a base or amino acid to one end and both ends of the sequence.

In this specification, "eels" refers to fishes belonging to the genus Anguilla in the family Anguillidae. Examples of eels include the Japanese eel (Anguilla japonica), giant eel (Anguilla marmorata), European eel (Anguilla anguilla), and American eel (Anguilla rostrata), of which the Japanese eel is preferred. Eels may be farmed or wild fish, and there are no limitations on their growth stage.

The eels of the present invention have reduced or lost function of the thyroid hormone receptor α (TRα) A gene and the TRαB gene.
The "TRα gene" is a gene encoding thyroid hormone receptor α, which is a type of nuclear receptor and regulates the expression of thyroid hormone target genes as a transcriptional regulator. There are two known isoforms of the TRα gene in eels: the TRαA gene and the TRαB gene. For example, the TRαA gene of the Japanese eel is registered in GenBank ([www.ncbi.nlm.nih.gov/genbank/]) as AB678206.1, and consists of the base sequence shown in SEQ ID NO: 1 and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The TRαB gene of the Japanese eel is registered in GenBank as AB678207.1, and consists of the base sequence shown in SEQ ID NO: 3 and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 4.

Specific examples of the TRαA gene in the present invention include the following:
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO:1;
(b) a polynucleotide consisting of a nucleotide sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the nucleotide sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα;
(c) a polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα.
Of these, the TRαA gene is preferably a polynucleotide consisting of the base sequence shown in SEQ ID NO:1.
Specific examples of the TRαB gene in the present invention include the following:
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO:3;
(e) a polynucleotide consisting of a nucleotide sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the nucleotide sequence shown in SEQ ID NO: 3, and encoding a protein that functions as TRα;
(f) A polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:3, and encoding a protein that functions as TRα.
Of these, the TRαB gene is preferably a polynucleotide consisting of the base sequence shown in SEQ ID NO:3.

Specific examples of proteins encoded by the TRαA gene in the present invention include the following:
(g) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(h) a protein consisting of an amino acid sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the amino acid sequence shown in SEQ ID NO: 2, and functioning as TRα;
(i) A protein which has an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added relative to the amino acid sequence shown in SEQ ID NO:2 and which functions as TRα.
Among these, the protein encoded by the TRαA gene is preferably TRα consisting of the amino acid sequence shown in SEQ ID NO:2.
Specific examples of proteins encoded by the TRαB gene in the present invention include the following:
(j) a protein consisting of the amino acid sequence represented by SEQ ID NO:4;
(k) a protein having an amino acid sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the amino acid sequence shown in SEQ ID NO: 4, and functioning as TRα;
(l) A protein having an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted or added relative to the amino acid sequence shown in SEQ ID NO:4, and which functions as TRα.
Among these, the protein encoded by the TRαB gene is preferably TRα having the amino acid sequence shown in SEQ ID NO:4.

The functions of the TRαA gene and the TRαB gene refer to functions that thyroid hormone receptors inherently have, such as ligand binding ability and regulation of expression of target genes of thyroid hormone.
"The functions of the TRαA gene and the TRαB gene are reduced" means that the functions of the TRαA gene and the TRαB gene are reduced in the eels of the present invention compared to wild-type eels. Here, "reduced functions" refers to the functions in the eels of the present invention being reduced to, for example, 50% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less of the functions in the wild-type eels. In one example, "the functions of the TRαA gene and the TRαB gene are reduced" means that the expression levels of the TRαA gene and the TRαB gene in the eels of the present invention are reduced to, for example, 50% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less, respectively, compared to wild-type eels. Here, "gene expression" may refer to either the expression of mRNA from the gene or the expression of a protein, but is preferably the expression of a protein. The functions and expression levels of the TRαA gene and the TRαB gene can be measured by known methods. For example, the expression levels of the genes can be measured by conventional techniques such as Northern blotting and quantitative PCR. The expression level of the protein can be measured by ordinary techniques such as Western blotting, colorimetry, ELISA, etc. Wild-type eels are eels having normal TRαA genes and normal TRαB genes, and preferably eels having the same genetic constitution as the eels of the present invention other than the TRαA genes and TRαB genes.
Furthermore, "having lost the functions of the TRαA gene and the TRαB gene" means that in the eels of the present invention, the function of the TRαA gene is below the detection limit and the function of the TRαB gene is below the detection limit. In one example, "having lost the functions of the TRαA gene and the TRαB gene" means, for example, that the expression levels of the TRαA gene and the TRαB gene are each below the detection limit in the eels of the present invention. The functions and expression levels of the TRαA gene and the TRαB gene can be measured by known methods as described above.

Eels in which the functions of the TRαA gene and the TRαB gene are reduced or lost can be obtained, for example, by introducing a loss-of-function mutation (hereinafter, sometimes simply referred to as a mutation) into the TRαA gene and the TRαB gene of the eel. A "loss-of-function mutation" means a mutation in which the function of the gene into which the mutation is introduced is reduced or lost. In other words, the eels of the present invention preferably have a loss-of-function mutation in each of the TRαA gene and the TRαB gene. The eels into which the mutation is introduced may be wild-type eels, eels artificially bred from the wild-type eels, or mutant eels or mutants in which the base sequence in the genome has been deleted, inserted, substituted, or added. Among these, wild-type eels are preferred. The type of mutation to be introduced is not particularly limited, and may be, for example, at least one type of mutation selected from deletion, insertion, substitution, and addition to the base sequence of the gene. The number of bases deleted, inserted, substituted or added may be at least one base, and is preferably the number of bases that causes a frameshift. The position at which the mutation is introduced is not particularly limited, and may be, for example, any of the coding region, non-coding region, promoter, enhancer, and other expression control regions of the gene. Of these, it is preferable to introduce the mutation into the coding region of the gene, and more preferably to introduce the mutation further upstream (translation initiation side) in the coding region. In a preferred example, in the eel of the present invention, a part or all of the TRαA gene is deleted and a part or all of the TRαB gene is deleted. In a more preferred example, in the eel of the present invention, a part of the TRαA gene is deleted and a part of the TRαB gene is deleted. The eel of the present invention may have a mutation in at least one allele, and preferably has the mutation in both alleles. In addition, in the eel of the present invention, it is sufficient that a mutation is introduced into at least some of the cells constituting the individual as long as the effect of the present invention is exhibited, and it is preferable that a mutation is introduced into all of the cells constituting the individual. On the other hand, it is preferable that the eels of the present invention do not have any foreign genes integrated into their genome.

As a means for introducing mutations, known methods can be used. Specific methods include, for example, mutation induction using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, and nitrous acid, or physical mutagens such as ultraviolet light, X-rays, gamma rays, and ion beams, and site-specific mutagenesis. Examples of methods for site-specific mutagenesis include homologous recombination, splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), ODA (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), Kunkel (Kunkel, T.A., Proc. Natl. Acad. Sci. USA, 1985, 82, 488), and genome editing using artificial DNA cleavage enzymes (artificial DNA nucleases or programmable nucleases). Alternatively, a commercially available site-specific mutagenesis kit can be used.

Genome editing is a technology that specifically cuts double-stranded DNA at target loci in the genome and modifies the genome in a site-specific manner by inducing deletion, insertion or substitution of nucleotides during the repair process of the cut DNA, or by inserting an exogenous polynucleotide. Such techniques are known as TALEN (transcription activator-like effector nuclease), ZFN (zinc-finger nuclease), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9, Homing endonuclease, compact designer TALEN, etc. (Nature Reviews Genetics, 2014, 15:321-334; Nucleic Acids Research, 2011, 39:e82; Nucleic Acids Research, 2011, 39:e82). Acids Research, 2006, 34: e149, Nature communications, 2013, 4: 1762). Kits for genome editing based on these techniques are commercially available and can be purchased, for example, from Life technologies, Takara Bio Inc.

From the viewpoint of efficiency of mutation introduction, introduction of mutations into the eel TRαA gene and TRαB gene is preferably carried out by genome editing, and more preferably by using CRISPR/Cas9.
Mutations introduced into the TRαA gene and TRαB gene of eels by CRISPR/Cas9 can be carried out, for example, as follows. That is, single-stranded guide RNA (sgRNA) for target genes (TRαA gene and TRαB gene) and CRISPR-associated protein 9 (Cas9) are introduced into fertilized eggs of eels. The introduced fertilized eggs are cultured and hatched, and then raised according to the usual method of raising eels. Whether or not the desired mutation has been introduced can be determined by taking a sample from the obtained eel individual and analyzing it by a conventional method, confirming the base sequence of the target gene, or measuring the expression level of the target gene or the protein encoded by the target gene.

The sgRNA for the target gene used in CRISPR/Cas9 includes a CRISPR RNA (crRNA) specific to the target gene sequence and a transactivating crRNA (tracrRNA), and the crRNA consists of about 20 bases upstream of the proto-spacer adjacent motif (PAM) in the target gene. The sgRNA can be designed using a known design tool such as CRISPRdirect, and the designed sgRNA can be obtained by synthesis. The sgRNA and Cas9 can be introduced into a target cell such as a fertilized egg in the form of an RNP complex consisting of the Cas9 protein and the sgRNA, an expression plasmid containing the sgRNA sequence and the Cas9 gene, a viral vector containing the sgRNA sequence and the Cas9 gene, or sgRNA and Cas9 mRNA. Of these, from the viewpoint of reducing off-target effects, it is preferable to introduce sgRNA and Cas9 into target cells as an RNP complex. The introduction method may be appropriately selected depending on the form of introduction, and examples include electroporation, transfection, and microinjection. After introduction, the RNP complex binds to the target sequence on the genome to break the double strand of DNA three bases upstream of PAM (double-strand break; DSB). After introduction of the DSB, it is immediately repaired, but at that time, insertion or deletion (insertion-deletion; Indel) of several bases to several tens of bases occurs by the non-homologous end-joining (NHEJ) pathway (Indel mutation). Since a frameshift mutation can be introduced into the target gene by this Indel, the protein encoded by the target gene cannot be expressed normally and loses function, so that the target gene can be efficiently knocked out.

The sgRNA used to introduce mutations into the TRαA gene and TRαB gene of eels may be designed appropriately according to the base sequence of the gene. The sgRNA may be designed for each of the TRαA gene and the TRαB gene, or an sgRNA common to the TRαA gene and the TRαB gene may be designed with a common sequence of the TRαA gene and the TRαB gene as a target sequence. Specific examples of the sgRNA include, but are not limited to, an sgRNA whose target sequence is the base sequence of the antisense strand common to the TRαA gene and the TRαB gene as shown in SEQ ID NO: 5.

In the case of mutation introduction by genome editing into fertilized eggs, the mutation is introduced into the cells of the fertilized egg or early embryo (preferably, one-cell stage embryo). When mutations are introduced into both alleles in the fertilized egg, the individual becomes genetically uniform, but in many cases, the individual becomes a mosaic individual in which the mutation pattern differs depending on the cell of the early embryo. By crossing the genetically uniform individual of the F0 generation in which a mutation has been introduced, or a mosaic individual in which a mutation has been introduced into a germ cell, with a wild-type individual, an F1 hetero individual can be obtained, and by crossing the F1 hetero individuals with each other, an F2 homo individual can be obtained. For breeding and rearing of eels, methods commonly used in this field may be adopted. For rearing, feed known in this field can be used (for example, JP-A-11-253111, JP-A-2005-13116, JP-A-2017-55674, JP-A-2018-153147). As shown in the examples below, the eels of the present invention exert the effects of the present invention from mosaic individuals of the F0 generation. Therefore, the eels of the present invention include all mosaic individuals, heterozygous individuals, and homozygous individuals with loss-of-function mutations in the TRαA gene and the TRαB gene, so long as the functions of the TRαA gene and the TRαB gene are reduced or lost. In the case of mosaic individuals and heterozygous individuals, the TRαA gene and the TRαB gene are knocked down, and in the case of homozygous individuals, the genes are knocked out.

By the above procedure, it is possible to produce eels of the present invention in which the functions of the TRαA gene and the TRαB gene are reduced or lost. Thus, the present invention also provides a method for producing eels. The method includes a step of introducing a loss-of-function mutation into the TRαA gene and the TRαB gene. The eels obtained by this step may be mosaic individuals containing loss-of-function mutations in the TRαA gene and the TRαB gene. The method may further include a step of mating the eels obtained in the loss-of-function mutation introduction step with wild-type eels. The eels obtained by this step may be heterozygous individuals containing loss-of-function mutations in the TRαA gene and the TRαB gene. Furthermore, the method may further include a step of mating multiple eels obtained in the mating step. The eels obtained by this step may be homozygous individuals containing loss-of-function mutations in the TRαA gene and the TRαB gene. Whether or not an eel has a loss-of-function mutation in the TRαA gene and the TRαB gene can be determined by taking a sample from an individual eel and analyzing it by a conventional method to confirm the base sequence of the TRαA gene and the TRαB gene or to measure the expression level of the gene or the protein encoded by the gene. For example, the expression level of the gene can be measured by a conventional method such as Northern blotting or quantitative PCR. The expression level of the protein can be measured by a conventional method such as Western blotting, colorimetry, or ELISA. Therefore, the method of the present invention may include, following each of the above steps, a step of selecting eels having a loss-of-function mutation in the TRαA gene and the TRαB gene based on the base sequence of the TRαA gene and the TRαB gene or the expression level of the gene or the protein encoded by the gene.

As shown in the Examples below, the eels of the present invention have a significantly shorter larval period and a significantly improved survival rate during the larval period compared to wild-type eels. In addition, the mutation introduction rate (gene editing rate) into the TRαA gene and the mutation introduction rate (gene editing rate) into the TRαB gene in the eels of the present invention each show a negative correlation with the number of days until the start of metamorphosis, so the effect of shortening the larval period is achieved by introducing loss-of-function mutations into at least either the TRαA gene or the TRαB gene, preferably both genes. Furthermore, the eels of the present invention achieve such an effect from the stage of the F0 generation in which the mutation is introduced, that is, the mosaic individual. Here, the "larval period" refers to the period of the leptocephalus from hatching to metamorphosis. In addition, the "survival rate during the larval period" refers to the probability that a population of eels survives a certain period up to a certain point in the larval period. In this way, the eels of the present invention have genetic characteristics that are suitable for efficient seed production. Through the raising and breeding of these eels, it will be possible to instill these acquired genetic characteristics in large-scale farmed eel populations in the future.
The aforementioned Non-Patent Document 2 reports that a mutation was introduced into the TRα gene of amphibians and functional analysis was performed, but does not mention or suggest the effect of the mutation on the survival rate. Therefore, the effect of improving the survival rate of larval fish obtained by the present invention is a completely unexpected effect.

The present invention also provides a method for improving the survival rate of eels during the larval stage. The details of this method are the same as those of the production method described above.

The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1: Creation of TRαA gene and TRαB gene modified eel By genome editing, TRαA gene and TRαB gene modified eel were created. The target sequence of sgRNA was 18 bases CAGGGCTCATCCTTCTCC (SEQ ID NO: 5) at the 5' end of the PAM sequence (AGG) among the antisense sequences common to the Japanese eel TRαA gene and TRαB gene. Phosphate buffered saline containing sgRNA at 100 ng/μL, Cas9 RNA at 100 ng/μL, Cas9 protein (Fasmac) at 500 ng/μL, and phenol red at 500 ng/μL was microinjected into Japanese eel one-cell stage embryos (mutation introduction group). The control group was microinjected with phosphate buffered saline containing the same concentration of other than sgRNA. The fertilized eggs microinjected into one-cell stage embryos were individually placed in each well of a 48-well culture plate filled with 1 mL of seawater containing antibiotics, and maintained in an incubator at 25°C and 100% humidity until 6 days after hatching.
In order to examine the proportion of individuals with mutations introduced, about 10 larvae two days after hatching were frozen at minus 30°C. The frozen larvae were dissolved at 55°C in 10 mM Tris buffer containing 0.1% Nonidet P-40 and protease at a concentration of 2 mg/mL. Primers were designed with sequences specific to the TRαA gene and the TRαB gene so that the region containing the target sequence of the sgRNA in the genomic DNA could be amplified by PCR (sense primer for TRαA: TTCCTCCTCCCCTAGTGCTCA (SEQ ID NO: 6), antisense primer: CACACGTCACCTTGCAG (SEQ ID NO: 7), sense primer for TRαB: CCTAGTTCTGAGCCTCTCTG (SEQ ID NO: 8), antisense primer: AAGCGTGTGGCTTGAGCTCT (SEQ ID NO: 9)). The lysate of the larvae was subjected to PCR using primers specific to the TRαA gene or the TRαB gene. The amplified PCR products were subjected to heteroduplex mobility analysis using an acrylamide gel, and the presence or absence of a gene mutation was determined based on the presence or absence of a smear-like band, thereby investigating the proportion of individuals in which the mutation was introduced.
On the sixth day after hatching, the hatched larvae of the mutation-introduced group and the control group were housed in separate breeding tanks (acrylic Kreisel tanks, water volume approximately 5.7 L), and diluted seawater with a salinity adjusted to 16-17 psu was adjusted to 23±0.5°C. After that, ultraviolet-sterilized breeding water was poured in at 0.9-1.0 L/min per minute, and the larvae were raised while constantly changing the water. Feeding was performed five times a day from the seventh day after hatching at 9, 11, 13, 15, and 17:00, with a combination of eel larvae suspension feed described in Table 1 of JP-A-2018-153147, at 10-15 mL per time. When feeding, the water injection was stopped, the feed was spread on the bottom of the tank using a pipette, and the feed was maintained in that state for 15 minutes to encourage the larvae to feed, and then water injection was resumed to discharge the remaining feed outside the tank, and the breeding water in the tank was maintained in a clean state. The illuminance was about 1,000 lx only during feeding, and 10 lx or less during other times. Every day, after the final feeding, the larvae were transferred to a new tank, and the used tank was cleaned and thoroughly dried, and used again the next day. Dead fish were removed from the tank, and their ages were recorded. At 50 and 100 days of age, 20 or more larvae were randomly selected from each tank, anesthetized in diluted seawater adjusted to 400 ppm 2-phenoxyethanol, and then photographed using a digital camera, and phenotypic data of total length and body height were recorded from the image data. In addition, the total number of surviving tails was counted, and the survival rate at each age was calculated using the number of tails at the time of accommodation (6 days old) as the denominator.
Individuals showing signs of metamorphosis (such as anal position moving forward, body height decreasing, etc.) were removed from the breeding tank, anesthetized and photographed in the same manner as above, and phenotypic data regarding age at the start of metamorphosis and body shape were recorded. After that, they were individually housed in 250 mL polycarbonate containers, filled with breeding water, and maintained without feeding to observe the progress of metamorphosis. The point at which the body shape completely changed to that of an elver eel was defined as the completion of metamorphosis, and the age at the completion of metamorphosis was recorded.
The above procedure was carried out on five lots of Japanese eels.
In order to investigate the correlation between the gene editing rate per individual and the number of days until the start of metamorphosis, for the second to fifth lots, glass eels after the completion of metamorphosis were frozen at -80 ° C. Genomic DNA was extracted from the frozen glass eels using a commercially available kit for genomic DNA extraction (DNeasy Blood & Tissue Kit, Qiagen). Using the extracted DNA as a template, a primer specific to the TRαA gene (SEQ ID NO: 6 and antisense primer: TGAATGCGTGCGTCTCCGT (SEQ ID NO: 10)) that can amplify the region containing the target sequence of the sgRNA shown above by PCR, and a primer specific to the TRαB gene (SEQ ID NOs: 8 and 9) were used to amplify the PCR product, which was subjected to sequence analysis using a next-generation sequencer. The proportion of sequences in which mutations different from the original sequence were introduced among all PCR products whose sequences were determined was taken as the gene editing rate (%), and the correlation with the number of days until the start of metamorphosis was examined.

For each lot of eels obtained, the number at the start of rearing, the mutation introduction rate, the number of eels that had started metamorphosis, the number of eels that had completed metamorphosis, the survival rate at 100 days, and the age at the start of metamorphosis (preanal length/total length < 70%) (age of the first individual, average age) are shown in Table 1 below.

Data from five lots, Lots 1 to 5, were compiled, and the age until the onset of metamorphosis was compared between the control group and the mutation-introduced group using survival time analysis by the Kaplan-Meier method. For individuals that died before the onset of metamorphosis after the age of 100 days, the age at death was used as censored data and was included in the survival time analysis. The relationship between the age at the onset of metamorphosis and the metamorphosis initiation rate is shown in Table 2 and Figure 1. Log-rank and Wilcoxon tests were used to test the differences between the mutation-introduced and control groups, with a p-value of <0.05 considered significant.

As shown in Tables 1 and 2 and Figure 1, the age at the onset of metamorphosis was shorter in the TRαA gene and TRαB gene mutation-introduced groups than in the control group. Comparing the median age at the onset of metamorphosis in both groups, taking into account censoring due to death, the age was shorter in the mutation-introduced group than in the control group by approximately 44 days. In the log-rank test and Wilcoxon test between the mutation-introduced group and the control group, both had p values of <0.0001, indicating a significant difference between the two groups. In other words, the larval period was significantly shorter in the mutation-introduced group than in the control group.

Furthermore, data from five lots, Lots 1 to 5, were compiled, and the survival rates (mean (%) ± standard error) at 50 days of age, 100 days of age, and at the start of metamorphosis were calculated for the mutation-introduced group and the control group, with the survival rate at 6 days of age being set at 100 (%). The Kruskal-Wallis rank sum test was used to test the differences between the mutation-introduced group and the control group, with p<0.05 considered significant. The results are shown in Figure 2.
As shown in FIG. 2, in the mutation-introduced group, the survival rate at 50 days of age was improved, and the survival rates at 100 days of age and at the onset of metamorphosis were significantly improved compared to the control group.
These results demonstrate that the larval period can be shortened and the survival rate of the larval period can be improved by introducing loss-of-function mutations into the eel TRαA gene and TRαB gene.

Figure 3(A) shows the correlation between the TRαA gene editing rate per individual and the number of days until the onset of metamorphosis, and Figure 3(B) shows the correlation between the TRαB gene editing rate per individual and the number of days until the onset of metamorphosis. As shown in Figure 3, a negative correlation was observed between the gene editing rate and the number of days until the onset of metamorphosis for both the TRαA gene and the TRαB gene. This demonstrates that mutations in either the TRαA gene or the TRαB gene, or both, shorten the larval period.

Claims (9)

Eels in which the function of the thyroid hormone receptor αA (TRαA) gene and the thyroid hormone receptor αB (TRαB) gene are reduced or lost. The eel according to claim 1, which has loss-of-function mutations in the TRαA gene and the TRαB gene. The eel according to claim 1 or 2, wherein the TRαA gene is any one selected from the following (a) to (c), and the TRαB gene is any one selected from the following (d) to (f):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO:1;
(b) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein that functions as TRα;
(c) a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides have been deleted, inserted, substituted or added relative to the nucleotide sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα;
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO:3;
(e) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein that functions as TRα;
(f) A polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:3, and encoding a protein that functions as TRα. The eel according to any one of claims 1 to 3, which lacks a part or all of the TRαA gene and a part or all of the TRαB gene. A method for producing eels, comprising the step of introducing loss-of-function mutations into the TRαA gene and the TRαB gene. A method for improving survival rate of eels during the larval stage, comprising the step of introducing loss-of-function mutations into the TRαA gene and the TRαB gene. The method according to claim 5 or 6, wherein the TRαA gene is any one selected from the following (a) to (c), and the TRαB gene is any one selected from the following (d) to (f):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO:1;
(b) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein that functions as TRα;
(c) a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides have been deleted, inserted, substituted or added relative to the nucleotide sequence shown in SEQ ID NO:1, and encoding a protein that functions as TRα;
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO:3;
(e) a polynucleotide having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein that functions as TRα;
(f) A polynucleotide having a base sequence in which one or several bases have been deleted, inserted, substituted or added relative to the base sequence shown in SEQ ID NO:3, and encoding a protein that functions as TRα. The method according to any one of claims 5 to 7, wherein the introduction of a loss-of-function mutation results in the deletion of a part or all of the TRαA gene and a part or all of the TRαB gene. The method according to any one of claims 5 to 8, wherein the introduction of a loss-of-function mutation is carried out by genome editing.