Composition injection method, egg collection method, composition for use in injection method, and reagent for preparing said composition

Abstract

The present invention addresses the problem of providing a method for injecting a composition into aquatic crustaceans. The present invention solves said problem by means of a composition injection method that includes injecting a composition containing at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains into the ovaries of an individual that is an aquatic crustacean.

Description

Method for injecting a composition, method for collecting eggs, composition for use in the injection method, and reagent for preparing the composition

[0001] This specification discloses a method for injecting a composition into an individual belonging to aquatic crustaceans, a method for collecting eggs, a composition for use in the injection method, and a reagent for preparing the composition.

[0002] Aquatic crustaceans such as shrimp are widely consumed aquatic products around the world and occupy an economically important position. Based on this, the world production volume of aquatic crustaceans was 9.5 million tons in 2018, but increased to 12.75 million tons in 2022, and variety improvement technologies for enhancing production efficiency and added value are being demanded more strongly than ever. In such a situation, genome editing technology and genetic recombination technology have attracted attention as new methods in the variety improvement of aquatic products.

[0003] For example, by using a genome editing tool to knockout the myostatin (mstn) gene of red sea bream (Pagrus major), an improved variety with a 16% increase in skeletal muscle mass (edible part) has been developed (Non-Patent Document 1).

[0004] In addition, genome editing technology has also been applied to insects (cockroaches), and a technology for inoculating a CRISPR-Cas9 complex into the coelom of female adults and delivering a genome editing tool to oocytes has been reported (Non-Patent Document 2).

[0005] Kishimoto et al., Aquaculture 495, p415-427, 2018Shirai et al., Cell Reports Methods 2, 100215, May 23, 2022Jaglarz et al., Arthropod Structure & Development 43, p349-360, 2014Ahyong et al., Zootaxa 3148, p165-191, Dec. 23, 2011Sander et al., Insect Biochemistry and Molecular Biology 165, p104068, 2[[ID=##]] [[ID=##]]

[0006] While genome editing technology is advancing in fish, many challenges remain in its application to crustaceans. Generally, for genome editing targeting individuals such as mammals and fish, microinjection, a method in which genome editing tools are directly injected into fertilized eggs immediately after spawning using a glass needle, is widely used. However, crustacean eggs are extremely vulnerable to physical damage in the early stages of development, and often rupture and leak their contents when a glass needle is inserted, making the application of microinjection extremely difficult.

[0007] Furthermore, the method described in Non-Patent Document 2 targets species belonging to the subphylum Hexapods (hexapods), and it has been reported that the body structure, including the ovaries, of hexapods differs from that of aquatic crustaceans (Non-Patent Document 3).

[0008] The object of this invention is to provide a method for injecting a composition into aquatic crustaceans.

[0009] The present invention includes the following embodiments: 1. A method for injecting a composition into the ovary of an individual belonging to an aquatic crustacean, comprising injecting a composition comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains. 2. The injection method according to 1, wherein the target nucleotide chain is used for at least one purpose selected from the group consisting of genome editing, epigenome editing, transcription or translation regulation, enzyme-dependent genetic recombination, and enzyme-independent genetic recombination, and the target peptide chain is used for at least one purpose selected from the group consisting of genome editing, epigenome editing, transcription or translation regulation, and enzyme-dependent genetic recombination. 3. The injection method according to item 2, wherein the genome editing is performed using a genome editing tool comprising at least one system selected from the Clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) system, Obligate mobile element-guided activity (OMEGA) system, Tandem interspaced guide RNA-TIGR-associated protein (TIGR-Tas) system, Bridge RNA system, Zinc Finger (ZF) system, Transcription activator-like effector (TALE) system, and Pentatricopeptide repeat (PPR) system. Item 4. The injection method according to item 2, wherein the epigenome editing is performed using an epigenome editing tool comprising at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, Bridge RNA system, ZF system, TALE system, and PPR system.5. The injection method according to item 2, wherein the transcription or translation regulation is performed by a transcription or translation regulation tool comprising at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, bridge RNA system, ZF system, TALE system, PPR system, and RNAi system. 6. The injection method according to item 2, wherein the enzyme-dependent genetic recombination is performed by an enzyme-dependent genetic recombination tool comprising at least one system selected from the integrase system, transposase system, and recombinase system. 7. The injection method according to any one of items 3 to 5, wherein the CRISPR-Cas system is the CRISPR-Cas9 system. 8. The injection method according to item 1, wherein the aquatic crustacean is one species selected from the classification of the class Malacostraca (Malacostraca). 9. The aquatic crustaceans selected from the classification of the class Malacostraca mentioned above are: Decapoda (Decapoda), Stomatopoda (Stomachids), Mysida (Mysida), Leptostraca (Pachycephala), Bathynellacea (Eucarida), Anaspidacea (Anaspidea), Spelaeogriphacea (Speleogriphacea), Thermosbaenacea (Thermosbaenacea), Lophogastrida (Lophogastrida), Mictacea (Mictocariidae), Bochusacea (Bochusacea), Amphipoda (Amphipoda), Isopoda (Isopoda), Tanaidacea (Tanaids), Cumacea (Bears), and Stygimysida. The injection method described in item 8, which is one species selected from the classification of the order consisting of (Cave Crawlers), Euphausiacea (Krills), and Amphionidacea (Amphionides). Item 10. A method for collecting eggs of aquatic crustaceans, or individuals hatched from such eggs, comprising raising individuals injected with the composition by the injection method described in item 1 to lay eggs after injection.Item 11. A composition comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains, for use in the injection method described in Item 1. Item 12. A reagent comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains, for preparing the composition described in Item 11.

[0010] In one embodiment, the composition can be injected into the ovaries of aquatic crustaceans, and eggs that have undergone, for example, genome editing, epigenome editing, transcription / translation regulation, genetic recombination, etc., can be collected.

[0011] This shows the process of intraovarian injection into *Echinococcus senticosus*. (A) Image showing the injection of the composition into the ovary of *Echinococcus senticosus*. (B) Image showing the localization of the dye in the injected composition within the ovary. Representative examples of HMA analysis results of eggs laid by *Echinococcus senticosus* subjects that underwent intraovarian injection of Cas9-sgRNA RNPs targeting AbdB are shown. The numbers at the top of each lane indicate the egg sample number. WT indicates PCR products using genomic DNA taken from eggs (wild type) of subjects that did not undergo intraovarian injection as a template. * indicates lanes determined to be HMA+. HMA+ is a term that indicates that there is a difference in electrophoretic mobility from the wild-type band due to mutation introduction, etc., and that the band has shifted. The band position of DNA that did not show HMA+ is indicated by the arrowhead on the right. As a representative example of the results of subcloning sequencing and analysis of PCR products No. 20, 24, 25, 30, and 62, which showed HMA+, the alignment results of the subcloning sequencing are shown. WT is the sequence of the PCR product using genomic DNA obtained from eggs (wild-type) of subjects that did not undergo ovarian injection, and the number on the left indicates the length of the deletion mutation (bp). For the sgRNA target sequence, the spacer sequence is underlined, the PAM sequence is enclosed in a square box, and the cleavage position is indicated by an arrowhead. Representative examples of the results of amplicon sequencing analysis of PCR products No. 25, 26, 58, 59, and 62, which showed HMA+, are shown. (A) shows a representative sequence of the alignment result of the PCR product obtained from the egg sample No. 62. WT is the sequence of the PCR product using genomic DNA obtained from eggs (wild-type) of subjects that did not undergo ovarian injection, and the number on the left indicates the length of the deletion mutation (bp). The numbers on the right indicate the introduction rate of each mutation pattern. The target sequence of the sgRNA is indicated by an underline for the spacer sequence, a square box for the PAM sequence, and arrowheads for the cleavage location. (B) The proportion of insertion / deletion mutations for No. 25, 26, 58, 59, and 62, which showed HMA+, is shown. The horizontal axis is the sample number, and the vertical axis is the mutation introduction rate. Deletion mutations are shown as diagonal bar graphs, and insertion mutations are shown as solid bar graphs. Representative examples of HMA analysis results from eggs laid by subjects of *Echinococcus senticosus* that were injected intraovarianly with Cas9-sgRNA RNPs targeting white are shown.The numbers at the top of each lane indicate the egg sample number. WT shows PCR products using genomic DNA templated from eggs (wild-type) of subjects that did not undergo ovarian injection. * indicates lanes determined to be HMA+. The right-hand arrow indicates the band position of DNA where HMA+ did not occur. As a representative example of the results of subcloning sequencing analysis of PCR products No. 8, 9, 11, 14, 32, and 33, which showed HMA+ in Figure 5, the subcloning sequencing alignment results are shown. WT represents the sequence of PCR products using genomic DNA templated from eggs (wild-type) of subjects that did not undergo ovarian injection, and the number on the left indicates the deletion mutation length (bp). For sgRNA target sequences, the spacer sequence is underlined, the PAM sequence is enclosed in a square, and the cleavage position is indicated by an arrowhead. This figure shows representative examples of individuals with different eye coloration at two weeks post-hatching from eggs laid by subjects that received intraovarian injection of Cas9-sgRNA RNPs targeting white. "White sgRNA" refers to juvenile individuals born from individuals that received intraovarian injection of white-targeting Cas9-sgRNA RNPs, while "WT" refers to juvenile individuals born from eggs of subjects that did not receive intraovarian injection. The arrows in the figure indicate the areas where changes in color and structure were observed in the eye. The figure also shows HMA results for juvenile individuals hatched from eggs laid by subjects that received intraovarian injection of white-targeting Cas9-sgRNA RNPs. The "KO" lane shows the band of the PCR product templated from genomic DNA from individuals with altered eye coloration, while the "WT" lane shows the band of the PCR product templated from genomic DNA collected from eggs of subjects that did not receive intraovarian injection (wild type). The right-hand arrowhead indicates the band location of DNA where HMA+ was not observed. The results of sequence alignment of juvenile individuals whose eye coloration changed two weeks after hatching are shown. WT represents the sequence of PCR products using genomic DNA templated from eggs (wild-type) of subjects that did not undergo intraovarian injection. The numbers on the left indicate the length (bp) of the deletion mutation. For sgRNA target sequences, the spacer sequence is underlined, the PAM sequence is enclosed in a square box, and the cleavage position is indicated by an arrowhead. The percentages on the right show the results of a simplified measurement of the mutation introduction efficiency.This shows representative sequence alignment results for eggs laid by *Echinops japonica* subjects who underwent intraovarian injection of a Cas12a-crRNA RNP targeting white, in which mutations were confirmed. WT is the sequence of a PCR product using genomic DNA from eggs of subjects that did not undergo intraovarian injection (wild type), and the number on the left indicates the length of the deletion mutation (bp). The percentage on the right shows the result of a simplified measurement of the mutation introduction efficiency. For the crRNA target sequence, the spacer sequence is underlined, the PAM sequence is enclosed in a square box, and the cleavage position is indicated by an arrowhead. This shows the waveform results of representative sequences for eggs laid by *Echinops japonica* subjects who underwent intraovarian injection of a Base Editor (ABE8e) targeting white, in which single nucleotide substitutions were confirmed. WT is the sequence of a PCR product using genomic DNA from eggs of subjects that did not undergo intraovarian injection (wild type). The arrowheads correspond to the base substitution sites, and in eggs laid by subjects introduced with ABE8e, waveforms corresponding to the substituted guanine (G) base can be observed in addition to the WT adenine (A) base. The estimated base occupancy rate from the waveform of each base is shown below the waveform data. The results of the analysis of the white mRNA expression suppression effect in eggs laid by subjects of *Echinococcus senticosus* who received intraovarian injection of white dsRNA are shown using the RT-PCR (reverse transcription PCR) method. (A) Electrophoretic images of the RT-PCR amplification products of the housekeeping gene (ubiquitin) and white for eggs collected from subjects on Day 6 are shown. (B) The bar graph shows the results of the semi-quantitative analysis of white expression levels on Day 6, using the housekeeping gene (ubiquitin) as an internal standard. Error bars indicate the mean ± standard deviation (SD). An asterisk (*) is indicated for the two groups that showed a significant difference (p<0.05) in the p-value by Welch's t-test. These images show stereomicroscope images of the ovarian region of mystery crayfish before and after intraovarian injection. (A) shows the intraovarian injection site before injection (arrowhead). (B) shows the intraovarian injection site after injection (arrowhead).Representative examples of HMA analysis results from eggs laid by mystery crayfish subjects who underwent intraovarian injection of Cas9-sgRNA RNPs targeting MIH are shown. The numbers at the top of each lane indicate the egg sample number. WT represents PCR products using genomic DNA from eggs (wild-type) of subjects that did not undergo intraovarian injection as a template. * indicates lanes determined to be HMA+. The arrowheads on the right indicate the band positions of DNA that did not produce HMA+ mutations. The results of the T7E1 assay for HMA+ eggs of mystery crayfish are shown. WT represents PCR products using genomic DNA from eggs (wild-type) of subjects that did not undergo intraovarian injection as a template. The bands indicated by the arrowheads at the top of each lane represent uncut wild-type bands. The numbers indicate the sample number. The results of subcloning sequencing alignment of PCR products No. 83 and 96, which showed HMA+ in mystery crayfish, are shown. WT represents wild-type sequences, and the numbers to the left of the sequence indicate the sample number. The numbers in parentheses to the right of the sequence indicate the length (bp) of the deletion mutation. For sgRNA target sequences, the spacer sequence is underlined, the PAM sequence is enclosed in a square, and the cleavage position is indicated by an arrowhead. A representative example of HMA analysis results from eggs laid by mystery crayfish subjects that were intraovarian-injected with a SYNCAS composition containing Cas9-sgRNA RNPs targeting MIH is shown. The numbers at the top of each lane indicate the egg sample number. WT indicates PCR products using genomic DNA taken from eggs (wild-type) of subjects that were not intraovarian-injected as a template. * indicates lanes determined to be HMA+. The band position of DNA that did not produce HMA+ is indicated by an arrowhead to the right. A summary of the amplicon sequencing analysis results for sample No. 18 is shown. The vertical axis shows the percentage of reads (%) for each deletion base pattern, and the horizontal axis shows the name of each sample. WT is the egg (wild-type) of a subject that was not intraovarian-injected, and 18 is sample No. 18 in Figure 17. Each bar graph shows the number of deletions in each sample, categorized as follows: 0: no mutation, 68: 68 bp deletion, Others: other mutations.In the wild-type (WT) sample, sequences without deletions (0) accounted for 99.6%, whereas in sample No. 18, a 68 bp deletion (68) was detected in 28.2% of the samples. The sequence of the 68 bp deletion obtained by amplicon sequencing analysis of sample No. 18 is shown. WT is the wild-type sequence, and the number to the left of the sequence indicates the sample name. The number to the right of the sequence indicates the length of the deletion mutation (bp). For the sgRNA target sequence, the spacer sequence is underlined, the PAM sequence is enclosed in a square frame, and the cleavage position is indicated by an arrowhead. Stereofluorescence microscope images of unfertilized eggs laid by Pacific white shrimp subjects injected intraovarian with a fluorescent protein composition (RFP) are shown. BF shows a bright-field image under visible light, and RFP shows an image in the same field of view through a 600 nm LP fluorescence filter under excitation light of 500-540 nm. The arrows in the image point to unfertilized eggs that emitted red fluorescence.

[0012] One embodiment relates to a method for injecting a composition into aquatic crustaceans (hereinafter sometimes simply referred to as the "injection method"), a composition for use in the injection method, and a reagent for preparing the composition.

[0013] 1. The composition and the reagent composition for preparing the composition are compositions capable of achieving the purpose of injection. The composition comprises at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains. For example, if the purpose of injection is genome editing of biological cells, the composition is a composition capable of achieving genome editing of biological cells.

[0014] The target nucleotide chain is not limited as long as it is a nucleotide chain capable of performing the expected function, either alone or in combination with other substances. The nucleotide chain may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleotide chain may be of the natural type, artificially synthesized, or a mixture of natural and artificially synthesized types. The nucleotide chain may be modified with a modifying agent. A natural type nucleotide chain refers to one synthesized in vivo by an organism or cell. An artificially synthesized nucleotide chain refers to one synthesized in vitro by an enzymatic reaction, chemical reaction, etc. The target nucleotide chain may be single-stranded, double-stranded, or a mixture of single-stranded and double-stranded nucleotides. The nucleotides constituting the target nucleotide chain may be natural type nucleotides or artificial nucleotides. The nucleotides may be modified with a modifying agent. A natural type nucleotide refers to a nucleotide with a chemical structure that exists in nature. An artificial nucleotide refers to a nucleotide with a chemical structure that does not exist in nature. Modifying agents may include labeling agents.

[0015] The length of a nucleotide chain (number of nucleotides, or number of base pairs) is not limited. In this specification, if a nucleotide chain is single-stranded, its length is expressed in units of "nt" (the number of nucleotides linked by phosphodiester bonds). If a nucleotide chain is double-stranded, its length is expressed in units of "bp" (the number of base pairs of nucleotides linked complementary by phosphodiester bonds). If a nucleotide chain consists of both single-stranded and double-stranded portions, its length is expressed in units of "nt" (the sum of the number of nucleotides in the single-stranded portion and the number of nucleotides in one of the double-stranded portions attached to the single-stranded portion). The length of a nucleotide chain is not limited as long as it can perform the function of the target nucleotide chain. For example, the shortest length of a nucleotide chain is 2, 3, 4, 5, or 6. The longest length of a nucleotide chain is not limited as long as it can perform the function of the target nucleotide chain. For example, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0016] The target peptide chain is not limited as long as it is a peptide chain capable of exhibiting the expected function, either alone or in combination with other substances. A peptide chain is intended to be a chain in which amino acid residues are linked by peptide bonds. The peptide chain may be of the natural type, artificially synthesized, or a mixture of natural and artificially synthesized components. The peptide chain may be modified with modifying substances. A natural type peptide chain is intended to be one synthesized in vivo by an organism or cell. An artificially synthesized peptide chain is intended to be one synthesized in vitro by chemical reactions, etc. The amino acids constituting the peptide chain may be natural amino acids or artificial amino acids. Furthermore, the amino acids may be modified with modifying substances. An amino acid "residue" is a constituent unit of an amino acid that makes up a peptide, and represents a group obtained by removing a hydrogen atom from the amino group of the main chain and / or removing the -OH from the carboxyl group of the main chain from an amino acid. The length of the peptide chain is not limited as long as it can exhibit the function of the target peptide chain. For example, the shortest length of a peptide chain is 2, 3, 4, 5, or 6. The maximum length of the peptide chain is not limited as long as it allows the target peptide chain to perform its function. For example, it may be 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000.

[0017] The purpose of injecting the composition is not limited, but examples include genome editing, epigenome editing, transcription or translation regulation, enzyme-dependent genetic recombination, and enzyme-independent genetic recombination. In performing genome editing, epigenome editing, transcription or translation regulation, and enzyme-dependent genetic recombination, tools including specific systems (hereinafter, these may be collectively referred to simply as "systems") may be used. Tools for genome editing are called genome editing tools, tools for epigenome editing are called epigenome editing tools, tools for transcription or translation regulation are called transcription or translation tools, and tools for enzyme-dependent genetic recombination are called enzyme-dependent genetic recombination tools. Enzyme-independent genetic recombination is an example of an injection purpose of a composition that does not involve the aforementioned systems. Examples of enzyme-independent genetic recombination include site-directed mutagenesis and forced expression using homologous recombination, etc. Preferably, injection purposes for the treatment, surgery, and / or diagnosis of diseases in individuals are excluded.

[0018] The system for genome editing is not particularly limited, but examples include at least one system selected from the following: Clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) system, Obligate mobile element-guided activity (OMEGA) system, Tandem interspaced guide RNA-TIGR-associated protein (TIGR-Tas) system, Bridge RNA system, Zinc Finger (ZF) system, Transcription activator-like effector (TALE) system, Pentatricopeptide repeat (PPR) system, etc.

[0019] The system for epigenome editing is not particularly limited, but examples include at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, bridge RNA system, ZF system, TALE system, and PPR system.

[0020] The aforementioned transcription or translation regulation system is not particularly limited, but examples include at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, bridge RNA system, ZF system, TALE system, PPR system, and RNAi system.

[0021] The system for enzyme-dependent gene recombination is not particularly limited, but examples include at least one system selected from an integrase system, a transposase system, and a recombinase system.

[0022] Genome editing refers to a technique that modifies polynucleotides contained in target biological cells by introducing mutations into those polynucleotides.

[0023] The polynucleotides into which mutations are introduced include, but are not limited to, nuclear genomes, mitochondrial genomes, plasmids, artificial chromosomes, viral DNA genomes, mRNA, miRNA, lncRNA, and viral RNA genomes. Furthermore, a subset of these polynucleotides that are targeted may be referred to as the target sequence below.

[0024] Examples of mutations include, but are not limited to, insertions, deletions, substitutions, inversions, translocations, and duplications of bases in the target sequence. The length of a mutation is not limited as long as it can perform its function. For example, the shortest mutation length is 1, 2, 3, 4, 5, or 6. The longest mutation length is not limited as long as it can perform its function. For example, it could be 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0025] The above modifications include, but are not limited to, altering, adding, enhancing, reducing, or losing the genetic information and functions of an individual organism or cell.

[0026] Epigenome editing refers to techniques for manipulating epigenetic modifications of polynucleotides and polypeptides within biological cells. These epigenetic modifications are involved in the higher-order structure of chromosomes and the regulation of gene transcription and translation, and examples include DNA methylation, hydroxymethylation, histone methylation, acetylation, phosphorylation, and ubiquitination.

[0027] Transcriptional and translational regulation refers to the control of the process by which genetic information is transcribed and translated, and includes post-transcriptional regulation. Examples include the activation and repression of transcription, interference with the action of transcription factors and RNA polymerase, RNA degradation via RNA interference, repression of translation via antisense polynucleotides, and effects on translation regulators and ribosome binding sites.

[0028] Genetic modification refers to a general technique for altering, adding, enhancing, reducing, or losing the genetic information and function of an organism or cell by introducing foreign polynucleotides, rearranging host polynucleotides, or combining these methods. Genetic modification can be classified into enzyme-dependent genetic modification, which involves systems such as integrase, transposase, and recombinase, and enzyme-independent genetic modification, which does not rely on these systems.

[0029] RNAi (RNA interference) used for the purpose of injecting the above composition refers to an operation in which nucleic acid molecules such as double-stranded RNA having a sequence complementary to the base sequence of the RNA of the target gene are introduced into cells or organisms, thereby specifically degrading or repressing the translation of the target mRNA and suppressing gene expression.

[0030] The form of the RNAi-inducing molecule is not particularly limited and includes small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), or microRNA (miRNA) precursors. These may be chemically synthesized RNA molecules, RNA molecules synthesized by in vitro transcription, or introduced in the form of DNA molecules or other molecules that can express them in cells.

[0031] The CRISPR-Cas system used for the injection of the above composition refers to a system that uses all or part of the CRISPR-Cas system, which is a mechanism that bacteria and archaea use to identify and defend against foreign polynucleotides, or a system that has been modified or improved thereto, or any system that is artificially created to mimic the CRISPR-Cas system.

[0032] In nature, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are polynucleotide regions consisting of short repeat sequences and spacer sequences. RNA molecules such as crRNA (CRISPR RNA) transcribed from CRISPR, or a complex of crRNA and tracrRNA (trans-activating CRISPR RNA), can recognize, bind to, and, in some CRISPR-Cas systems, cleave target sequences by forming a complex with one or more related Cas (CRISPR-associated) proteins.

[0033] The above Cas proteins are not particularly limited as long as they are included in the CRISPR-Cas system, but include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas10d, Cas11, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Examples include Cas13, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, etc.

[0034] The CRISPR-Cas system described above involves a complex formed between crRNA or a complex of crRNA and tracrRNA, a complex of RNA molecules obtained by further fragmentation of these, a single-stranded polynucleotide (gRNA: guide RNA, also known as sgRNA: single-guide RNA) linked via a linker, or a modified or improved version of these RNA molecules, or an RNA molecule artificially generated to mimic these RNA molecules, and one or more Cas proteins to form an RNP (Ribonucleoprotein), which searches for and recognizes a sequence complementary to the target sequence. After the RNP binds to the target sequence, in some CRISPR-Cas systems, the polynucleotide cleavage domain of the Cas protein introduces a single-strand or double-strand break into the target sequence.

[0035] The CRISPR-Cas system described above is particularly preferably the CRISPR-Cas9 system. The CRISPR-Cas9 system is the most widely used system in genome editing. Furthermore, the Cas9 protein in the CRISPR-Cas9 system is not limited as long as it is classified as a Cas9 protein, a modified or improved version of the Cas9 protein, or an artificially generated Cas9 protein that mimics the Cas9 protein. However, the most widely used Cas9 protein is called SpCas9 or SpyCas9 (derived from Streptococcus pyogenes) after the species from which it is derived. In addition, other proteins such as SaCas9 or SauCas9 (derived from Staphylococcus aureus), FnCas9 or FnoCas9 (derived from Francisella novicida), StCas9 or SthCas9 (derived from Streptococcus thermophilus), NmCas9 or NmeCas9 (derived from Neisseria meningitidis) can also be used.

[0036] While there are no particular restrictions on modified or improved versions of the above Cas9 protein, examples of SpCas9 mutants include Cas9 nickase (D10A), Cas9 nickase (H840A), dead Cas9 (D10A&H840A), xCas9, SpCas9-NG, SpG, SpRY, SpRYc, etc. Furthermore, there are no limitations on other SpCas9 mutants, Cas9 from other species, or mutants consisting of artificially generated Cas9.

[0037] The CRISPR-Cas system described above is preferably the CRISPR-Cas12a system. The CRISPR-Cas12a system is a widely used system in genome editing. The Cas12a protein in the CRISPR-Cas12a system is not limited as long as it is classified as a Cas12a protein, or is a modified or improved version of a Cas12a protein, or is an artificially generated Cas12a protein that mimics a Cas12a protein. However, the most widely used Cas12a protein is called LbCas12a or LbaCas12a (derived from Lachnospiraceae bacterium ND2006) after the species from which it is derived. In addition, other proteins such as AsCas12a (derived from Acidaminococcus sp. (strain BV3L6)), FnCas12a, or FnoCas12a (derived from Francisella novicida) can also be used.

[0038] Cas12a is a CRISPR effector protein classified as Type VA, and the LbCas12a used in this example recognizes a T-rich PAM sequence called 5'-TTTV-3'. This enzyme forms a complex with a single short crRNA (approximately 42 nt) and is a genome editing enzyme that introduces staggered cuts (cuts that produce sticky ends) into the double strands of target DNA.

[0039] "Base editing technology" refers to a gene modification technique that directly converts specific bases on a target genome into other bases without substantially inducing DNA double-strand breaks (DSBs).

[0040] Typically, these proteins are composed of a fusion protein between a DNA-binding protein (e.g., an RNA-dependent nuclease such as Cas9 with lost or reduced nuclease activity, TALE, or zinc finger) and a base-modifying enzyme (deaminase, glycosylase, etc.).

[0041] The type of base editor is not particularly limited, and includes Cytosine Base Editor (CBE) that converts cytosine to thymine (or uracil), Adenine Base Editor (ABE) that converts adenine to guanine (or inosine), and the like.

[0042] In the examples described below, ABE8e, which is a type of base editor, is used. ABE8e is an adenine base editing enzyme that combines a highly active TadA-8e variant and nCas9 to convert adenine (A) at the target site to guanine (G) extremely efficiently and rapidly. Base editors containing ABE8e are used for purposes such as introducing point mutations in the coding region or regulatory region of a target gene, or gene disruption (knockout) by forming a stop codon.

[0043] In this specification, a variant of a Cas protein refers to a polypeptide in which a part of the polypeptide is modified, and while some function of the underlying Cas polypeptide is retained, some other function may be conferred, enhanced, decreased, or reduced.

[0044] The Obligate mobile element-guided activity (OMEGA) system used for the purpose of injecting the above composition is a type of RNA-guided DNA targeting system, and a protein called IscB, TnpB, Fanzor, etc. recognizes a target sequence with a non-coding RNA called omega RNA (ωRNA), or a system that recognizes and cleaves it. The components of the OMEGA system are not limited as long as they are classified as components of the OMEGA system, or those modified or improved based on the components of the OMEGA system, or those artificially generated by mimicking the components of the OMEGA system.

[0045] The Tandem interspaced guide RNA-TIGR-associated protein (TIGR-Tas) system used for the purpose of injecting the above composition is a kind of RNA-induced DNA targeting system. It is a system that recognizes or recognizes and cleaves a target sequence by a guide RNA composed of consecutive recognition sequences called TigRNA (Tandem Interspaced Guide RNA) and a Tas (TIGR-associated) protein that binds to it. The components of the TIGR-Tas system are not limited as long as they are classified as components of the TIGR-Tas system, or are modified or improved based on the components of the TIGR-Tas system, or are artificially generated by mimicking the components of the TIGR-Tas system.

[0046] The bridge RNA system used for the purpose of injecting the above composition is composed of a recombinase derived from the IS family transposable element and bridge RNA, and is a system for inserting a foreign polynucleotide into a target polynucleotide. The bridge RNA mediates the insertion of the foreign polynucleotide by recognizing both the foreign polynucleotide and the target sequence. The components of the bridge RNA system are not limited as long as they are classified as components of the bridge RNA system, or are modified or improved based on the components of the bridge RNA system, or are artificially generated by mimicking the components of the bridge RNA system.

[0047] The Zinc Finger (ZF) system used for the injection of the above composition is a system that recognizes target polynucleotides by a DNA-binding domain linked to zinc finger motifs, which are components of transcription factors. The ZF system alone has the activity to bind to target sequences, and can also exert any desired function by fusing it with any factor. For example, a protein (ZFN: Zinc Finger Nuclease) fused with the ZF system and a polynucleotide cleavage domain derived from FokI restriction enzymes, etc., can recognize and cleave target sequences. The components of the ZF system are not limited as long as they are classified as components of the ZF system, modified or improved versions of components of the ZF system, or artificially generated to mimic components of the ZF system.

[0048] The Transcription Activator-like Effector (TALE) system used for the injection of the above composition is a system that recognizes target polynucleotides by the DNA-binding domain of a TALE protein derived from plant pathogenic bacteria. The TALE system alone has the activity to bind to target sequences, and can also exert arbitrary functions by fusing it with arbitrary factors. For example, a protein (TALEN: TALE Nuclease) obtained by fusing the TALE system with a polynucleotide cleavage domain derived from FokI restriction enzymes, etc., can recognize and cleave target sequences. The components of the TALE system are not limited as long as they are classified as components of the TALE system, modified or improved versions of components of the TALE system, or artificially generated to mimic components of the TALE system.

[0049] The Pentatricopeptide repeat (PPR) system used for the injection of the above composition is a system that recognizes a target sequence by a polynucleotide binding domain formed by linking multiple pentatricopeptide repeats. The PPR system alone has the activity to bind to the target sequence, and can also exert any desired function by fusing it with any factor. A protein formed by fusing the PPR system with a polynucleotide cleavage domain derived from FokI restriction enzymes, etc., can recognize and cleave the target sequence. The components of the PPR system are not limited as long as they are classified as components of the PPR system, modified or improved versions of components of the PPR system, or artificially generated to mimic components of the PPR system.

[0050] The integrase system used for the injection of the above composition is a system that incorporates foreign polynucleotides into host polynucleotides using a recombinant enzyme (integrase) specific to a particular short polynucleotide, and examples include attP / attB site recombination by the integrase of φC31 phage and Bxb1 phage. The components of the integrase system are not limited as long as they are classified as components of an integrase system, modified or improved versions of components of an integrase system, or artificially generated to mimic components of an integrase system.

[0051] The transposase system used for the injection of the above composition is a system that uses transposase, an enzyme of DNA transposable elements (transposons), to insert foreign polynucleotides into the region called IR / DR(R), and examples include PiggyBac and Sleeping Beauty (SB). The components of the transposase system are not limited as long as they are classified as components of a transposase system, or are modified or improved versions of components of a transposase system, or are artificially produced to mimic components of a transposase system.

[0052] The recombinase system used for the injection of the above composition is a system that inserts foreign polynucleotides into the inside of a specific short DNA recognition sequence, such as a loxP site, using an enzyme (recombinase) that catalyzes site-directed recombination. Examples include Cre recombinase and Flp recombinase. The components of the recombinase system are not limited as long as they are classified as components of a recombinase system, modified or improved versions of components of a recombinase system, or artificially generated to mimic components of a recombinase system.

[0053] When the target double-stranded DNA is cleaved by the above system, the cell's DNA repair mechanisms, such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and homology-directed repair (HDR), including homologous recombination (HR), can be used to introduce mutations into the target sequence or insert specific sequences.

[0054] When RNA is targeted using the above system, the system itself or the fused factors can perform actions such as degradation of the target RNA, base substitution, modification such as methylation and demethylation, fluorescent labeling, splicing control, translation control, competitive inhibition of interacting molecules, and polyadenylation control.

[0055] The above system recognizes and binds to target nucleotide chains, and in some systems, it can cleave only one strand of double-stranded DNA, or cleave double-stranded DNA, or cleave RNA, after which it can perform base substitution, epigenome editing, transcription and translation regulation, etc.

[0056] When the system is used for the purpose of injecting the above composition, the target nucleotide chain can be a nucleotide chain for achieving the objective, a nucleotide chain containing a sequence encoding a polynucleotide for achieving the objective, etc. Examples of RNAs for achieving the objective include crRNA, tracrRNA, gRNA, ωRNA, TigRNA, and bridge RNA. Examples of nucleotide chains encoding a polynucleotide for achieving the objective include a nucleotide chain containing a sequence encoding crRNA, tracrRNA, gRNA, ωRNA, TigRNA, bridge RNA, etc., and a nucleotide chain encoding a protein included in the system. The nucleotide chain encoding the polynucleotide for achieving the objective may also include vector sequences, promoter sequences, poly-A sequences, etc., in addition to the sequences encoding crRNA, tracrRNA, gRNA, ωRNA, TigRNA, bridge RNA, etc. Furthermore, whether or not the system is used for the purpose of injecting the above composition, the target nucleotide chain can be a donor polynucleotide containing arbitrary mutations, or a polynucleotide that assists in the introduction or integration of the donor polynucleotide into genomic DNA (carrier polynucleotide, polynucleotide without sequence homology to the target sequence, etc.).

[0057] When the system is used for the purpose of injecting the above composition, the target peptide chain may include enzymes and other components of the system. Furthermore, whether or not the system is used for the purpose of injecting the above composition, the target peptide chain may include peptides that assist in the introduction of the target nucleotide chain or the target peptide chain (vitellogenin protein that induces vitellogenesis or a part thereof used as a peptide, cell-permeable peptides, etc.).

[0058] Whether or not the system is used for the injection of the above composition, other functional peptide chains can be cited as target peptide chains. These may include not only those introduced alone, but also those introduced co-introduced with the target nucleotide chain and / or the target peptide chain, those fused to the target nucleotide chain and / or the target peptide chain, and those recruited to the target peptide chain and / or the target nucleotide chain. Examples of other functional peptide chains include, but are not limited to, deaminases, reverse transcriptases, DNA and histone modifying enzymes, enzymes involved in transcription and translation regulation (transcription activators, transcription repressors, translation initiation factors, translation termination factors, etc.), DNA polymerases, RNA polymerases, factors related to genetic recombination, DNA repair, and the cell cycle (NHEJ-related factors, MMEJ-related factors, HR-related factors, etc.), or their domain portions and / or various signal sequences (nuclear localization signals, mitochondrial localization signals, etc.), various reporter proteins (fluorescent proteins, luminescent proteins), and various tag sequences (hereinafter, they may simply be referred to as "functional peptide chains"). Other functional peptide chain functions include, but are not limited to, functions such as converting specific bases, adding and / or removing epigenetic modifications to DNA or histones, increasing or decreasing the transcription and translation of target genes or proteins, efficiently introducing new sequence information into target sequences, and regulating desired mutation patterns or mutation efficiency.

[0059] Examples of the functional peptide chains mentioned above include, for example, APOBEC, CDA1, and TadA as deaminases; MMLV as a reverse transcriptase; DNMT, TET, HAT, and HDAC as DNA and histone modifying enzymes; VP64, VPR, and KRAB as enzymes involved in transcription and translation regulation; and Rad51, Mre11, and CtIP (RBBP8) as factors related to genetic recombination and DNA repair. Furthermore, any peptide chain having the necessary function to achieve the injection purpose of the above composition is acceptable, and is not limited to those exemplified herein.

[0060] Whether or not the system is used for the injection of the above composition, the target nucleotide chain and / or target peptide chain may be chemically modified or further fused with and / or recruited with other nucleotide chains. Examples of chemical modifications include, but are not limited to, those for the purpose of fluorescent labeling or stabilization. Examples of other nucleotide chains include, but are not limited to, aptamer sequences and ribozyme sequences having specific ligand-binding ability.

[0061] Examples of mechanisms for recruiting the above-mentioned functional peptide chains include, but are not limited to, combinations of an RNA stem-loop sequence derived from MS2 bacteriophage and the coat protein MCP, and SunTag, which is composed of a GCN4 peptide and the mini-antibody scFv that recognizes it. The number of functional peptide chains to be recruited may be one or multiple. The type of functional peptide chain to be recruited may be one or multiple.

[0062] When the purpose of injecting the above composition is transcription and translation regulation, the target nucleotide chain may include, but is not limited to, RNA such as siRNA, shRNA, dsRNA, miRNA, or a vector containing a sequence encoding said RNA, or a single-stranded polynucleotide having a sequence complementary to the gene targeted for transcription and translation regulation and having the function of suppressing protein translation, or a vector that forcibly transcribes and / or expresses a nucleotide chain or peptide chain.

[0063] Even if the system is not used for the purpose of injecting the above composition, site-directed mutagenesis and forced expression can be performed. Site-directed mutagenesis is a method of directly modifying a target sequence using a target nucleotide chain such as a single-stranded oligonucleotide that is complementary to the target sequence. Forced expression is a method of artificially increasing gene expression levels using plasmid vectors or the like.

[0064] When the purpose of injecting the above composition is genetic recombination, examples of target nucleotide chains include single-stranded linear polynucleotides, single-stranded cyclic polynucleotides, double-stranded linear polynucleotides, double-stranded cyclic polynucleotides containing plasmids or cosmids, etc. Furthermore, examples of sequences included in these target nucleotides include, but are not limited to, sequences for performing genetic recombination (e.g., donor sequences), sequences encoding proteins to be expressed by genetic recombination, as well as vector sequences, promoter sequences, polyA sequences, etc.

[0065] When the purpose of injecting the above composition is genetic recombination, the target peptide chain may include, but is not limited to, proteins used for genetic recombination (e.g., recombinase, transposase, integrase, etc.).

[0066] If the objective is protein transfection, the target peptide is the protein or polypeptide that is to be transfected.

[0067] The composition is not limited as long as it contains at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains, but it takes a form that can be injected into the ovary. The composition may be a mixed solution or the like, combining at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains with other components necessary for injection. The composition may also contain at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains, and other components that are not essential for injection but are preferable to include in the composition at the time of injection.

[0068] The aforementioned other components are preferably substances that are harmless or have low toxicity to aquatic crustacean individuals or cells within the ovaries. Specifically, examples include solvents, glycosides, low molecular weight compounds, high molecular weight carriers, transfection reagents (for target nucleotide chains, for target peptide chains), and components that promote ovarian maturation. The solvent may be hydrophilic or hydrophobic, but is preferably hydrophilic. Water can be an example of a hydrophilic solvent. The solvent may include salts such as potassium salts, sodium salts, and calcium salts; pH adjusters; chelating agents such as EDTA; protein stabilizers such as 2-mercaptoethanol and dithiothreitol; surfactants; dyes, etc. The dye is preferably one that allows the injection of the composition into the ovary to be directly or indirectly observed by the human eye with the naked eye or under a microscope. The dye may be visible in visible light or visible by fluorescence excitation. Examples of dyes include Acid Blue, Methylene Blue, Trypan Blue, Phenol Red, and Fast Green. Glycosides are compounds in which a sugar portion is linked to a non-sugar portion via a glycosidic bond, and they exhibit biological activity and functionality. Examples of glycosides include saponins, phenolic glycosides, and flavonoid glycosides. Low molecular weight compounds are substances with relatively small molecular weights that are relatively easy to synthesize and modify, and are used in a wide range of fields such as pharmaceuticals and industrial products. Examples of low molecular weight compounds include chloroquine and ammonium chloride (NH4Cl). High molecular weight carriers are carriers made of polymers or composite materials with large molecular weights, and are designed to enhance stability within the body and cells, as well as cell targeting function. Examples of high molecular weight carriers include polyethyleneimine (PEI), PAMAM dendrimers (polyamidoamine dendrimers), and chitosan. Transfection reagents are reagents used to introduce nucleotide chains and / or peptide chains into cells, and mainly consist of cationic lipids and cationic polymers. Examples of transfection reagents include Lipofectamine, FuGENE, BAPC, and cationic liposomes.

[0069] The above composition can be prepared from a reagent containing the target nucleotide chain and / or the target peptide chain. The reagent may be in liquid or dry state. The reagent may consist only of the target nucleotide chain and / or the target peptide chain, but may also contain other components. The components are as described above.

[0070] 2. The method for injecting the composition, and the method for collecting and injecting eggs, or individuals hatched from said eggs, comprises injecting a composition containing at least one selected from the group consisting of a target nucleotide chain and a target peptide chain into the ovary of an individual belonging to an aquatic crustacean (hereinafter sometimes simply referred to as "aquatic crustacean"). Preferably, in this embodiment, microinjection methods in which injection is performed directly into eggs after spawning are excluded. Also preferably, the injection method is excluded if it is intended for the treatment, surgery, and / or diagnosis of diseases in the individual.

[0071] The aforementioned individual is not limited as long as it has ovaries or tissue containing cells capable of forming eggs. The aforementioned individual may be one that is born female, or one that acquires female characteristics through sex reassignment.

[0072] In biological classification, aquatic crustaceans can be listed as individuals belonging to the class Malacostraca (skulls).

[0073] The classification of aquatic crustaceans belonging to the class Malacostraca is illustrated in Non-Patent Document 4.

[0074] Further classification of aquatic crustaceans selected from the above-mentioned Malacostraca class includes: Decapoda (Decapoda), Stomatopoda (Stomachids), Mysida (Mysida), Leptostraca (Pachycephala), Bathynellacea (Eucarida), Anaspidacea (Anaspidea), Spelaeogriphacea (Speleogriphacea), Thermosbaenacea (Thermosbaenacea), Lophogastrida (Lophogastrida), Mictacea (Mictocariida), Bochusacea (Bochusacea), Amphipoda (Amphipoda), Isopoda (Isopoda), Tanaidacea (Tanaids), Cumacea (Bears), and Stygimysida. Examples include the orders Euphausiacea (cave webworms), Euphausiacea (krills), and Amphionidacea (amphionides).

[0075] Aquatic crustaceans belonging to the above-mentioned Malacostraca class include, for example, the Japanese spiny lobster, the red-clawed swimming crab, the autumn mysid shrimp, the snow crab, the American ginkgo crab, the American crayfish, the American lobster, the Argentine red shrimp, the spiny lobster, the tiger prawn, the endeavor prawn, the Australian spiny lobster, the Australian tiger prawn, the giant snow crab, the giant freshwater prawn, the swimming crab, the tiger prawn, the kuruma prawn, the hairy crab, the Korean shrimp, the sakura shrimp, the monkey shrimp, the Shiba shrimp, the spotted prawn, the snow crab, the Taiwanese swimming crab, the king crab, and the Chinese spider. Kudzu crab, hornless krill, caddisfly prawn, spiny mantis shrimp, spiny mud crab, Toyama shrimp, tiger shrimp, Hanasaki crab, banana shrimp, Pacific white shrimp, red snow crab, northern red shrimp, true northern red shrimp, European red shrimp, European ginkgo crab, European lobster, Yoshi shrimp, red stone crab, red shrimp, red-spotted crab, red-spotted minnow, Asahi crab, mud shrimp, African giant pygmy crab, African southern spiny lobster, Amami spiny lobster, reticulated mud crab, Amur shrimp, Isaza mysid shrimp, stone crab, thorny crab, Iba False crab, thorny shrimp, signal crayfish, slipper lobster, false slipper lobster, Ezo thorny crab, large slipper lobster, large three-spined broad-legged shrimp, spotted spiny lobster, barnacle, chestnut crab, black prawn, hairy spiny lobster, five-colored shrimp, knobby slipper lobster, Sagami red shrimp, Sagami spiny lobster, Sakhalin spotted shrimp, freshwater crab, striped stone crab, striped spiny lobster, mantis shrimp, Japanese crab, white shrimp, white shrimp, human-like shrimp, striped shrimp, snow crab, slipper lobster, slipper lobster, giant spider crab, long-armed shrimp, spiny chestnut crab, spiny prawn, spiny shrimp Snow crab, spiny flat shrimp, common pipefish, ornate shrimp, Japanese mysid shrimp, Panama mino shrimp, long-horned shrimp, Higoro shrimp, Himeamae shrimp, flat-clawed crab, flat-clawed freshwater shrimp, thick-clawed shrimp, brown spiny lobster, red stone crab, spot prawn, Hokkai shrimp, Hokkoku shrimp, Hong Kong stone crab, Matsuba crab, Marugoshi mino shrimp, southern red shrimp, southern spiny lobster, southern slipper lobster, southern freshwater shrimp, mountain barnacle, mino shrimp, long-legged crab, freshwater shrimp, Chinese mitten crab, spiny red shrimp, spotted stone crab, coconut crab, red stonefish,Red-striped shrimp, red-horned squat lobster, red-clawed crab, spiny shrimp, spiny hermit crab, stone shrimp, stone hermit crab, Izumi shrimp, Indian Heike crab, thin-striped pipefish, sea slug, long-armed shrimp, dimpled slipper lobster, branched horned crab, shrimp, shrimp, area-dwelling squirrel crab, Enkou crab, Ouston crab, Oiran hermit crab, giant enkou crab, giant sea cucumber, giant isopod, Kajiwara shrimp, light stone crab, light stone hermit crab, Kawari stone crab, Kishie shrimp, Kishinoue shrimp, Kitazako shrimp, Northern horned shrimp, Kushinohakudahige shrimp, Kubo shrimp, Kubochu squat shrimp, Hairy hermit crab, Hairy shore crab, Hairy shore crab, Small-legged pipefish, Small gill crab, Kodakakudahige shrimp, Kounaga crab, Black hermit crab, Black barnacle, Black crane crab, Hairy ash crab, Goto hermit crab, Knobbed crab, Fiddler crab, Salmon louse, Shark-skinned fan crab, Shark-skinned heike crab, Eleven-spined knotfish, Beaded beard shrimp, Scarlet shrimp, Shojin crab, Smooth shrimp, Smooth manju crab, Striped mantis shrimp, Senju shrimp, So Denashikarappa, Sobagaragani, Sorihashihimekudahigeebi, Taiwanese Ryomaebi, Tatejimakobushi, Chigogani, Chochinkobushi, Tsunonagakobushi, Tsunonagashinkaikoshioriebi, Terao Botanebi, Tenjikujinkenebi, Toyokoshioriebi, Toyo Homora, Togeashigani, Togeebijako, Nakienkougani, Nanatogekobushi, Nanseisaruebi, Niseoniteppouebi, Nihonsunamoguri, Nukaebi, Nokogirigani, Hakusenebi, Higegani, Higenagakudahigeebi, Higenagachuukoshioriebi, Hishigani, Himeakaigani, Hira Horned shrimp, Biwa crab, Two-spined shrimp, Two-spotted stone crab, Two-spotted pistol shrimp, Helichrysum puellaris, Slender-armed crab, Slender shrimp, Japanese hermit crab, Round-necked caprellid, Three-horned crab, Three-pronged shrimp, Southern marsh shrimp, Myoga clam, Mutsuhaogi crab, Mutsuhasake shrimp, Menko crab, Menkohishi crab, Mogi shrimp, Peacock mantis shrimp, Yasaoki shrimp, Yamato stone shrimp, Yamato fiddler crab, Yamato crab, Yamato shrimp, Yumon crab, Four-spined mantis shrimp, Last-ban hermit crab, Ryoma shrimp, Hexagonal knotweed, Buzzy seaweed,Examples include Parhyale hawaiensis, Exopalaemon carinicauda, ​​and the mystery crayfish.

[0076] Preferably, the aquatic crustaceans are species that are used for food. More preferably, the species can be reared after the composition is injected. Among the aquatic crustaceans belonging to Malacostraca (class Malacostraca) that are used for food, preferably include the Japanese spiny lobster, the red-clawed swimming crab, the autumn mysid shrimp, the snow crab, the American ginkgo crab, the American crayfish, the American lobster, the Argentine red shrimp, the spiny lobster, the tiger prawn, the endeavor prawn, the Australian spiny lobster, the Australian tiger prawn, the giant snow crab, the giant freshwater prawn, the swimming crab, the tiger prawn, the kuruma prawn, the hairy crab, the Korean shrimp, the sakura shrimp, the monkey shrimp, the Shiba shrimp, and the spotted prawn. Snow crab, Taiwanese swimming crab, king crab, Chinese mitten crab, hornless krill, caddisfly prawn, spiny mantis shrimp, spiny swimming crab, Toyama shrimp, tiger shrimp, Hanasaki crab, banana shrimp, Pacific white shrimp, red snow crab, northern red shrimp, true northern red shrimp, European red shrimp, European ginkgo crab, European lobster, Yoshi shrimp, red stone crab, red shrimp, red spotted crab, red spotted minnow, Asahi crab, mud shrimp, African giant pygmy crab, African southern spiny lobster, Amami Spiny lobster, reticulated swimming crab, Amur shrimp, mysid shrimp, stone crab, thorny crab, false thorny crab, thorny shrimp, signal crayfish, slipper lobster, false slipper lobster, Ezo thorny crab, large slipper lobster, large three-spined broad-spined shrimp, spotted spiny lobster, barnacle, chestnut crab, black shrimp, hairy spiny lobster, five-colored shrimp, knobby slipper lobster, Sagami red shrimp, Sagami spiny lobster, Sakhalin spotted shrimp, freshwater crab, striped stone crab, striped spiny lobster, mantis shrimp, Japanese crab, white shrimp, white shrimp, Japanese prawn, Striped shrimp, snow crab, slipper lobster, giant spiny lobster, Japanese spider crab, long-armed shrimp, spiny crab, spiny snow crab, spiny flat shrimp, common pipefish, ornate shrimp, Japanese mysid shrimp, Panama shrimp, long-horned shrimp, red shrimp, sweet shrimp, flat-clawed crab, flat-clawed long-armed shrimp, thick-clawed shrimp, brown spiny lobster, red stone crab, spot prawn, Hokkaido shrimp, northern shrimp, Hong Kong stone crab, Matsuba crab, round-legged shrimp, southern red shrimp, southern spiny lobster,Examples include the southern slipper lobster, southern long-armed shrimp, mountain barnacle, minnow, long-legged crab, freshwater shrimp, Japanese mitten crab, spiny red shrimp, spotted stone crab, coconut crab, and Exopalaemon carinicauda. Among the aquatic crustaceans belonging to the above-mentioned Malacostraca (class), edible species that can be reared after injection of the composition are preferably American crayfish, tiger prawn, giant freshwater prawn, swimming crab, kuruma prawn, kuruma shrimp, Korean shrimp, Taiwanese swimming crab, Chinese mitten crab, caddisfly prawn, spiny swimming crab, banana shrimp, Pacific white shrimp, mantis shrimp, spiny lobster, reticulated swimming crab, signal crayfish, freshwater prawn, barnacle, red shrimp, red-clawed swimming crab, American ginkgo crab, American lobster, Argentine red shrimp, European red shrimp, European ginkgo crab, European lobster, Asahi crab, Exopalaemon carinicauda, ​​etc.

[0077] The ovaries of aquatic crustaceans are covered on the outside by a membrane. This membrane separates the tissue containing gamete-forming cells such as oogonia and oocytes from the body cavity. There is no uniform name for this membrane, but in some literature, it is called the ovarian sac, ovarian sheath, ovarian envelope, ovarian wall, or ovarian membrane, depending on the author of the literature. In this specification, for convenience, the membrane covering the outside of the ovary may be referred to as the "ovarian exodia," but this term comprehensively includes the membrane structures that separate the ovary from the body cavity, such as the ovarian sac, ovarian sheath, ovarian envelope, ovarian wall, and ovarian membrane.

[0078] In the injection method, the composition described in 1. above is injected into the ovary via a capillary needle or syringe-attached injection needle that has been punctured from the outside of the ovary through the ovarian outer membrane, thereby delivering the composition to the ovary. Therefore, the embodiment is not intended to be a microinjection method that directly injects the composition into the cells within the ovary.

[0079] Specifically, the injection of the composition into the ovary of an aquatic crustacean receiving the composition (hereinafter also referred to as "test subject") involves puncturing the ovary with a capillary needle or injection needle (hereinafter sometimes simply referred to as "injection needle, etc.") filled with the composition, either from above the exoskeleton, or, if the exoskeleton is hard, by partially peeling off the exoskeleton of the cephalothorax (cephalothorax carapace), and then puncturing the ovarian outer membrane with the tip of the injection needle, etc., reaching beneath the ovarian outer membrane, and injecting the composition from the injected injection needle, etc., or from a syringe or tube connected to the injection needle, etc., into the ovary. The puncture site of the exoskeleton with the injection needle, etc., is not limited as long as the injection needle, etc., can be punctured into the ovary. For example, the puncture site may be the dorsal side of the thorax. For example, the puncture site may be the ventral side of the abdomen. For example, the puncture site may be the lateral side of the thorax. For example, the puncture site may be the lateral side of the abdomen. The composition inside the capillary needle can be injected by blowing air into the capillary needle or by operating the syringe of an injector connected to the capillary needle. An example of an injector is a microinjector, preferably an IM-12 microinjector (Narishige Group). The composition inside the syringe or tube connected to the injection needle can be injected by pressing the plunger inserted into the syringe connected to the injection needle or the syringe connected to the tube in the cylinder axis direction. An example of a syringe is a needle-equipped syringe, preferably a Terumo syringe made of PP (polypropylene) with an injection needle attached.

[0080] The amount of composition injected into the ovary in a single injection can be determined according to the size of the subject's ovary, within an amount that can be injected and achieve the objective. For example, the amount of composition injected is approximately 0.05 μL to 5,000 μL in absolute terms. For example, if the subject is an amphipod or the like, and its body length is approximately 2 mm to 15 mm, then approximately 0.05 μL to 1.0 μL, preferably 0.3 μL to 0.7 μL of composition, can be injected. For example, if the subject is a mystery crayfish or the like, and its body length is approximately 5 cm to 15 cm, then approximately 5 μL to 20 μL, preferably 10 μL to 15 μL of composition, can be injected. For example, if the subject is a Pacific white shrimp or the like, and its body length is approximately 15 cm to 25 cm, then approximately 100 μL to 2,000 μL, preferably 200 μL to 1,000 μL of composition, can be injected. For example, if the body length of the subject exceeds 25 cm, an absolute amount of the composition can be injected in the range of approximately 200 μL to 5,000 μL, preferably 500 μL to 2,000 μL, but is not limited to this.

[0081] The amount of target nucleotide chain injected into the ovary in a single injection can be determined according to the body length of the subject, within an amount that can be injected and achieve the objective. For example, the amount of target nucleotide chain injected is approximately 10 ng to 10 mg in absolute terms. For example, if the subject is an amphipod or similar organism with a body length of approximately 2 mm to 15 mm, an absolute amount of approximately 10 ng to 500 ng, preferably 20 ng to 100 ng, of the target nucleotide chain can be injected. For example, if the subject is a mystery crayfish or similar organism with a body length of approximately 5 cm to 15 cm, an absolute amount of approximately 5 μg to 100 μg, preferably 10 μg to 50 μg, of the composition can be injected. For example, if the subject is a Pacific white shrimp or similar organism with a body length of approximately 15 cm to 25 cm, an absolute amount of approximately 100 μg to 1 mg, preferably 200 μg to 900 μg, of the composition can be injected. For example, if the body length of the subject exceeds 25 cm, an absolute amount of the composition can be injected, preferably about 1 mg to 10 mg, more preferably about 1 mg to 9 mg.

[0082] The amount of target peptide chain injected into the ovary in a single injection can be determined according to the body length of the subject, within an amount that can be injected and achieve the objective. For example, the amount of target peptide chain injected is approximately 30 ng to 10 mg in absolute terms. For example, if the subject is an amphipod or similar organism with a body length of approximately 2 mm to 15 mm, an absolute amount of approximately 30 ng to 1,500 ng, preferably 100 ng to 1,000 ng of the target peptide chain, can be injected. For example, if the subject is a mystery crayfish or similar organism with a body length of approximately 5 cm to 15 cm, an absolute amount of approximately 10 μg to 1,000 μg, preferably 30 μg to 700 μg of the composition, can be injected. For example, if the subject is a Pacific white shrimp or the like, and its body length is about 15 cm to 25 cm, then an absolute amount of the composition of about 50 μg to 1,000 μg, preferably about 100 μg to 900 μg, can be injected. For example, if the body length of the subject exceeds 25 cm, then an absolute amount of the composition of about 100 μg to 10 mg, preferably about 200 μg to 5 mg, can be injected, but is not limited to this.

[0083] It is preferable to expose the subject to cold water or ice water, or to apply a fish or crustacean anesthetic, etc., to immobilize it before injection.

[0084] Furthermore, to promote ovarian maturation, ovarian maturation promoters such as serotonin may be administered from about one year to the day before the composition is injected. Methods for administering ovarian maturation promoters are well known; for example, the literature Kulkarni et al., Invertebrate Reproduction & Development, 1992, reports examples using crayfish. The ovarian maturation promoter can be administered, for example, by injecting an absolute amount of 0.001 mg to 5 mg into the peritoneal cavity of the subject. The dosage of the ovarian maturation promoter can be adjusted according to the body length of the subject. In addition, the ovarian maturation promoter may be administered multiple times.

[0085] Immediately after injection, the subjects can be returned to their rearing environment for rearing. Preferably, to prevent the injected solution from leaking into the rearing water, they may be reared overnight on a tissue or similar material moistened with rearing water, and then returned to their rearing environment the next day. The rearing environment can be selected according to the species of the subject.

[0086] One embodiment relates to a method for inducing egg-laying in a subject after injection and collecting the eggs.

[0087] The term "egg" as used above refers to eggs in a broad sense, encompassing all stages of egg development from a biological perspective, including unfertilized and post-fertilized states, as well as all pre-embryonic stages such as oocytes present in the ovary.

[0088] After injection, the subjects can be reared for at least one day and then mate with a male or induce parthenogenesis to produce eggs. The eggs laid by the subjects, or the individuals hatched from the eggs, can be collected according to conventional methods.

[0089] By the injection method described above, eggs that have achieved the objective, or eggs that are in line with the objective, or individuals that have hatched from eggs, can be collected.

[0090] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited to the examples.

[0091] 1. Materials and Methods (1) The species of organism used and the reared amphipod Hyale barbicornis were collected from the coast of Shio-Ashiya, Hyogo Prefecture. Hyale barbicornis is a close relative of Parhyale hawaiensis (P. hawaiensis). For rearing, 3% artificial seawater maintained at a water temperature of approximately 23°C ± 2°C or approximately 23°C ± 3°C was used. The light-dark cycle was set to 14 hours:10 hours (light period: dark period). Salted wakame seaweed and abalone Ace (manufactured by Nippon Nosan Kogyo) were given as food. Crossbreeding was induced by rearing one male and one female individual in the same cup. Amphipods are suitable as model organisms because they are relatively easy to rear and maintain, have a short period from hatching to sexual maturity, and their eggs hatch in about 10 days.

[0092] Mystery crayfish (Procambarus virginalis) were kept in a laboratory setting. Water was maintained at a temperature of 23°C ± 2°C for rearing. The light-dark cycle was 14 hours:10 hours (light:dark). They were fed Vital Prawn Growth Formula (Higashimaru) every two to three days.

[0093] Female Litopenaeus vannamei shrimp, approximately 20 cm in length and mature enough to have their ovaries visible through their body surface, were used for rearing. Natural seawater maintained at a temperature of 27°C ± 2°C was used. No adjustments were made to the light-dark cycle, and the shrimp were reared under natural light. They were fed Vital Prawn (Higashimaru) at least twice a day, and for two weeks prior to the egg collection experiment, they were fed live food such as squid, oysters, krill, and polychaetes in five divided portions daily.

[0094] (2) Determination of the AbdB coding sequence and design of sgRNA in P. hawaiensis Although the AbdB gene sequence of P. hawaiensis has not been identified, since P. hawaiensis is a close relative, we referred to the sequence of the Hox gene AbdB of P. hawaiensis (Reference: Martin et al., Current Biology, 2016) and prepared the primer set AbdB_seq_F (SEQ ID NO: 13), AbdB_seq_R (SEQ ID NO: 14), and KOD FX (Toyobo, Cat#KFX-101) shown in the sequence information at the end of this document as primers to amplify the AbdB coding sequence of P. hawaiensis. Using this primer set, amplification was performed by PCR for 40 cycles, with one cycle consisting of 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds. The obtained PCR products were subjected to Sanger sequencing using AbdB_seq_F and AbdB_seq_R as sequencing primers. The sequencing analysis was outsourced to Eurofins Genomics K.K. Subsequently, based on the determined coding sequence information of the AbdB gene derived from *Echinococcus senticosus*, sgRNAs targeting the AbdB gene were designed and synthesized.

[0095] (3) Determination of the white coding sequence and design of sgRNA in Hyalella barbicornis The white gene sequence from Hyalella barbicornis, which corresponds to the white gene (Mackenzie et al., Biochimica et Biophysica Acta, 1999) known to be involved in eye pigment formation in Drosophila, was identified. First, the white gene from Hyalella azteca, a close relative of Hyalella barbicornis, was used as a reference sequence, and the predicted mRNA sequence (accession no. XM_018160141.2) was obtained from the NCBI RefSeq database. Based on this reference sequence, a primer set (White_seq_F (sequence number 27), White_seq_R (sequence number 28)) was designed to amplify the coding region of the white gene from Hyalella barbicornis, and this was used in PCR. KOD FX DNA polymerase (Toyobo Co., Ltd., Cat. No. KFX-101) was used for the PCR reaction, and the amplification reaction was performed for 40 cycles under the conditions of 15 seconds at 98°C, 15 seconds at 58°C, and 15 seconds at 68°C per cycle. The obtained PCR products were sequenced using Sanger sequencing with White_seq_F and White_seq_R as sequencing primers. The sequencing analysis was outsourced to Eurofins Genomics K.K. Subsequently, based on the determined coding sequence information of the white gene derived from *Echinococcus senticosus*, sgRNA targeting the white gene was designed and synthesized.

[0096] (4) Preparation of Cas9-sgRNA RNPs intraovarian injection compositions targeting the AbdB gene of Hyale barbicornis and the MIH gene of the mystery crayfish. The spacer sequences (20 bases excluding the PAM sequence) of the sgRNAs used are shown in the sequence information as AbdB_spacer (Hyale barbicornis) (SEQ ID NO: 7) and MIH_spacer (Procambarus virginalis) (SEQ ID NO: 8). sgRNA synthesis was performed using the CUGA® 7 gRNA Synthesis Kit (Nippon Gene, Model No. 314-08691), following the manufacturer's recommended protocol. AbdB_sgRNA_oligo_a (SEQ ID NO: 9), MIH_sgRNA_oligo_a (SEQ ID NO: 10), oligo_b (SEQ ID NO: 11), and oligo_c (SEQ ID NO: 12), as shown in the sequence information, were used as primers for synthesis. Furthermore, the sgRNA scaffold sequence is shown as sgRNA_scaffold (sequence number 48) in the sequence information shown in Table 3.

[0097] Alt-R® SpCas9 Nuclease V3 (Integrated DNA Technologies, Cat#1081059) was used as the Cas9 protein. In genome editing of *Echinococcus mutabilis*, Cas9 protein and sgRNA were mixed to final concentrations of 750 ng / μL and 150 ng / μL, respectively, to form Cas9 RNPs. In genome editing of *Mystery Crayfish*, Cas9 protein and sgRNA were mixed to final concentrations of 3.3 μg / μL and 1.3 μg / μL, respectively, to form Cas9 RNPs. Acid Blue (Tokyo Chemical Industries, B0790) was added to the RNP solution containing the prepared RNPs to make them easier to identify during injection. Finally, 300 mM KCl was added to adjust the concentration of Cas9 RNPs (final concentration 30–100 mM) to prepare the ovarian injection composition.

[0098] (5) Preparation of Cas9-sgRNA RNPs intraovarian injection composition targeting the white gene of Hyale barbicornis The spacer sequence (20 bases excluding the PAM sequence) of the white sgRNA used is shown in the sequence information as white_spacer_1 (Hyale barbicornis) (SEQ ID NO: 32). The sgRNA was synthesized and purified using the CUGA® 7 gRNA Synthesis Kit (Nippon Gene, model number 314-08691) according to the manufacturer's recommended protocol. White_sgRNA_1_oligo_a (SEQ ID NO: 29), oligo_b (SEQ ID NO: 11), and oligo_c (SEQ ID NO: 12), as shown in the sequence information, were used as primers for synthesis. The sgRNA scaffold sequence is also shown in the sequence information as sgRNA_scaffold (SEQ ID NO: 48).

[0099] Alt-R® SpCas9 Nuclease V3 (Integrated DNA Technologies, Cat#1081059) was used as the Cas9 protein. Cas9 protein and sgRNA were mixed to final concentrations of 750 ng / μL and 150 ng / μL, respectively, to form Cas9 RNPs. Acid Blue (Tokyo Chemical Industries, Ltd., Catalog No. B0790) was added to the RNP solution containing the prepared RNPs to make it easier to identify during injection. Finally, 300 mM KCl was added to adjust the concentration of Cas9 RNPs (final concentration 30–100 mM) to prepare the ovarian injection composition.

[0100] (6) Preparation of Cas12a-sgRNA RNPs targeting the white gene of *Echinococcus senticosus* Alt-R® LbCas12a (Cpf1)Ultra (Integrated DNA Technologies, Cat#10001272) was used as the Cas12a protein to be injected into *Echinococcus senticosus*. Nuclease-resistant modified Alt-R LbCas12a crRNA was used as the crRNA. The spacer sequence of the crRNA targeting white is shown as white_crRNA_spacer (SEQ ID NO: 35) at the end of this document. In genome editing of *Echinococcus senticosus*, the Cas12a protein and crRNA were mixed to a final concentration of 2 μg / μL each to form Cas12a RNPs. Acid Blue (Tokyo Chemical Industries, B0790) was added to the RNP solution containing the prepared RNPs to make them easier to identify during injection. Finally, IDT Duplex Buffer (100 mM Potassium Acetate, 30 mM HEPES) was added to adjust the concentration of Cas12a RNP, and the ovarian injection composition was prepared.

[0101] (7) Preparation of Base Editor ABE8e-sgRNA RNPs targeting white As the ABE8e protein to be injected into *Echinococcus senticosus*, ABE8e (Cat. No. RC00010) from GenScript was used. As sgRNAs, in addition to White_sgRNA_1_oligo_a (SEQ ID NO: 29), which targets the coding sequence of the white gene, sgRNAs synthesized from White_sgRNA_2_oligo_a (SEQ ID NO: 30) and White_sgRNA_3_oligo_a (SEQ ID NO: 31) were used. In addition to each oligo_a, oligo_b (SEQ ID NO: 11) and oligo_c (SEQ ID NO: 12) were used to synthesize sgRNAs. The spacer sequences (20 bases excluding the PAM sequence) of the sgRNAs used are shown in the sequence information as white_spacer_1 (SEQ ID NO: 32), white_spacer_2 (SEQ ID NO: 33), and white_spacer_3 (SEQ ID NO: 34). The sgRNA scaffold sequence is also shown in the sequence information as sgRNA_scaffold (SEQ ID NO: 48). ABE8e protein was mixed at a final concentration of 860 ng / μL, and the three types of sgRNA were mixed at 150 ng / μL each to form ABE8e-sgRNA RNPs. Acid Blue (Tokyo Chemical Industries, Ltd., product code B0790) was added to the solution containing the prepared RNPs to improve visibility during the injection procedure. Furthermore, IDT Duplex Buffer was added to adjust the concentration of ABE8e-RNPs, creating the intraovarian injection composition.

[0102] (8) Preparation of dsRNA targeting white mRNA of *Echinococcus mutabilis* For use as dsRNA for RNA interference (RNAi) to be injected into *Echinococcus mutabilis*, a primer set for amplifying the coding region of the white gene derived from *Echinococcus mutabilis* (White_seq_F (SEQ ID NO: 27), White_seq_R (SEQ ID NO: 28)) and a primer set with T7 sequences attached to each (T7_White_seq_F (SEQ ID NO: 36), T7_White_seq_R (SEQ ID NO: 37)) were used. KOD FX DNA polymerase (Toyobo, Cat. No. KFX-101) was used for the PCR reaction. The sets of (White_seq_F (SEQ ID NO: 27) and T7_White_seq_R (SEQ ID NO: 37), and T7_White_seq_F (SEQ ID NO: 36) and White_seq_R (SEQ ID NO: 28) were used, and 40 amplification cycles were performed under conditions of 15 seconds at 94°C, 15 seconds at 58°C, and 15 seconds at 68°C, respectively. After purifying the PCR products using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250), double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX Express RNAi System (Promega; P1700) according to the manufacturer's recommended protocol. For ovarian injection of dsRNA targeting *Echinococcus senticosus* white mRNA, dsRNA was used. The solution was adjusted to a final concentration of 2.5 μg / μL, and Acid Blue (manufactured by Tokyo Chemical Industry Co., Ltd., B0790) was added to color it for easier identification during injection, thus creating the ovarian injection composition.

[0103] (9) Preparation of a SYNCAS-like intraovarian injection composition containing Cas9-sgRNA RNPs targeting the mystery crayfish MIH gene The spacer sequence of the sgRNA used (20 bases excluding the PAM sequence) is shown in the sequence information as MIH_spacer (Procambarus virginalis) (SEQ ID NO: 8). The scaffold sequence of the sgRNA is also shown in the sequence information as sgRNA_scaffold (SEQ ID NO: 48). As a SYNCAS-like intraovarian injection composition containing Cas9-sgRNA RNPs targeting the mystery crayfish MIH gene (sometimes simply referred to as a SYNCAS-like intraovarian injection composition), Cas9 protein and sgRNA were first mixed to final concentrations of 3.3 μg / μL and 1.3 μg / μL, respectively, to form Cas9 RNPs. Acid Blue (manufactured by Tokyo Chemical Industry Co., Ltd., product code B0790) was added to the solution containing the prepared RNPs to make it easier to identify during injection. The concentration of Cas9 RNP was adjusted by adding 300 mM KCl (final concentration 30–100 mM). Furthermore, BAPC (BAPtofect-25 0.5 mg Kit; PHOREUS BIOTECH, B25-005) and Saponin (Tokyo Chemical Industries, Ltd.; S0019) were mixed to final concentrations of 500 ng / μL and 300 ng / μL, respectively, and allowed to stand at room temperature for 5 minutes. Finally, calcium chloride was mixed to a final concentration of 500 ng / μL to prepare a SYNCAS-like intraovarian injection composition.

[0104] In this invention, a composition used in a technique called SYNCAS (Non-Patent Literature 5), a type of genome editing method for insects, was used for intraovarian injection. SYNCAS is a method that enables efficient genome editing in the next generation without embryonic manipulation by administering a CRISPR-Cas9 complex containing saponin and branched-chain amphiphilic peptide capsules (BAPCs) into the coelom of adult female insects of the parental generation. However, there have been no reports of intraovarian injection in aquatic crustaceans.

[0105] (10) Preparation of peritoneal injection solution for inducing ovarian maturation and injection: The serotonin solution (2 mg / mL) used for peritoneal injection of mystery crayfish was prepared by dissolving 4 mg of serotonin hydrochloride (Sigma-Aldrich; Cat#H9523) in 2 mL of crayfish saline (modified Van's solution: NaCl 12.0 g / L, KCl 0.4 g / L, CaCl2 (anhydrous) 1.5 g / L, MgCl2・6H2O 0.5 g / L). A 27G needle and a 1 mL syringe were used for injection. The prepared composition was injected into the interpleural space between the second and third abdominal appendages of mystery crayfish at a rate of 0.25 mL / individual, every two days, followed by intraovarian injection one day later, or by one injection between the abdominal appendages followed by intraovarian injection on the same day.

[0106] (11) Intraovarian injection of Cas9-sgRNA RNPs targeting the AbdB gene of *Echinococcus septempunctatus* From among *Echinococcus septempunctatus* reared in captivity, those in which pre-mating guarding (male embracing female) was observed were isolated in separate containers, and female subjects in which mating and egg-laying were confirmed were used in the experiment. The egg-laying day was designated as Day 0, and from Day 3 to Day 10, three different subjects were incapacitated each day using FA100 (Bussan Animal Health Co., Ltd.), and then Cas9-sgRNA RNPs targeting the AbdB gene were injected into the ovaries by puncturing the exoskeleton on the dorsal side of the thorax (Figure 1). The injection was performed under a microscope using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. Each injection volume was approximately 0.3 μL (final KCl concentration: 50–300 mM), and one injection was performed per subject. Whether or not Cas9-sgRNA RNPs targeting the AbdB gene were injected into the ovary was confirmed by the localization of the dye.

[0107] To prevent the injected solution from leaking into the rearing water, the subjects were kept overnight on tissue paper moistened with rearing water, and the following day they were returned to the tank and mated again with male individuals. Eggs were then collected 2-3 days after the subjects that received the injection laid eggs, and mutation analysis was performed. The number of eggs laid in a single spawning was 3-20.

[0108] (12) Intraovarian injection of Cas9-sgRNA RNPs targeting the white gene of *Scutellaria japonica* From among *Scutellaria japonica* reared in captivity, those in which pre-mating guarding (male embracing female) was observed were isolated in a separate container, and female subjects in which mating and egg-laying were confirmed were used in the experiment. The egg-laying day was designated as Day 0, and individuals from Day 7 to Day 10 were collected. After immobilizing them using FA100 (Bussan Animal Health Co., Ltd.), Cas9-sgRNA RNPs targeting the white gene were injected into the ovaries by puncturing the exoskeleton on the dorsal side of the thorax. The injection was performed under a microscope using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. The injection volume per injection was approximately 0.1–0.3 μL (final KCl concentration: 50–300 mM), and one injection was performed per subject. Whether or not Cas9-sgRNA RNPs targeting the white gene were injected into the ovary was confirmed by the localization of the pigment. Subjects that received the injection were immediately returned to the rearing water and mated again with males on the same day. Eggs were then collected 1-2 days after the subjects that received the injection laid eggs, and genome extraction and mutation analysis were performed. The number of eggs laid in a single spawning was 3-7.

[0109] (13) Intraovarian injection of Cas12a-crRNA RNPs targeting the white gene of *Echinococcus mutabilis* From among *Echinococcus mutabilis* reared in captivity, those that showed pre-mating guarding were isolated in a separate container, and female subjects in which mating and egg-laying were confirmed were used in the experiment. The egg-laying day was designated as Day 0, and individuals from Day 7 to Day 10 were collected. After immobilizing them using FA100 (Bussan Animal Health Co., Ltd.), Cas12a-crRNA RNPs targeting the white gene were injected into the ovaries by puncturing the exoskeleton on the dorsal side of the thorax. The injection was performed under a microscope using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. The injection volume was approximately 0.1 to 0.3 μL, and one injection was performed per subject. Whether or not the Cas12a-crRNA RNPs targeting the white gene were injected into the ovaries was confirmed by the localization of the pigment. The subjects that received the injection were immediately returned to the rearing water and mated again with male individuals on the same day. Eggs were then collected 1-2 days after the injected subjects laid eggs, and genome extraction and mutation analysis were performed. The number of eggs laid in each spawning was 3-7.

[0110] (14) From a population of *Scutellaria* species reared with an intraovarian injection of a Base Editor targeting the *Scutellaria* white gene, individuals exhibiting pre-mating guard were isolated in separate containers, and female individuals in which mating and egg-laying were confirmed were used as test subjects for the experiment. For these test subjects, after suppressing their movement using FA100 (Bussan Animal Health Co., Ltd.), the exoskeleton on the dorsal side of the thorax was punctured, and ABE8e-sgRNA RNPs targeting the white gene were injected into the ovaries. The injection procedure was performed under a microscope using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. The injection volume per injection was approximately 0.1 to 0.3 μL, and one injection was performed per test subject. Whether or not the ABE8e-sgRNA RNPs targeting the white gene were injected into the ovaries was confirmed by the localization of a dye administered simultaneously. The subjects that received the injection were immediately returned to the rearing water and mated again with male individuals on the same day. Eggs were then collected 1-2 days after the injected subjects laid eggs, and genome extraction and mutation analysis were performed. The number of eggs laid in each spawning was 3-7.

[0111] (15) Intraovarian injection of dsRNA targeting white mRNA of *Scutellaria japonica* From a population of *Scutellaria japonica* reared in captivity, individuals exhibiting pre-mating guarding were isolated in separate containers, and female individuals in which mating and egg-laying were confirmed were used as test subjects for the experiment. For these test subjects, after suppressing their movement using FA100 (Bussan Animal Health Co., Ltd.), the exoskeleton on the dorsal side of the thorax was punctured, and dsRNA targeting white mRNA was injected into the ovaries. The injection procedure was performed under a microscope using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. The injection volume per injection was approximately 0.1 to 0.3 μL, and each test subject received only one injection. Individuals serving as mock controls were similarly injected with Acid Blue diluted with water to the same concentration (control solution). The injected test subjects were immediately returned to the rearing water and mated again with male individuals on the same day. Subsequently, eggs were collected six days after the subjects who received the injection laid eggs (Day 6).

[0112] (16) Intraovarian injection of a Cas9-sgRNA RNPs composition targeting MIH into mystery crayfish Mystery crayfish with a total length of approximately 4 cm were used for the intraovarian injection experiment. The day before the experiment, a portion of the left and right cephalothorax of the subjects was removed to make the ovaries visible from the outside. Immediately before injection, the subjects were exposed to ice water to stop their movement. The injection was performed using a microinjector (IM-12; Narishige Group) equipped with a glass capillary needle. Approximately 10 μL of the intraovarian injection solution was injected into the ovary from either the left or right side of the subject. The injected subjects were kept alive, and all eggs laid were subjected to mutation introduction analysis.

[0113] (17) Intraovarian injection of SYNCAS-like composition targeting MIH into mystery crayfish In the intraovarian injection experiment of a SYNCAS-like composition containing Cas9-sgRNA RNPs targeting MIH in mystery crayfish, individuals approximately 4 cm in total length were used. First, serotonin solution was injected at a rate of 0.25 mL / individual between the second and third pleopods of the mystery crayfish, and then the composition was injected into the ovaries on the same day. Approximately 5 μL of intraovarian injection composition solution was injected into the ovaries from either the left or right side of the individual. The injected subjects were kept alive, and all eggs laid were subjected to mutation analysis.

[0114] (18) Intraovarian injection of fluorescent protein into Pacific white shrimp A composition of red fluorescent protein was injected into the ovaries of Pacific white shrimp. A 25G needle and a 1 mL syringe were used for the intraovarian injection of the composition. The amount injected per injection was approximately 500 μL. After immobilizing the injected Pacific white shrimp using FA100 (Bussan Animal Health Co., Ltd.), the needle containing the composition was inserted between the dorsal segments, and the RFP composition was injected into the ovaries. The injected subjects were kept in a tank with males for several hours, then moved to individual tanks and kept until the following morning. Red fluorescence was confirmed in the unfertilized eggs laid using a stereofluorescence microscope.

[0115] (19) Recombinant RFP protein (BIOSS; bs-33011P) diluted to a final concentration of 100 μg / mL was used as the red fluorescent protein injected into the Pacific white shrimp.

[0116] (20) Genomic DNA Extraction of Eggs of the Eggplant Species: Genomic DNA was extracted from eggs laid by subjects that underwent injection of genomic DNA into eggs using the following method. 50 μL of lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 8.0) was placed in a 0.2 mL tube, and eggs were placed one by one into the tube. The eggs were then crushed, and the contents of the eggs were suspended in the lysis buffer. These 0.2 mL tubes were incubated at 95°C for 10 minutes, and then 50 μL of reaction stop buffer (40 mM Tris-HCl, pH 8.0) was added and mixed well. This was used as a template for genomic PCR.

[0117] (21) Mutation analysis of test eggs of *Echinococcus senticosus* injected with AbdB-targeted Cas9-sgRNA-RNPs. For heteroduplex mobility assay (HMA) to detect band shifts caused by mutations, a microchip electrophoresis system (Shimadzu Corporation; MCE-202 or Agilent; M5310AA) was used. For PCR amplification in HMA, KOD FX (Toyobo: Cat#KFX-101) and HMA primer sets AbdB_HMA_F (SEQ ID NO: 15) and AbdB_HMA_R (SEQ ID NO: 16) which amplify the target region of the AbdB gene were used. The reaction was performed for 40 cycles, with one cycle consisting of 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds.

[0118] For subcloning sequencing analysis, after agarose gel electrophoresis of the PCR product, the gel fragment from which the target product was excised was purified using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250), and then TArget Clone TMPCR amplification fragments were subcloned using -Plus- (Toyobo; TAK-201). Sanger sequencing was performed on this plasmid using M13_F (SEQ ID NO: 5), and the resulting sequences were aligned with the genome sequence of a wild-type individual. Sanger sequencing was outsourced to Eurofins.

[0119] Amplicon sequencing analysis was performed as follows: The region where mutations were to be confirmed was PCR-amplified using KOD FX (Toyobo; Cat#KFX-101). Amplification was performed in two steps: 1st PCR and 2nd PCR. For the first PCR, a pair of primers, AbdB_NGS_F1 (SEQ ID NO: 17), AbdB_NGS_F2 (SEQ ID NO: 18), AbdB_NGS_F3 (SEQ ID NO: 19), AbdB_NGS_F4 (SEQ ID NO: 20), AbdB_NGS_F5 (SEQ ID NO: 21), AbdB_NGS_F6 (SEQ ID NO: 22), AbdB_NGS_F7 (SEQ ID NO: 23), AbdB_NGS_F8 (SEQ ID NO: 24), AbdB_NGS_F9 (SEQ ID NO: 25), and AbdB_NGS_R (SEQ ID NO: 26), were used, each containing an adapter sequence and a barcode sequence for separating the mixed sample, attached to the target region sequence. The PCR was performed for 35 cycles, with each cycle consisting of 98°C for 15 seconds, 58°C for 15 seconds, and 68°C for 15 seconds. The second PCR was performed for 17 cycles, with each cycle consisting of 98°C for 15 seconds followed by 68°C for 60 seconds, using a sequencing adapter and primers (provided by the National Institute of Biotechnology) with sample identification indices. The PCR products were mixed according to their barcodes and subjected to amplicon sequencing analysis. The resulting sequences were analyzed using CRISPResso (Pinello et al., Nature Biotechnology, 2016). The amplicon sequencing analysis was commissioned to the National Institute of Biotechnology.

[0120] (22) Mutation analysis of test eggs of *Echinococcus senticosus* injected with Cas9-sgRNA RNPs targeting the white gene. For heteroduplex mobility assay (HMA) to detect band shifts caused by mutations, a microchip electrophoresis system (Shimadzu Corporation; MCE-202 or Agilent; M5310AA) was used. For PCR amplification in HMA to amplify the target region of the white gene, KOD FX (Toyobo: Cat#KFX-101) and HMA primers (sequence information: White_seq_F (SEQ ID NO: 27), White_seq_R (SEQ ID NO: 28)) were used. The reaction consisted of 40 cycles, with one cycle being 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds.

[0121] For subcloning sequencing analysis, after agarose gel electrophoresis of the PCR product, the gel fragment from which the target product was excised was purified using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250), and then TArget Clone TM PCR amplification fragments were subcloned using -Plus- (Toyobo; TAK-201). Sanger sequencing was performed using the primer White_seq_R (SEQ ID NO: 28), and the resulting sequences were aligned with the genome sequence of the wild-type individual. Sanger sequencing was outsourced to Eurofins Genomics K.K.

[0122] (23) Phenotypic confirmation and mutation analysis of eggs of *Echinococcus mutabilis* subjects injected with Cas9-sgRNA RNPs targeting the white gene. Two weeks after hatching, the eye color of juvenile individuals hatched from eggs laid by individuals that had been administered Cas9-sgRNA RNPs targeting the white gene intraovum was observed using a stereomicroscope. Genomic DNA was extracted from these juvenile individuals, and heterozygous double-strand mobility analysis (HMA) was performed on the region surrounding the target sequence of the sgRNA targeting the white gene. Subsequently, nucleotide sequences were analyzed by Sanger sequencing. Mutation efficiency was estimated by comparing the waveform of the Sanger sequence with that of a control wild-type sequence and decomposing the mixed signal. Sanger sequencing was commissioned to Eurofins Genomics K.K.

[0123] (24) Mutation analysis of test eggs of *Echinococcus mutabilis* injected with Cas12a-crRNA RNPs targeting the white gene. For Sanger sequencing analysis to detect waveform disturbances caused by mutations, KOD FX (Toyobo: Cat#KFX-101) and HMA primers that amplify the target region of the white gene (sequence information: White_seq_F (SEQ ID NO: 27), White_seq_R (SEQ ID NO: 28)) were used for PCR amplification. The reaction consisted of 40 cycles of 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds. The nucleotide sequence was analyzed using Sanger sequencing with the PCR product. Mutation efficiency was estimated by comparing the Sanger sequence waveform with the control wild-type sequence and decomposing the mixed signal. Sanger sequencing was commissioned to Eurofins Genomics K.K.

[0124] (25) Mutation analysis of test eggs of *Echinococcus mutabilis* injected with ABE8e-sgRNA RNPs targeting the white gene. For detecting the waveform of the Sanger sequence resulting from the base substitution mutation of white_spacer_1 (SEQ ID NO: 32), KOD FX (Toyobo: Cat#KFX-101) and HMA primers that amplify the target region of the white gene (sequence information: White_seq_F (SEQ ID NO: 27), White_seq_R (SEQ ID NO: 28)) were used for PCR amplification. The reaction was performed for 40 cycles, with one cycle consisting of 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds. Sanger sequencing was commissioned to Eurofins Genomics K.K. To estimate the base substitution efficiency of the target sequence, calculations were performed on the waveform occupancy rate of each base in the target region.

[0125] (26) RNAi analysis targeting the white gene of *Echinops japonica* Eggs laid by individuals injected with dsRNA targeting the white gene and a control solution into the ovaries were collected from Day 3 to Day 6, with the egg-laying day designated as Day 0. Eggs collected on the same day were divided into sets of 5 from the dsRNA-injected individuals and 5 from the control solution-injected individuals, and RNA extraction was performed using NucleoSpin® RNA Plus XS (Takara Bio; 740990.50) according to the manufacturer's recommended protocol. Using the extracted RNA, a reverse transcription reaction was performed using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo; FSQ-301) according to the product protocol to synthesize cDNA. The cDNA concentration was measured using Quant-iT® 1X dsDNA Assay Kits, high sensitivity (HS) (Thermo Fisher Scientific, Q33232), and after dilution adjustment to equalize the concentration among the samples, RT-PCR was performed. For PCR amplification in RT-PCR, KOD FX (Toyobo: Cat#KFX-101) and primer sets RT-White_seq_F (SEQ ID NO: 38) and RT-White_seq_R (SEQ ID NO: 39), which are specific to the white gene, were used. Furthermore, as an endogenous control, the ubiquitin gene region, presumed to be a housekeeping gene in Hyalella azteca, was used. Therefore, the predicted mRNA sequence (accession no. XM_018162205.2) was obtained from the NCBI RefSeq database using the ubiquitin gene from the closely related species Hyalella azteca as the reference sequence. Based on this reference sequence, primer sets RT-ubiquitin_F (SEQ ID NO: 40) and RT-ubiquitin_R (SEQ ID NO: 41) were designed to amplify the coding region of the ubiquitin gene from Hyalella azteca. The PCR reaction was performed for 40 cycles, with one cycle consisting of 98°C for 15 seconds followed by 68°C for 15 seconds.For semi-quantitative measurement of PCR products, a capillary electrophoresis apparatus (Agilent; M5310AA) was used, and the estimated concentration of each peak in the band was utilized. For semi-quantitative RT-PCR analysis, the PCR concentration of the white gene was averaged with the PCR product concentration of the ubiquitin gene, a housekeeping gene. The standard deviation (SD) was calculated using the mean values ​​of each white gene PCR concentration. Furthermore, to examine the difference in mean values ​​between the two groups, Welch's t-test was used, and significance (p-value) was calculated using a two-tailed test.

[0126] (27) Mutation analysis of mystery crayfish test eggs into which the intraovarian injection composition was introduced. Genomic PCR was amplified for 40 cycles, with one cycle consisting of 98°C for 15 seconds - 58°C for 15 seconds - 68°C for 15 seconds, using KOD FX (Toyobo; Cat#KFX-101) and primer sets Pvir_1_F (SEQ ID NO: 1) and Pvir_1_R (SEQ ID NO: 2). Mutations were detected by HMA by electrophoresis of the PCR product and by T7 endonuclease I (T7E1) assay. A microchip electrophoresis system (Shimadzu Corporation; MCE-202) was used for electrophoresis. Samples in which mutations were detected by HMA were further subjected to T7E1 assay and subcloning sequencing analysis.

[0127] The T7E1 assay is performed by GeneArt TM The Genomic Cleavage Detection Kit (Thermo Fisher Scientific, A24372) was used. Two μL of the PCR product used for HMA was treated with T7 endonuclease I, which recognizes and cleaves only DNA containing mismatches, according to the manufacturer's recommended protocol. The treated product was subjected to electrophoresis on an agarose gel to check for nuclease cleavage.

[0128] For subcloning sequencing analysis, genomic PCR was performed using KOD FX (Toyobo; Cat#KFX-101) and primer sets Pvir_2_F (SEQ ID NO: 3) and Pvir_2_R (SEQ ID NO: 4). The amplification reaction consisted of 40 cycles, with each cycle running at 98°C for 15 seconds, followed by 58°C for 15 seconds, and then 68°C for 15 seconds. The PCR product was then electrophoresed on an agarose gel, and the gel portion corresponding to the expected amplification length (795 bp) was excised. After purification using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250), the product was processed using Target Clone. TM Subcloning was performed using -Plus- (Toyobo; TAK-201). Sanger sequencing was performed on this plasmid using M13_F (SEQ ID NO: 5) and M13_R (SEQ ID NO: 6), and the resulting sequences were aligned. Sanger sequencing was outsourced to Eurofins.

[0129] (28) Mutation analysis of mystery crayfish test eggs injected with a SYNCAS-like intraovarian injection composition targeting MIH. Genomic PCR for amplicon sequencing analysis was performed using KOD FX (Toyobo; Cat#KFX-101) and primers (sequence information: Pvir_3_F (SEQ ID NO: 42), Pvir_1_R (SEQ ID NO: 2)) for 35 cycles of amplification, with each cycle consisting of 98°C for 15 seconds - 64°C for 15 seconds - 68°C for 15 seconds. The PCR product was subjected to electrophoresis on an agarose gel, and the gel portion with the expected amplification length (229 bp) was excised and purified using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250). Using this product as a template, an amplification reaction was performed for 20 cycles, with each cycle consisting of 98°C for 15 seconds, 64°C for 15 seconds, and 68°C for 15 seconds, using a primer set to which a barcode for sample identification had been added again (sequence information: Pvir_NGS_F (SEQ ID NO: 43), Pvir_NGS_R1 (SEQ ID NO: 44), or Pvir_NGS_R2 (SEQ ID NO: 45)). Using this product as a template, an amplification reaction was performed for 10 cycles, with each cycle consisting of 98°C for 15 seconds, 60°C for 30 seconds, and 68°C for 30 seconds, using a primer set to which a sequence for next-generation sequencing had been added again (sequence information: NGS_2nd_F (SEQ ID NO: 46), NGS_2nd_R (SEQ ID NO: 47)). This product was purified using NucleoSpin® Gel and PCR Clean-up (Takara Bio; 740609.250). These PCR products were mixed according to their barcodes and subjected to amplicon sequencing analysis. The resulting sequences were analyzed using CRISPResso (Pinello et al., Nature Biotechnology, 2016). The amplicon sequencing of these PCR products was commissioned to the National Institute of Biotechnology.

[0130] 2. Results 2-1. *Echinops japonica* 2-1-1. Injection of Cas9-sgRNA RNPs targeting the AbdB gene into the ovaries of *Echinops japonica* Based on the confirmed AbdB coding sequence, sgRNA targeting AbdB in *Echinops japonica* was synthesized, and RNPs containing this were directly injected into the ovaries from the dorsal side of the *Echinops japonica*. Figure 1 shows images of the injection of Cas9-sgRNA RNPs into the ovaries of *Echinops japonica*. As shown in Figure 1(A), a glass needle was inserted into the ovary visible on the dorsal side in the center of the thorax, and the RNP solution (containing a blue dye) was directly injected into the ovary. In individuals where the injection was successful, it was observed that the dye remained in the ovaries (Figure 1(B)).

[0131] The genomes of all eggs laid by subjects after injection of Cas9-sgRNA RNPs targeting the AbdB gene into the ovary were individually extracted, and HMA was performed using the region surrounding the sgRNA target sequence. HMA is a simple method for confirming the introduction of mutations through genome editing, and it detects mutations by utilizing the band shift of heterodouble-stranded DNA formed by the wild-type allele and the introduced allele. Figure 2 shows some of the analysis results. Table 1 shows a summary of the HMA analysis results. Table 1 shows the date of ovarian injection counted from the last egg-laying date before ovarian injection, the number of eggs laid, the number of HMA+ eggs, and the percentage of HMA+ eggs (number of HMA+ eggs / total number of eggs laid). Changes in DNA band mobility (HMA positive, HMA+) were confirmed in 20 out of 128 individuals (15.6%). Furthermore, eggs from individuals that underwent ovarian injection 8 to 10 days after oviposition prior to injection showed a high rate of HMA+ due to HMA, and in particular, mutation introduction was suggested in 7 out of 13 individuals (53.8%) of those injected on day 8 (Table 1). On the other hand, although there were differences in frequency, the appearance of mutant individuals was suggested over a long period of at least Day 4 to Day 10. This demonstrates that the ovarian injection method of the present invention can introduce Cas9-sgRNA RNPs into ovarian cells regardless of the injection timing.

[0132]

[0133] Next, some of the PCR products identified as HMA+ (No. 20, 24, 25, 30, 62) were subcloned, and their nucleotide sequences were analyzed by Sanger sequencing. Figure 3 shows the alignment results. As a result, deletion mutations ranging in length from 11 bp to 59 bp were confirmed (Figure 3). Multiple types of microhomology sequences (CGGCGGA, GGCGG, GGTGGCGGCG (SEQ ID NO: 84), etc.) were present in the target region, and it is presumed that a repair mechanism mediated by microhomology-mediated end joining (MMEJ) was activated based on these sequences, resulting in the introduction of relatively long deletion mutations.

[0134] Figure 4 shows the results of amplicon sequencing analysis performed on representative HMA+ samples (No. 25, 26, 58, 59, 62). Similar to the results shown in Figure 3, mutations were confirmed. Specifically, the most common mutation in individual No. 62 was an 11 bp deletion, with this mutation pattern accounting for 14.5% of cases, and the overall mutation rate, including various mutation patterns, was 25.6% (Figure 4(A)). Mutations were similarly confirmed in the other PCR products, but most of the mutations were deletion mutations (Figure 4(B)).

[0135] 2-1-2. Injection of Cas9-sgRNA RNPs targeting the white gene into *Echinops spp.* and mutation analysis. After injecting Cas9-sgRNA RNPs targeting the white gene into the ovaries of subjects, the genomes of all eggs laid by the subjects were individually extracted, and HMA was performed using the region surrounding the sgRNA target sequence. Figure 5 shows some of the analysis results. The total number of eggs laid was 33, and the number of HMA+ eggs was 6. As a result, the percentage of HMA+ eggs (number of HMA+ eggs / total number of eggs laid) was 19%.

[0136] Next, subcloning was performed on some of the PCR products identified as HMA+ (No. 8, 9, 11, 14, 32, 33), followed by nucleotide sequence analysis using Sanger sequencing. The alignment results are shown in Figure 6. As a result, multiple deletion mutations ranging in length from 10 bp to 38 bp were identified in the target region.

[0137] 2-1-3. Phenosystem Analysis of White Gene Modification in *Echinops japonica* The eye color of juvenile individuals hatched from eggs obtained from individuals administered Cas9-RNP mixed with white sgRNA into the ovaries was observed under a microscope. As a result, individuals with altered eye color and incomplete structures were identified (Figure 7 shows an example of the results). Next, the genomes of juvenile individuals with incomplete eyes were extracted and HMA was performed. The results showed a band shift (HMA+) in the HMA in individuals with altered eye color (Figure 8). These individuals were subjected to Sanger sequencing, and deletion mutations with lengths ranging from 10 bp to 24 bp were identified in the target region (Figure 9). Some of these deletion mutations (-10 deletion mutations) are presumed to involve a repair mechanism mediated by microhomology-mediated end joining (MMEJ), while some mutations thought to be due to non-homologous end joining (NHEJ), where the use of microhomology sequences is not clear, were also identified.

[0138] 2-1-4. Intraovarian injection of Cas12a targeting the white gene of *Echinococcus mutabilis* and mutation analysis. The genome of eggs laid by subjects after injection of Cas12a-crRNA RNPs targeting the white gene into the ovary was extracted, and PCR was performed to amplify the region surrounding the crRNA target sequence. Subsequently, the nucleotide sequences of the PCR products obtained by Sanger sequencing were analyzed. The alignment results are shown in Figure 10. As a result, multiple deletion mutations with a length of 24 bp to 27 bp were confirmed in the target region of the Cas12a-crRNA targeting the white gene. Thus, deletion mutations caused by intraovarian injection of Cas12a-crRNA RNPs were confirmed.

[0139] 2-1-5. Intraovarian injection and mutation analysis of a base editor (ABE8e) targeting the white gene of *Echinococcus senticosus* Genomic DNA was extracted from eggs laid by subjects who had been injected with ABE8e-sgRNA RNPs targeting the white gene into their ovaries. Next, the target region was amplified by PCR targeting the region surrounding the sgRNA target sequence, and then the nucleotide sequence was analyzed for all samples using Sanger sequencing. As a result, when comparing the waveform of the WT (Whole World) and the waveform of the ABE8e-injected subjects in the target region of the sgRNA targeting the white gene, a mutation in which adenine (A) was partially replaced with guanine (G) was confirmed (Figure 11, ABE8e). Furthermore, by calculating the occupancy rate of these waveforms, it was confirmed that the occupancy rate of G in the waveform of the ABE8e-injected subjects increased from 1% to 51% compared to the WT waveform. From the above, base substitution due to intraovarian injection of ABE8e-sgRNA RNPs was confirmed.

[0140] 2-1-6. RNAi targeting white mRNA in *Echinococcus senticosus* RNA was individually extracted from eggs laid by subjects who had dsRNA and a control solution injected into their ovaries, on Day 6, and RT-PCR of white mRNA was performed. RT-PCR is a highly sensitive gene detection and analysis technique that uses "reverse transcriptase" with RNA as a template to synthesize complementary DNA (cDNA) and amplifies that DNA by PCR, and is a method for semi-quantitatively measuring the mRNA expression level of a subject. As a result, amplification was observed in all ubiquitin regions, but the expression level decreased on Day 6 in eggs from subjects injected with dsRNA targeting white mRNA (Figure 12 (A), (B)). From this, the phenomenon of RNAi was confirmed in the late stages of embryonic development, and it was suggested that dsRNA can be introduced into laid eggs by directly injecting dsRNA into the ovary.

[0141] The results above demonstrate that in adult females of the amphipod Hyale barbicornis, mutations can be introduced into targeted regions of juvenile organisms by directly injecting CRISPR-Cas9-related, CRISPR-Cas12-related, and Base Editor-related genes and proteins into the ovary. Furthermore, it was shown that the amount of target gene transcripts in juvenile organisms can be regulated by directly injecting dsRNA for RNAi into the ovary.

[0142] 2-2. Mystery Crayfish 2-2-1. Injection of an intraovarian injection composition containing Cas9-sgRNA RNPs into the ovaries of mystery crayfish Subsequently, an intraovarian injection composition containing Cas9-sgRNA RNPs was injected into adult mystery crayfish, which are larger than the Japanese dwarf crayfish but capable of oogenesis through parthenogenesis without mating.

[0143] The target for genome editing in mystery crayfish was molt-inhibiting hormone-like (MIH).

[0144] First, to induce ovarian maturation in mystery crayfish, serotonin was administered intraperitoneally to the subjects, following the example of Kulkarni et al., Invertebrate Reproduction & Development, 1992. After serotonin administration, an intraovarian injection composition containing Cas9-sgRNA RNPs, which include sgRNA targeting MIH, was directly injected into the ovaries of the mystery crayfish. Figure 13 shows images of the injection of the intraovarian injection composition into the ovaries of mystery crayfish. As shown in Figure 13(A), a glass needle was inserted into the ovary of a subject with its carapace removed, and the composition (containing a blue dye) was directly injected into the ovary. In individuals where the injection was successful, the dye was observed to remain in the ovary (Figure 13(B)).

[0145] Of the subjects who underwent intraovarian injection of the composition, one subject laid eggs 20 days after injection. The genomes of all 104 eggs from this subject were individually extracted and subjected to HMA. As a result, HMA+ was confirmed in two eggs (2 / 102, 1.96%) (Figure 14).

[0146] Next, to confirm that the HMA+ in these two samples (No. 83, 96) was due to the formation of heterodouble-stranded DNA, a T7E1 assay was performed. The results are shown in Figure 15. In the two eggs in which HMA suggested a change in DNA band mobility, cleavage of the PCR product by T7 endonuclease I was confirmed, confirming the presence of mutations.

[0147] Next, to confirm the mutation patterns of these fragments, the PCR products were subcloned and their nucleotide sequences were analyzed by Sanger sequencing. The results are shown in Figure 16. The mutation pattern of the nucleotide sequences in both fragments was confirmed to be the same seven-nucleotide deletion. Several types of microhomology sequences (e.g., GTGG) were present in the target region, and it was hypothesized that the deletion mutation was introduced by MMEJ repair based on these sequences.

[0148] These results demonstrate that, in mystery crayfish, mutations can be introduced into the target region of the offspring by directly injecting CRISPR-Cas9-related genes and proteins into the ovary.

[0149] 2-2-2. Injection and Mutation Analysis of a SYNCAS-like Intraovarian Injection Composition Containing Cas9-sgRNA RNPs Targeting MIH in Mystery Crayfish One subject of mystery crayfish that underwent intraovarian injection of a SYNCAS-like intraovarian injection composition containing Cas9-sgRNA RNPs targeting MIH laid eggs 13 days after injection. The genomes of all 19 eggs from this subject were individually extracted and subjected to HMA. As a result, HMA+ was confirmed in one egg (No. 18, 1 / 19, 5.26%) (Figure 17).

[0150] 2-2-3. Injection of a SYNCAS-like intraovarian injection composition containing Cas9-sgRNA RNPs targeting MIH in Mystery Crayfish and Mutation Analysis Next, to confirm the mutation pattern of eggs that produced HMA+, the nucleotide sequence of PCR product No. 18 was submitted for amplicon sequencing analysis. The distribution of mutation patterns in the total reads obtained is shown in Figure 18. The mutation pattern was a deletion of 68 nucleotides (Figure 18). One type of microhomology sequence (GTACAC) was present in the target region, and it was presumed that the deletion mutation was introduced by MMEJ repair based on this sequence (Figure 19).

[0151] 2-3. Injection of fluorescent protein into the ovaries of Pacific white shrimp Of the Pacific white shrimp subjects that underwent intraovarian injection of RFP solution, one subject laid eggs without mating from the night of the injection to the following day. Some of these unhatched eggs were collected and observed under a fluorescence microscope. The results are shown in Figure 20. One egg was observed to fluoresce under RFP excitation light. It was presumed that this egg fluoresced because the injected RFP had migrated into the egg. This result indicates that in Pacific white shrimp, proteins can be introduced into spawning individuals by directly injecting the protein into the ovaries.

[0152] The experiments described above demonstrated that the target nucleotide chain and target peptide chain can be delivered to cells in the ovaries of aquatic crustaceans according to the present invention, and that the functions of the target nucleotide chain and target peptide chain can be exercised.

[0153] <Sequence Information>

Claims

1. A method for injecting a composition, comprising injecting the composition into the ovary of an individual belonging to an aquatic crustacean, the composition comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains.

2. The injection method according to claim 1, wherein the target nucleotide chain is used for at least one purpose selected from the group consisting of genome editing, epigenome editing, transcription or translation regulation, enzyme-dependent genetic recombination, and enzyme-independent genetic recombination, and the target peptide chain is used for at least one purpose selected from the group consisting of genome editing, epigenome editing, transcription or translation regulation, and enzyme-dependent genetic recombination.

3. The injection method according to claim 2, wherein the genome editing is performed using a genome editing tool comprising at least one system selected from the following: Clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) system, Obligate mobile element-guided activity (OMEGA) system, Tandem interspaced guide RNA-TIGR-associated protein (TIGR-Tas) system, Bridge RNA system, Zinc Finger (ZF) system, Transcription activator-like effector (TALE) system, and Pentatricopeptide repeat (PPR) system.

4. The injection method according to claim 2, wherein the epigenome editing is performed using an epigenome editing tool comprising at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, bridge RNA system, ZF system, TALE system, and PPR system.

5. The injection method according to claim 2, wherein the transcription or translation regulation is performed by a transcription or translation regulation tool comprising at least one system selected from the CRISPR-Cas system, OMEGA system, TIGR-Tas system, bridge RNA system, ZF system, TALE system, PPR system, and RNAi system.

6. The injection method according to claim 2, wherein the enzyme-dependent genetic recombination is performed by an enzyme-dependent genetic recombination tool comprising at least one system selected from an integrase system, a transposase system, and a recombinase system.

7. The injection method according to claim 3, wherein the CRISPR-Cas system is a CRISPR-Cas9 system.

8. The injection method according to claim 1, wherein the aquatic crustacean is a species selected from the classification of the class Malacostraca (Malacostraca).

9. The aquatic crustaceans selected from the classification of the class Malacostraca mentioned above are: Decapoda (Decapoda), Stomatopoda (Stomachids), Mysida (Mysida), Leptostraca (Pachycephala), Bathynellacea (Eucarida), Anaspidacea (Anaspidea), Spelaeogriphacea (Speleogriphacea), Thermosbaenacea (Thermosbaenacea), Lophogastrida (Lophogastrida), Mictacea (Mictocariida), Bochusacea (Bochusacea), Amphipoda (Amphipoda), Isopoda (Isopoda), Tanaidacea (Tanaids), Cumacea (Bears), and Stygimysida. The injection method according to claim 8, wherein the insect is one selected from the classification of the order (Cave-web order), Euphausiacea (Krill order), and Amphionidacea (Amphionidea order).

10. A method for collecting eggs of aquatic crustaceans, or individuals hatched from such eggs, comprising injecting the composition by the injection method described in claim 1, and then rearing and allowing the individuals injected with the composition to lay eggs.

11. A composition for use in the injection method according to claim 1, comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains.

12. A reagent comprising at least one selected from the group consisting of one or more target nucleotide chains and one or more target peptide chains, for preparing the composition according to claim 11.

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