Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/60—New or modified breeds of invertebrates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01B—MECHANICAL TREATMENT OF NATURAL FIBROUS OR FILAMENTARY MATERIAL TO OBTAIN FIBRES OF FILAMENTS, e.g. FOR SPINNING
  • D01B7/00—Obtaining silk fibres or filaments

Definitions

• the present invention relates to genetically modified lepidopteran insects and methods for producing the same.
• the silk gland of the silkworm (Bombyx mori) has the ability to synthesize a large amount of protein in a short period of time.
• the silk gland of the silkworm is a large organ, so it is easy to extract, and the synthesized protein is stored in the lumen of the silk gland, so it is easy to recover. Therefore, transgenic silkworms that express a target protein in the silk gland are considered to be promising as a mass production system for proteins.
• the silk gland of a silkworm is a pair of organs, one on the left and one on the right, each of which is composed of three regions, the anterior silk gland, the middle silk gland, and the posterior silk gland.
• fibroin H chain (hereinafter often abbreviated as "Fib H")
• Fib L fibroin L chain
• fibrohexamarin also called p25/FHX
• sericin a gelatin-like protein that is a coating component of silk thread.
• Non-Patent Document 1 When using the silkworm silk gland as a protein expression system, a gene expression system that is specifically expressed in the middle or posterior silk gland may be used.
• Non-Patent Document 2 When silkworm silk glands are used as a protein expression system, the GAL4/UAS system (Non-Patent Document 2) and a mass expression method using a system combining the sericin 1 promoter and Hr3 enhancer (Non-Patent Document 3) have been reported as recombinant protein expression systems, but currently the GAL4/UAS system is widely used due to its superiority in terms of protein expression amount.
• the GAL4/UAS system is a gene control system that utilizes a combination of the yeast-derived transcription factor GAL4 and the UAS regulatory sequence.
• a GAL4 line expressing the GAL4 gene under the promoter control of a gene specifically expressed in the middle or posterior silk gland and a UAS line expressing a target protein gene under the control of the UAS regulatory sequence are independently established by genetic recombination using piggyBac, and then the two lines are crossed to construct an expression system that expresses a target protein in the silk gland.
• Non-Patent Document 4 discloses various methods that have been implemented in the past to express recombinant proteins in the silk gland of silkworms. However, the previously reported methods had problems such as low expression levels of fusion proteins and loss of activity of proteins fused with silk proteins. Therefore, there is a need for new methods for stable and large-scale production of target proteins and new methods for efficiently producing silk proteins fused with target proteins.
• the object of the present invention is to provide a new expression system for stable and large-scale production of a target protein in a lepidopteran insect such as a silkworm, and a new method for producing functional silk containing a silk protein fused with a target protein.
• the present inventors came up with the idea of constructing a new expression system for expressing a target gene by directly utilizing the promoter activity and enhancer activity of the endogenous gene, by knocking in a target gene encoding a target protein fused to the C-terminus of an endogenous signal peptide into an exon sequence encoding a signal peptide in a sericin gene, a fibroin gene, or the like.
• the present inventors attempted to knock-in a target gene sequence into an exon sequence by cutting the genome not in the exon sequence into which the target gene sequence is introduced, but in an intron sequence adjacent to the exon sequence.
• the inventors used the above knock-in technology to introduce a fluorescent protein gene into the exon sequence of an endogenous fibroin gene, and produced cocoons containing a fusion protein of a fluorescent protein and full-length fibroin as a constituent fiber.
• a genetically modified lepidopteran insect In an exon sequence encoding a signal peptide or a functional fragment thereof of an endogenous gene, the exon sequence includes a gene sequence encoding a protein of interest or a fragment thereof, The genetically modified lepidopteran insect as described above, wherein the target protein or a fragment thereof is fused to the C-terminus of the signal peptide or a functional fragment thereof.
• a double-stranded circular DNA for introducing a target gene sequence into a genomic cleavage site in an intron sequence in an endogenous gene of a genetically modified lepidopteran insect comprising:
• the endogenous gene is (a) a first spacer sequence adjacent to the 5'-terminus of the genome cleavage site; (b) a second spacer sequence adjacent to the 3'-terminus of the genome cleavage site; (c) a first recognition sequence recognized by a first genome editing enzyme at the 5' end of the first spacer sequence; and (d) a second recognition sequence recognized by a second genome editing enzyme at the 3' end of the second spacer sequence;
• the double-stranded circular DNA comprises the first recognition sequence, the second spacer sequence, the first spacer sequence, a genome homologous sequence, and a target gene sequence in this order;
• the genome-homologous sequence comprises a base sequence homologous to a genome sequence from the second recognition sequence to an exon sequence or a partial sequence thereof located on
• a donor nucleic acid for producing a genetically modified lepidopteran insect by homologous recombination comprising:
• the homologous recombination method includes cleaving a genomic cleavage site within an intron sequence in an endogenous gene with a genome editing enzyme,
• the donor nucleic acid is (a) a first genomic homologous sequence and a second genomic homologous sequence derived from the endogenous gene; and (b) a gene of interest sequence disposed therebetween,
• the first genome homologous sequence is a base sequence homologous to a genome sequence from a base located on the 5'-end side of the genome cleavage position on the genome to an exon sequence or a partial sequence thereof located on the 3'-end side of the intron sequence, and has a mutation in a recognition sequence for the genome editing enzyme;
• the second genomic homologous sequence is a base sequence homologous to a genomic sequence located on the 3'-terminal side of the exon sequence or a partial sequence thereof
• a donor nucleic acid for producing a genetically modified lepidopteran insect by homologous recombination comprising:
• the homologous recombination method includes cleaving a genomic cleavage site within an intron sequence in an endogenous gene with a genome editing enzyme,
• the donor nucleic acid is (a) a first genomic homologous sequence and a second genomic homologous sequence derived from the endogenous gene; and (b) a gene of interest sequence disposed therebetween,
• the first genomic homologous sequence is a nucleotide sequence homologous to a genomic sequence from a nucleotide located on the 5'-terminal side of the intron sequence to an exon sequence or a partial sequence thereof located on the 5'-terminal side of the intron sequence
• the second genome homologous sequence is a base sequence homologous to a genome sequence from a base located on the 3'-end side of the exon sequence or a partial sequence thereof and on the 5'-end side of the
• a method for producing a genetically modified lepidopteran insect comprising the steps of: (6) The double-stranded circular DNA according to (6).
• the method includes an introduction step of introducing the first genome editing enzyme or a nucleic acid encoding the first genome editing enzyme in an expressible state, and the second genome editing enzyme or a nucleic acid encoding the second genome editing enzyme in an expressible state into an egg of a lepidopteran insect by microinjection.
• a method for producing a genetically modified lepidopteran insect comprising the steps of: The method includes an introduction step of introducing the donor nucleic acid according to any one of (7) to (10), and the genome editing enzyme, or a nucleic acid encoding the genome editing enzyme in an expressible state, into an egg of a lepidopteran insect by microinjection.
• the present invention further provides the following.
• a genetically modified lepidopteran insect In an exon sequence encoding a signal peptide or a functional fragment thereof of an endogenous gene, the exon sequence includes a gene sequence encoding a protein of interest or a fragment thereof, The genetically modified lepidopteran insect, wherein the target protein or a fragment thereof is fused between the signal peptide or a functional fragment thereof and a mature protein or a C-terminal fragment thereof obtained by cleaving the signal peptide from a precursor protein encoded by the endogenous gene.
• the target protein is selected from the group consisting of a fluorescent protein, an antibody, an antigenic polypeptide, an enzyme, a cytokine, and an antibacterial polypeptide.
• a donor nucleic acid for producing a genetically modified lepidopteran insect by homologous recombination comprising:
• the homologous recombination method includes cleaving a genomic cleavage site within an intron sequence in an endogenous gene with a genome editing enzyme,
• the donor nucleic acid is (a) a first genomic homologous sequence and a second genomic homologous sequence derived from the endogenous gene; and (b) a gene of interest sequence disposed therebetween,
• the first genome homologous sequence is a base sequence homologous to a genome sequence from a base located on the 5'-end side of the genome cleavage position on the genome to an exon sequence or a partial sequence thereof located on the 3'-end side of the intron sequence, and has a mutation in a recognition sequence for the genome editing enzyme;
• the second genomic homologous sequence is a base sequence homologous to a genomic sequence located on the 3'-terminal side of the exon sequence or a partial sequence thereof
• a donor nucleic acid for producing a genetically modified lepidopteran insect by homologous recombination comprising:
• the homologous recombination method includes cleaving a genomic cleavage site within an intron sequence in an endogenous gene with a genome editing enzyme,
• the donor nucleic acid is (a) a first genomic homologous sequence and a second genomic homologous sequence derived from the endogenous gene; and (b) a gene of interest sequence disposed therebetween,
• the first genomic homologous sequence is a nucleotide sequence homologous to a genomic sequence from a nucleotide located on the 5'-terminal side of the intron sequence to an exon sequence or a partial sequence thereof located on the 5'-terminal side of the intron sequence
• the second genome homologous sequence is a base sequence homologous to a genome sequence from a base located on the 3'-end side of the exon sequence or a partial sequence thereof and on the 5'-end side of the
• a donor nucleic acid described in (6) or (7) comprising a nuclease recognition sequence at the end opposite the target gene sequence of the first genome homologous sequence and/or the second genome homologous sequence.
• the donor nucleic acid described in (8) wherein the nuclease recognition sequence is a recognition sequence for the genome editing enzyme or a restriction enzyme recognition sequence.
• a method for producing a genetically modified lepidopteran insect comprising: The method includes an introduction step of introducing the donor nucleic acid according to (6) or (7), and the genome editing enzyme or a nucleic acid encoding the genome editing enzyme in an expressible state into an egg of a lepidopteran insect by microinjection.
• a fusion protein comprising a protein of interest or a fragment thereof, and fibroin, sericin, or fibrohexamarin.
• a cocoon or silk thread comprising the fusion protein according to (13) or (14).
• Cocoons or silk threads The cocoon or silk thread, wherein one or more silk proteins selected from the group consisting of fibroin H chain, fibroin L chain, sericin 1, sericin 2, sericin 3, and fibrohexamarin contained therein are composed of the fusion protein described in (13) or (14).
• FIG. 1 shows the introduction of a target gene sequence having a stop codon into an endogenous gene.
• the target gene sequence is introduced into an exon sequence encoding a signal peptide in the endogenous gene.
• the target protein encoded by the target gene sequence is fused to the C-terminal side of the signal peptide encoded by the endogenous gene.
• 2A and 2B are diagrams showing the genomic cleavage positions in endogenous genes in the production of knock-in lines. The genomic cleavage positions are designed within the intron sequence located at the 5' end of the exon sequence into which the target gene sequence is introduced.
• FIG. 2A shows the sericin 1 gene
• FIG. 2B shows the fibroin H gene
• FIG. 2C shows the fibroin L gene.
• This is a diagram showing an outline of gene knock-in using the TAL-PITCh method.
• Figure 3A shows the position in endogenous genes from which each sequence used to construct donor nucleic acid used in the TAL-PITCh method originates.
• Figure 3B shows the structure of double-stranded circular DNA used in the TAL-PITCh method.
• Figure 3C shows the structure of knock-in gene obtained by knocking in a target gene sequence using the TAL-PITCh method.
• FIG. 1 shows a method for gene knock-in using homologous recombination. 1 shows the results of observation of silk glands and cocoons in 5th instar larvae of the SP(FibH)-EGFP knock-in line.
• FIG. 7 shows GM-CSF production in a silkworm line in which the GM-CSF gene sequence was knocked into the second exon of the endogenous fibroin H gene.
• Figure 7A shows the knock-in of the GM-CSF gene sequence into the fibroin H gene.
• Figure 7B shows the results of detecting GM-CSF by Western blotting.
• FIG. 8 shows IgG production in silkworm strains in which gene sequences encoding IgG H chain and IgG L chain were knocked into the second exon of the endogenous fibroin H gene and the third exon of the endogenous fibroin L gene, respectively.
• Figure 8A shows the knock-in of the IgG H chain gene sequence into the fibroin H gene.
• Figure 8B shows the knock-in of the IgG L chain gene sequence into the fibroin L gene.
• Figure 8C shows the amount of IgG produced.
• FIG. 1 shows a method for gene knock-in using homologous recombination.
• the results of EGFP protein detection by Western blotting are shown in Fig. 11A. Wild-type line (WT) and EGFP-FibL knock-in line.
• Fig. 11B Wild-type line (WT), SP(Ser1)-EGFP knock-in line, and EGFP-Ser1 knock-in line.
• Photographs of cocoons obtained from EGFP-FibL knock-in lines containing the EGFP-FibL knock-in gene in a heterozygous or homozygous state were taken under normal white light. Photographs of cocoons produced by heterozygotes and homozygotes of the EGFP-FibL knock-in line produced by the method of the present invention, and cocoons produced using the conventional piggyBac system, observed under white light or fluorescent light, are shown.
• the results of knock-in were performed by designing the genome cleavage site within an intron sequence or exon sequence.
• Figure 14A shows the results of performing homologous recombination by cleaving the genome within the intron sequence of the Fib H gene.
• Figure 14B shows the results of performing homologous recombination by cleaving the genome within the exon sequence of the Fib H gene. Less than 2% of individuals developed into adults capable of mating, and more than 95% of the injected generations showed incomplete pupation or showed naked pupae or thin cocoons.
• the first aspect of the present invention is a genetically modified Lepidoptera insect.
• the genetically modified Lepidoptera insect of the present invention contains a target gene sequence in an exon sequence encoding a signal peptide or a functional fragment thereof of an endogenous gene, and expresses a target protein or a fragment thereof fused to the C-terminus of the signal peptide or the functional fragment thereof.
• the genetically modified Lepidoptera insect of this aspect can stably and mass-produce a target protein.
• lepidoptera insects refers to insects belonging to the taxonomic order Lepidoptera, including butterflies and moths.
• Butterflies include insects belonging to the families Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae.
• Moths include insects belonging to the families Saturnidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, Archtiidae, Noctuidae, Pyralidae, and Sphingidae.
• moths include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically, silkworms, mulberry silkworms (Bombyx mandarina), Samia cynthia (including Samia cynthia ricini and hybrids of Samia cynthia and Samia ricini), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma.
• the lepidopteran insects as hosts for the transformant of the present invention are not limited to these, silkworms, which have high industrial applicability, are particularly preferred as hosts.
• the term "genetically modified lepidopteran insect” refers to a genetically modified lepidopteran insect carrying foreign DNA produced using recombinant gene technology, or its progeny.
• the genetically modified lepidopteran insect particularly refers to a genetically modified insect obtained by introducing foreign DNA into a lepidopteran insect egg by microinjection.
• the term “silk gland” refers to a tubular organ that is a modified salivary gland that has the function of producing, accumulating, and secreting liquid silk. Silk glands are usually present in pairs on the left and right sides of insects that can spin silk thread, mainly along the digestive tract of the larvae, and each silk gland is composed of three regions: the anterior, middle, and posterior silk gland.
• the posterior silk gland produces and secretes fibroin, a fiber component of silk thread.
• the middle silk gland also produces and secretes sericin, a coating component, and accumulates in its lumen together with fibroin that has migrated from the posterior silk gland.
• the term "endogenous gene” refers to a gene derived from a lepidopteran insect that is present a priori on the genome of that lepidopteran insect.
• an endogenous gene is, in principle, a gene that encodes a protein having a signal peptide. Therefore, in the present specification, an endogenous gene is, in principle, a gene that encodes a secretory protein or a membrane protein.
• the secretory protein may be, for example, any protein that constitutes silk thread.
• any protein that constitutes silk thread is often referred to as a "silk protein," and a gene that encodes a silk protein is referred to as a "silk gene.”
• Specific examples of endogenous genes in lepidopteran insects include genes that encode fibroin, sericin, and fibrohexamarin.
• exogenous gene or “foreign gene” refers to a foreign gene that is acquired later through artificial manipulation or the like and is not present in the genome of a wild-type lepidopteran insect.
• “Fibroin” is a protein that constitutes the fiber component of silk thread.
• Silkworm fibroin is mainly composed of three proteins, namely, fibroin H chain (Fib H), fibroin L chain (Fib L), and fibrohexamerin. Fibrohexamerin is also called p25/FHX as mentioned above.
• "Sericin" is a protein that covers the outer layer of the fibers formed by fibroin in silk threads.
• sericin is synthesized in the cells of the middle silk gland and secreted into the lumen of the middle silk gland after synthesis. The functions of sericin are known to be adhesion between fibroin fibers and protection of fibroin fibers from external stimuli.
• Silkworms can spin silk immediately after hatching, but the protein components of silk threads spun at each age and silk threads from cocoons are different, and the sericin variant composition contained therein is also different.
• sericin protein variants sericin 1A', sericin 1C, sericin 1D, sericin 2, sericin 3, and sericin 4
• Ser1, Ser2, Ser3, and Ser4 sericin genes
• a "signal peptide” or “secretion signal” refers to an extracellular transport signal required for secreting a protein biosynthesized by gene expression outside the cell. After translation, the signal peptide is cleaved and removed by a signal peptidase before being secreted outside the cell. In addition, in this specification, the signal peptide is often written as "SP", and the endogenous gene name from which the signal peptide is derived is written in parentheses.
• a signal peptide is usually a relatively short peptide sequence of several tens of amino acids or less, and is characterized by a sequence that is rich in hydrophobicity.
• the sequence of a signal peptide can be predicted based on the amino acid sequence of a protein using a prediction tool such as signalP, but a structure prediction provided on a database can also be used.
• the sequence region of the signal peptide can also be determined based on the sequence annotation provided on databases such as KAIKObase and KAIKOcDNA available in the Agricultural and Livestock Products Genome Information Database.
• a "functional fragment" of a signal peptide refers to a fragment consisting of a partial sequence of a signal peptide and retaining extracellular localization signal activity.
• a functional fragment of a signal peptide may retain, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or the same or more, of the extracellular localization signal activity of the full-length signal peptide.
• the amino acid length of the functional fragment is not particularly limited as long as it retains the activity of the full-length signal peptide, and may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the full length.
• full length means the entire amino acid sequence corresponding to a protein that is synthesized and functions in a living body, or the entire base sequence in a gene that codes for it.
• the full length gene corresponds to the start codon to the stop codon
• the full length protein corresponds to a polypeptide or peptide consisting of the amino acid sequence encoded by the full length gene.
• the endogenous signal peptide contained on the N-terminus side is cleaved and removed during the secretion process and is not ultimately included.
• the "full length” does not need to include the signal peptide.
• the full length protein before the signal peptide is cleaved and removed during the secretion process is called a "precursor protein”
• the full length protein after the signal peptide is cleaved and removed is called a "mature protein”.
• “exon” means a region of the base sequence of a gene that remains in a mature transcript. In general, in eukaryotes, a gene is transcribed as a primary transcript, and then an intervening region called an "intron” is removed by splicing, and exons are linked to each other to form a mature transcript.
• exon sequence means a base sequence corresponding to an exon
• intron sequence means a base sequence corresponding to an intron.
• the exon sequence and intron sequence of any gene can be determined by comparing the genome sequence and cDNA sequence of the gene, but the exon/intron structure can also be predicted by obtaining sequence information published on databases such as the National Center for Biotechnology Information (NCBI), or by using genome analysis tools available in the technical field. For example, in the case of silkworm gene information, exon sequences and intron sequences can be searched for using databases such as KAIKObase and KAIKOcDNA available in the Agricultural and Livestock Products Genome Information Database.
• target gene sequence refers to a gene sequence that codes for a target protein or a fragment thereof.
• the target gene sequence may be a gene sequence derived from a genome or a gene sequence consisting of cDNA, and may or may not contain an intron within the target gene sequence.
• the target gene sequence may or may not contain a stop codon in addition to the gene sequence that codes for the target protein or a fragment thereof, and may or may not contain a transcription termination sequence downstream of the stop codon.
• target protein refers to a desired protein encoded by a target gene. The type of target protein is not important.
• the target protein may be either a structural protein or a functional protein.
• structural proteins include collagen, actin, myosin, fibroin and other fibrous proteins, keratin, histone, and the like.
• functional proteins include peptide hormones (insulin, calcitonin, parathormone, growth hormone, and the like), cytokines (granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin (IL), interferon (IFN), tumor necrosis factor ⁇ (TNF- ⁇ ), transforming growth factor ⁇ (TGF- ⁇ ), and the like), transcription factors (including GAL4), antibodies (immunoglobulin, and the like), serum albumin, hemoglobin, enzymes, fluorescent proteins, pigment synthesis proteins, luminescent proteins, and the like.
• GM-CSF granulocyte-macrophage colony-stimulating factor
• EGF epidermal growth factor
• FGF fibroblast growth factor
• IFN interleukin
• the immunoglobulin may be any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).
• the fluorescent protein is not limited and may be, for example, CFP, AmCyan, RFP, DsRed, YFP, GFP (including derivatives such as EGFP and EYFP).
• the pigment synthesis protein may be, for example, a protein involved in the biosynthesis of melanin pigments (including dopamine melanin), ommochrome pigments, or pteridine pigments.
• the luminescent protein may be, for example, aequorin or luciferase.
• the target protein may be either a wild-type protein or a mutant protein.
• a "fragment" of a protein refers to a polypeptide or peptide that includes a partial region of a full-length protein.
• the fragment preferably retains activity.
• the fragment may retain 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or the same or more, of the activity of the full-length protein.
• the amino acid length of the fragment is not particularly limited as long as it retains the activity of the full-length protein, but may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the full length.
• transcription termination sequence refers to a sequence that can terminate the transcription of a gene, and is also called a terminator.
• the type of transcription termination sequence is not particularly limited. It is preferably a terminator derived from the same species as the genetically modified Lepidoptera insect. For example, in the case of an insect such as a silkworm, an hsp70 terminator, an SV40 terminator, etc. can be used.
• the term “plurality” refers to an integer of 2 or more, for example, an integer of 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.
• the "identity" of a base sequence refers to the percentage of matching bases in the entire length of two base sequences when the two base sequences are aligned by inserting appropriate gaps into one or both of the base sequences as necessary to maximize the number of matching bases.
• the term “homologous sequence” refers to a base sequence having an identity of about 60% or more to a reference sequence.
• the identity of a homologous sequence to a reference sequence may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more.
• genomic sequence refers to a base sequence having any of the above-mentioned identities with the genome sequence of a lepidopteran insect as a reference sequence
• genomic sequence refers to a sequence having 100% identity to the corresponding base sequence in the genome.
• amino acid identity refers to the percentage (%) of the number of matching amino acid residues out of the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when the sequences are aligned by inserting appropriate gaps into one or both of the sequences as necessary to maximize the number of matching amino acid residues.
• amino acid substitution refers to substitution within a conservative amino acid group that has similar properties such as charge, side chain, polarity, aromaticity, etc., among the 20 types of amino acids that constitute natural proteins.
• substitutions within the uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) with a low polarity side chain the branched chain amino acid group (Leu, Val, Ile), the neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), the neutral amino acid group (Asn, Gln, Thr, Ser, Tyr, Cys) with a hydrophilic side chain, the acidic amino acid group (Asp, Glu), the basic amino acid group (Arg, Lys, His), and the aromatic amino acid group (Phe, Tyr, Trp) can be mentioned.
• the terms “5'-end” and “3'-end” refer to the 5'-end and 3'-end, respectively, of a transcription product transcribed from an endogenous gene, unless otherwise specified.
• upstream and downstream refer to the upstream and downstream directions of a gene, respectively, based on the transcription direction of an endogenous gene, unless otherwise specified.
• the genetically modified lepidopteran insect of the present invention comprises a target gene sequence encoding a target protein or a fragment thereof in an exon sequence encoding a signal peptide or a functional fragment thereof of an endogenous gene.
• the target gene sequence is contained in the exon sequence such that the target protein or a fragment thereof is fused to the C-terminus of the signal peptide or the functional fragment thereof.
• exon sequence encoding a signal peptide or a functional fragment thereof of an endogenous gene (hereinafter often referred to as "target exon sequence") is not limited to an exon sequence encoding a signal peptide in an endogenous gene.
• a signal peptide is usually encoded by the first exon located most upstream in an mRNA transcribed from the endogenous gene, or by a plurality of exon sequences including the first exon, but the target exon sequence may be any exon sequence.
• the target exon sequence may be the first exon, the second exon, the third exon, or the fourth exon.
• the target exon sequence may be, for example, an exon encoding the C-terminal amino acid residue of a signal peptide, or an exon adjacent to the 5'-terminal side thereof.
• the gene sequence of interest is inserted into the target exon sequence so that the protein of interest or a fragment thereof encoded by the gene sequence of interest is fused to the C-terminus of the signal peptide or a functional fragment thereof of the endogenous gene.
• the gene sequence of interest is linked in frame to the 3'-terminus of the base sequence encoding the signal peptide or a functional fragment thereof in the target exon sequence of the endogenous gene.
• the N-terminus of the protein of interest or a fragment thereof is fused to the C-terminus of the signal peptide or a functional fragment thereof of the endogenous gene, and a fusion gene encoding a fusion polypeptide containing the signal peptide or a functional fragment thereof of the endogenous gene and the protein of interest or a fragment thereof is constructed in the locus of the endogenous gene.
• the signal peptide or a functional fragment thereof and the protein of interest or a fragment thereof may be directly linked, or an amino acid sequence other than the signal peptide encoded by the target exon sequence (for example, an amino acid sequence located at the N-terminus of the mature protein described below) may be inserted between them.
• the endogenous gene encodes a protein that constitutes silk thread.
• the protein that constitutes silk thread is not particularly limited and may be, for example, fibroin, sericin, and/or fibrohexamarin.
• the fibroin may be a fibroin H chain and/or a fibroin L chain.
• the sericin is not particularly limited and may be, for example, sericin 1.
• the precursor protein including the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 1
• the mature protein excluding the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 2.
• the signal peptide of the fibroin H chain consists of the amino acid sequence of positions 1 to 21 in SEQ ID NO: 1.
• the signal peptide is encoded by the first and second exons, and the C-terminal amino acid residue of the signal peptide is encoded by the second exon (FIG. 2B).
• the first exon is at positions 1001 to 1042, the first intron is at positions 1043 to 2013, the second exon includes positions 2014 to at least 17763, and the region encoding the signal peptide in the second exon is at positions 2014 to 2034.
• the precursor protein including the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 4
• the mature protein excluding the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 5.
• the signal peptide of the fibroin L chain consists of the amino acid sequence of positions 1 to 16 in SEQ ID NO: 4.
• the signal peptide is encoded by the first, second, and third exons, and the C-terminal amino acid residue of the signal peptide is encoded by the third exon (FIG. 2C).
• the first exon of the fibroin L-chain gene is at positions 574 to 889
• the first intron is at positions 890 to 966
• the second exon is at positions 967 to 1036
• the second intron is at positions 1037 to 8976
• the third exon is at positions 8977 to 9059
• the region encoding the signal peptide in the third exon is at positions 8977 to 8988.
• a precursor protein including a signal peptide has the amino acid sequence shown in SEQ ID NO: 7, and a mature protein excluding the signal peptide has the amino acid sequence shown in SEQ ID NO: 8.
• the signal peptide has the amino acid sequence of positions 1 to 19 in SEQ ID NO: 7.
• the signal peptide of the above isoform is encoded by the first and second exons, and the C-terminal amino acid residue of the signal peptide is encoded by the second exon ( FIG. 2A ).
• the first exon of the above isoform is at positions 947 to 1039
• the first intron is at positions 1040 to 3051
• the second exon is at positions 3052 to 3082
• the region encoding the signal peptide in the second exon is at positions 3052 to 3069.
• the precursor protein including the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 10
• the mature protein excluding the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 11.
• the signal peptide of fibrohexamarin consists of the amino acid sequence of positions 1 to 17 in SEQ ID NO: 10.
• the signal peptide is encoded by exon 1, and the C-terminal amino acid residue of the signal peptide is encoded by exon 1.
• the first exon of the fibrohexamarin gene is at positions 918 to 1052, the first intron is at positions 1053 to 1536, the second exon is at positions 1537 to 1756, and the region encoding the signal peptide in the first exon is at positions 1001 to 1051.
• the target gene sequence may be introduced into a single endogenous gene or into multiple endogenous genes.
• the genetically modified lepidopteran insect of the present invention may have an exon sequence containing the target gene sequence in a heterozygous or homozygous form.
• the types of target gene sequences introduced into the multiple endogenous genes may be the same or different.
• the multiple endogenous genes into which the gene sequence of interest has been introduced may be genes encoding a fibroin H chain and a fibroin L chain, or genes encoding a fibroin H chain and sericin 1, or genes encoding a fibroin H chain, a fibroin L chain, and sericin 1.
• the gene of interest sequence has a stop codon.
• the targeted exon sequence comprises a transcription termination sequence 3' to the stop codon of the gene of interest sequence.
• the gene sequence of interest does not have a stop codon.
• the protein of interest or a fragment thereof is fused between a signal peptide or a functional fragment thereof and a mature protein (a protein in which the signal peptide has been cleaved and removed from a precursor protein) or a C-terminal fragment thereof (e.g., a fragment in which a part of the N-terminal sequence encoded by the target exon sequence in the mature protein is deleted) encoded by an endogenous gene.
• a mature protein a protein in which the signal peptide has been cleaved and removed from a precursor protein
• a C-terminal fragment thereof e.g., a fragment in which a part of the N-terminal sequence encoded by the target exon sequence in the mature protein is deleted
• the N-terminus of the protein of interest or a fragment thereof is fused to the C-terminus of the signal peptide or a functional fragment thereof of the endogenous gene
• the C-terminus of the protein of interest or a fragment thereof is fused to the N-terminus of the mature protein or a C-terminal fragment thereof.
• a fusion gene encoding a fusion protein comprising the signal peptide or a functional fragment thereof of the endogenous gene, the protein of interest or a fragment thereof, and the mature protein or a C-terminal fragment thereof is constructed at the locus of the endogenous gene.
• the protein of interest is a fluorescent protein, an antibody, an antigen polypeptide, an enzyme, a cytokine, or an antimicrobial polypeptide.
• the protein of interest is an antibody
• the heavy chain gene and the light chain gene constituting the antibody may be introduced into different endogenous genes.
• the genetically modified lepidopteran insect of the present invention is capable of stably and mass-producing a target protein encoded by a target gene introduced into a target exon sequence.
• the GAL4 gene and the UAS regulatory sequence are introduced into random positions on the genome, which can cause large variations in the expression level of the target protein.
• the second aspect of the present invention is a double-stranded circular DNA.
• the double-stranded circular DNA of this aspect allows a target gene sequence to be introduced into a target exon sequence located on the 3'-end side of a genomic cleavage site in an intron sequence in an endogenous gene of a lepidopteran insect.
• the double-stranded circular DNA of this aspect can be used for knocking in a target gene based on, for example, the TAL-PITCh (precise integration into target chromosome) method.
• the term "double-stranded circular DNA” refers to a circular double-stranded DNA molecule that contains at least a target gene sequence for introducing the target gene sequence into an endogenous gene of a lepidopteran insect.
• the double-stranded circular DNA is preferably a vector that can be maintained and/or replicated in bacterial cells such as E. coli, and may contain, for example, a sequence necessary for maintenance or replication in the cell (such as a replication origin and/or a gene encoding an antibiotic resistance protein).
• the double-stranded circular DNA may be, for example, a plasmid vector.
• gene editing refers to a gene targeting technique that uses a DNA repair mechanism associated with double strand break (DSB) caused by a DNA cleavage enzyme to insert a foreign gene (knock-in) or destroy a target gene (knock-out) at any position on the genome.
• Genome editing techniques include the zinc finger nuclease (ZFN) method, the TALEN method, and the CRISPR/Cas method, and any of these methods may be used in this specification.
• TALEN Transcription Activator-Like Effector Nuclease
• TALE Transcription Activator-Like Effector Nuclease
• TALEN is a genome editing technology using an artificial DNA cleavage enzyme in which a TAL effector (TALE) protein derived from a plant pathogenic bacterium, the genus Xanthomonas, is fused with a non-specific endonuclease domain.
• TALEN is a protein consisting of a TALE domain containing repeated DNA binding units as a DNA binding domain and a non-specific endonuclease domain such as the nuclease domain of FokI.
• the nuclease domain having the enzymatic activity of cleaving DNA functions as a dimer, so that the TALEN functions as a dimer consisting of a polypeptide (often referred to herein as "Left-TALEN”) that recognizes the DNA sequence near the upstream side (5' side) of the double strand break (DSB) site in the target base sequence and a polypeptide (often referred to herein as "Right-TALEN”) that recognizes the DNA sequence near the downstream side (3' side) of the DSB site.
• Left-TALEN polypeptide
• DSB double strand break
• the DNA binding unit constituting the TALE domain has mutations in the amino acid residues at positions 12 and 13 from the N-terminus, and each pair of two amino acids can specifically recognize each of the four bases (A: adenine, G: guanine, C: cytosine, T: thymine) that constitute DNA.
• A adenine
• G guanine
• C cytosine
• T thymine
• the number of repeats of the DNA binding unit can vary depending on the base length of the target base sequence.
• the gene knockout method using the TALEN method in lepidopteran insects such as silkworms is a known technique.
• the method described in Takasu Y., et al., 2013, PLoS One 8, e73458 may be referred to.
• the "Zinc Finger Nuclease (ZFN) method” is a genome editing technology that uses an artificial DNA cleavage enzyme consisting of a zinc finger domain as a DNA binding domain and a non-specific endonuclease domain such as the nuclease domain of FokI. Since one zinc finger motif can recognize three bases and bind to a target nucleic acid, by linking multiple zinc finger motifs, it specifically recognizes and binds to three times the number of bases linked.
• CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins
• Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins
• crRNA CRISPR RNA
• the crRNA binds to a Cas protein having nuclease activity directly or via a transactivating RNA (tracrRNA) to form a CRISPR/Cas complex.
• the CRISPR/Cas complex binds to and cleaves a target DNA or RNA sequence having a complementary base sequence via the crRNA.
• genome editing enzyme refers to a protein having the activity of specifically cutting and editing a target site on a genome.
• genome editing proteins include TALEN (transcription activator-like effector nuclease), Cas9 (CRISPR associated protein 9), and ZFN (zinc finger nuclease) that can be used for the above-mentioned genome editing.
• TALEN transcription activator-like effector nuclease
• Cas9 CRISPR associated protein 9
• ZFN zinc finger nuclease
• the genome editing protein is a TALEN
• Left TALEN and Right TALEN can be used as TALEN that can function as a dimer.
• Cas9 a guide RNA such as the above-mentioned crRNA is required to perform genome editing. 2-3.
• the double-stranded circular DNA of this embodiment includes a first recognition sequence, a second spacer sequence, a first spacer sequence, a genome homologous sequence, and a target gene sequence in this order.
• the first recognition sequence, the second spacer sequence, the first spacer sequence, and the genome homologous sequence are derived from the base sequence of a genome region including an endogenous gene that is a target for introducing the target gene sequence.
• the double-stranded circular DNA of this embodiment includes a second recognition sequence on the 5'-end side of the genome homologous sequence.
• a double-stranded cleavage site is generated in an intron sequence on the genome using two genome editing enzymes (hereinafter referred to as the "first genome editing enzyme” and the “second genome editing enzyme") in the target endogenous gene.
• this double-stranded cleavage site is referred to as the "genome cleavage position”.
• the genome cleavage position can be set at any position within the intron sequence of the endogenous gene, but it is preferable to place it at a position other than the functional sequences such as the splice donor sequence and splice acceptor sequence required for splicing, and the branch site.
• each element sequence constituting the double-stranded circular DNA is specified based on the position assumed as the genome cleavage position, and the position where the above-mentioned two genome editing enzymes actually cleave the genome and the double-stranded circular DNA is not limited to that position, but may be a position nearby (for example, any position in the first spacer sequence and/or the second spacer sequence).
• the first recognition sequence, the second spacer sequence, and the first spacer sequence contained in the double-stranded circular DNA of this embodiment, as well as the second recognition sequence located on the 5'-end side in the genome homologous sequence, are derived from a base sequence located near the genome cleavage position in the genome sequence of the endogenous gene.
• the "first recognition sequence” and the “second recognition sequence” are located on the 5'-end side and the 3'-end side, respectively, of the genome cleavage position in the genome sequence of the endogenous gene, and are identical to the base sequences recognized and bound by the first genome editing enzyme and the second genome editing enzyme, respectively.
• the base length of the first recognition sequence and the second recognition sequence varies depending on the type of genome editing enzyme, but is usually 8 to 30 bases long, for example, 10 to 25 bases long, 12 to 20 bases long, or 14 to 18 bases long.
• the "first spacer sequence” is derived from a sequence adjacent to the 5'-end side of the genome cleavage position in the genome sequence of the endogenous gene, and is derived from a base sequence located between the first recognition sequence and the genome cleavage position.
• the "second spacer sequence” is derived from a sequence adjacent to the 3'-end of the genome cleavage position in the genome sequence of the endogenous gene, and is derived from a base sequence located between the genome cleavage position and the above-mentioned second recognition sequence.
• the base length of the first spacer sequence and the second spacer sequence varies depending on the type of genome editing enzyme, but is usually 6 to 30 bases long, for example, 8 to 25 bases long, 10 to 20 bases long, or 12 to 15 bases long.
• the endogenous gene includes the first recognition sequence, the first spacer sequence, the second spacer sequence, and the second recognition sequence in order from the upstream side of the gene, whereas the double-stranded circular DNA of this embodiment is characterized in that the arrangement of the first spacer sequence and the second spacer sequence is reversed.
• the genome-homologous sequence contained in the double-stranded circular DNA of this embodiment includes the above-mentioned second recognition sequence on its 5'-end side, and is composed of a base sequence homologous to the genome sequence from the second recognition sequence to the exon sequence or a partial sequence thereof located on the 3'-end side of the intron sequence including the genome cleavage position.
• the exon sequence or a partial sequence thereof located on the 3'-end side of the intron sequence including the genome cleavage position may be an exon sequence adjacent to the 3'-end side of the intron sequence including the genome cleavage position.
• the exon sequence or a partial sequence thereof included on the 3'-end side of the genome-homologous sequence encodes the C-terminal side of a signal peptide or a functional fragment thereof, and is linked in frame to the target gene sequence located on the 3'-end side thereof.
• the genome-homologous sequence is not particularly limited as long as it includes the second recognition sequence of the genome editing enzyme.
• the base length of the genome homologous sequence may be, for example, 15 to 20,000 bases, 20 to 10,000 bases, 50 to 5,000 bases, 100 to 2,000 bases, or 500 to 1,000 bases.
• the region from the first recognition sequence to the second recognition sequence in the genome homologous sequence consists of the first recognition sequence, the second spacer sequence, the first spacer sequence, and the second recognition sequence in the genome homologous sequence.
• the gene of interest sequence in the double-stranded circular DNA of this aspect contains a stop codon.
• the double-stranded circular DNA of this aspect contains a transcription termination sequence on the 3'-terminal side of the gene of interest sequence.
• the double-stranded circular DNA of this aspect contains a marker gene for identifying an individual into which a gene sequence of interest has been introduced into an endogenous gene.
• the marker gene can be located on the 3'-end side of the transcription termination sequence located on the 3'-end side of the gene sequence of interest.
• the type of genome editing enzyme that recognizes the first recognition sequence and the second recognition sequence contained in the double-stranded circular DNA of this embodiment is not limited, and may be TALEN, ZFN, and/or Cas9.
• the genome editing enzyme that recognizes the first recognition sequence and the second recognition sequence may be TALEN.
• the two genome editing enzymes that recognize the first recognition sequence and the second recognition sequence may be Left-TALEN and Right-TALEN that function as a dimer.
• multiple exons, including the first exon encode a signal peptide. 2-4.
• the double-stranded circular DNA of this embodiment can be used in the TAL-PITCh method.
• the third aspect of the present invention is a donor nucleic acid.
• the donor nucleic acid of this aspect can introduce a target gene sequence into a target exon sequence located on the 3'-end or 5'-end side of a genomic cleavage site in an intron sequence in an endogenous gene of a lepidopteran insect.
• the donor nucleic acid of this aspect can be used, for example, for knocking in a target gene based on homologous recombination.
• the term "donor nucleic acid” refers to a nucleic acid for introducing a target gene sequence into an endogenous gene of a lepidopteran insect.
• the form of the donor nucleic acid is not limited, and it may be, for example, a double-stranded circular DNA such as a plasmid vector or a linear DNA.
• the donor nucleic acid of this embodiment includes a first genome homologous sequence, a second genome homologous sequence, and a target gene sequence disposed therebetween.
• the first genome homologous sequence and the second genome homologous sequence are derived from the base sequence of a genomic region including an endogenous gene to be a target for introducing the target gene sequence.
• a genome editing enzyme that recognizes a sequence in the vicinity of an intron sequence on the genome is used to generate a double-stranded cleavage site in the intron sequence on the genome in the target endogenous gene.
• this double-stranded cleavage site is also referred to as a "genome cleavage position" in this embodiment.
• the genome cleavage position may not be accurately identified.
• each element sequence constituting the donor nucleic acid is specified based on the position assumed as the genome cleavage position, and the position where the genome editing enzyme actually cleaves the genome is not limited to that position, but may be a position nearby it.
• the genome cleavage position can be set at any position within the intron sequence of the endogenous gene, but it is preferable to place it at a position other than the splice donor sequence, splice acceptor sequence, branch site, and other sequences necessary for splicing.
• the sequence recognized by this genome editing enzyme is referred to as a "genome editing enzyme recognition sequence" or simply a "recognition sequence”.
• the specific configurations of the first genome homologous sequence and the second genome homologous sequence contained in the donor nucleic acid of this embodiment differ between when the genome cleavage site is located on the 5'-end side of the target exon sequence into which the target gene sequence is introduced and when they are located on the 3'-end side of the genome cleavage site, and therefore will be described separately below.
• the first genome homologous sequence is a base sequence that is homologous to the genome sequence from a base located on the 5'-end side of the genome cleavage position on the genome (e.g., a base located 10 bases, 20 bases, 50 bases, 100 bases, 500 bases, 1,000 bases or more upstream from the genome cleavage position) to a target exon sequence or a partial sequence thereof located on the 3'-end side of an intron sequence including the genome cleavage position (or adjacent).
• a base located 10 bases, 20 bases, 50 bases, 100 bases, 500 bases, 1,000 bases or more upstream from the genome cleavage position to a target exon sequence or a partial sequence thereof located on the 3'-end side of an intron sequence including the genome cleavage position (or adjacent).
• the genome sequence corresponding to the first genome homologous sequence has a mutation in its recognition sequence so as not to be cleaved by a genome editing enzyme that cleaves the above-mentioned genome cleavage position.
• the type of the mutation is not particularly limited, and may be, for example, a substitution, deletion, and/or insertion of a base in the recognition sequence.
• the number of mutated bases in the recognition sequence is not particularly limited. For example, one or more bases may be substituted, deleted, and/or inserted, more specifically, one or more, two or more, three or more, four or more, five or more, or six or more bases may be substituted, deleted, and/or inserted.
• the second genome-homologous sequence consists of a base sequence that is homologous to a genome sequence located on the 3'-terminal side of the target sequence or a partial sequence thereof.
• the base length of the first and second genome homologous sequences is not particularly limited, and may be, for example, 100 to 20,000 bases, 200 to 10,000 bases, 500 to 5,000 bases, or 1,000 to 2,000 bases.
• the first genomic homologous sequence is composed of a base sequence homologous to a genomic sequence from a base located on the 5'-terminal side of an intron sequence including the genomic cleavage site (specifically, a base located on the 5'-terminal side of the position in the target exon sequence where the target gene sequence is inserted, for example, a base located 100 bases, 200 bases, 500 bases, 1,000 bases, 5,000 bases, or 10,000 bases or more upstream from the position where the target gene sequence is inserted) to a target exon sequence or a partial sequence thereof located on the 5'-terminal side of (or adjacent to) the intron sequence (for example, to a base encoding the C-terminal amino acid residue of a signal peptide or a functional fragment thereof in the target exon sequence, or
• the second genome homologous sequence is a base sequence homologous to the genome sequence from a base located on the 3'-end side of the target exon sequence or a partial sequence thereof and on the 5'-end side of the genome cleavage position (e.g., a base encoding an amino acid residue further C-terminal than the C-terminal amino acid residue of a signal peptide or a functional fragment thereof in the target exon sequence) to a base located on the 3'-end side of the genome cleavage position (e.g., a base located 10 bases, 20 bases, 50 bases, 100 bases, 500 bases, 1,000 bases, 5,000 bases, or 10,000 bases or more downstream from the genome cleavage position).
• a base located on the 3'-end side of the target exon sequence or a partial sequence thereof and on the 5'-end side of the genome cleavage position e.g., a base encoding an amino acid residue further C-terminal than the C-terminal amino acid residue of a signal peptide
• the genome sequence corresponding to the second genome homologous sequence has a mutation in its recognition sequence so as not to be cleaved by a genome editing enzyme that cleaves the above-mentioned genome cleavage position.
• the type of the mutation and the number of mutated bases are not particularly limited, and may be, for example, a substitution, deletion, and/or insertion of one or more bases in the recognition sequence, as in (1) above.
• the base length of the first and second genome homologous sequences is not particularly limited, and may be, for example, 100 to 20,000 bases, 200 to 10,000 bases, 500 to 5,000 bases, or 1,000 to 2,000 bases.
• the target exon sequence or a partial sequence thereof contained in either the first or second genomic homologous sequence encodes a signal peptide or a functional fragment thereof or a sequence on the C-terminal side thereof, and is linked in frame to a target gene sequence located on the 3'-terminal side thereof via a base sequence encoding, in some cases, multiple amino acid residues.
• the gene of interest sequence in the donor nucleic acid of this aspect includes a stop codon.
• the donor nucleic acid of this aspect includes a transcription termination sequence on the 3' end of the gene of interest sequence.
• the donor nucleic acid of this aspect contains a marker gene for identifying a transgenic Lepidoptera insect in which a gene sequence of interest has been introduced into an endogenous gene.
• the type of genome editing enzyme used in the homologous recombination method using the donor nucleic acid of this embodiment is not limited, and may be TALEN, ZFN, and/or Cas9.
• the donor nucleic acid of this aspect comprises a nuclease recognition sequence at the end opposite to the target gene sequence of the first genome homologous sequence and/or the second genome homologous sequence.
• the nuclease recognition sequence is not particularly limited, and may be the recognition sequence of a genome editing enzyme that cuts the above-mentioned genome cutting position, or may be a restriction enzyme recognition sequence that can be cut by any restriction enzyme different from the genome editing enzyme.
• the nuclease recognition sequence is a TALEN recognition sequence
• the nuclease recognition sequence may be a combination of two recognition sequences recognized by Left TALEN and Right TALEN. 3-3.
• the fourth aspect of the present invention is a method for producing a genetically modified Lepidopteran insect.
• the double-stranded circular DNA described in the second aspect or the donor nucleic acid described in the third aspect is introduced into an egg of a Lepidopteran insect by microinjection to introduce a target gene sequence into the exon sequence of an endogenous gene, thereby producing a genetically modified Lepidopteran insect.
• the production method of this embodiment includes an essential step of introducing a double-stranded circular DNA or a donor nucleic acid into an egg of a lepidopteran insect by microinjection, and includes an egg obtaining step and a genetically modified lepidopteran insect selecting step. The configuration of each step will be described below.
• the "egg obtaining step” is a step of obtaining eggs by allowing adult female parent silkworms to lay eggs.
• the method of obtaining eggs may be a conventional method in the field.
• Egg laying is initiated by providing an egg-laying mat to the mated female parent silkworm.
• the temperature during egg laying is 23 to 28°C, preferably around 25°C.
• female silkworms start laying eggs several hours after mating.
• microinjection In order to incorporate the DNA introduced into the eggs into the nucleus, microinjection must be performed within 2 to 8 hours, preferably 3 to 6 hours, after laying.
• the "introducing step” is a step of introducing a double-stranded circular DNA or a donor nucleic acid into an egg of a lepidopteran insect by microinjection.
• the configuration of the double-stranded circular DNA is as described in aspect 2.
• the double-stranded circular DNA described in aspect 2, the first genome editing enzyme described in aspect 2 or a nucleic acid encoding the first genome editing enzyme in an expressible state, and the second genome editing enzyme described in aspect 2 or a nucleic acid encoding the second genome editing enzyme in an expressible state are introduced into an egg of a lepidopteran insect by microinjection.
• the introduction step in the production method of this aspect involves introducing a donor nucleic acid into an egg
• the configuration of the donor nucleic acid is as described in aspect 3.
• the donor nucleic acid described in aspect 3, the genome editing enzyme described in aspect 3, or a nucleic acid encoding the genome editing enzyme in an expressible state is introduced into an egg of a lepidopteran insect by microinjection.
• the term "expressible state” refers to a state in which a gene to be expressed is located downstream of a promoter under the control of the promoter.
• the nucleic acid encoding the genome editing enzyme in an expressible state may be RNA such as mRNA, or DNA such as plasmid DNA or linear DNA.
• the DNA encoding the genome editing enzyme in an expressible state contains a promoter that can be expressed in the eggs of a lepidopteran insect in addition to a base sequence encoding the first or second genome editing enzyme, and may contain components such as a marker gene (selection marker), an enhancer, a terminator, a replication origin, and a polyA signal, as necessary.
• the microinjection method may be performed by a method known in the art. For example, the double-stranded circular DNA or donor nucleic acid, and the genome editing enzyme or the nucleic acid encoding the genome editing enzyme in an expressible state are dissolved or diluted with a solvent such as water or a buffer to an appropriate concentration to prepare an injection solution.
• the amount of nucleic acid to be introduced is not particularly limited. It may be appropriately determined depending on the type, properties, and purpose of the nucleic acid. Usually, 50 nL to 30 nL is sufficient. After the introduction, the lepidopteran insect eggs may be incubated under appropriate conditions, for example, at 25°C, until hatching.
• the "genetically modified Lepidoptera insect selection step" is a step of selecting a genetically modified Lepidoptera insect from hatched Lepidoptera insects. This step may also be carried out by a method known in the art.
• the desired genetically modified Lepidoptera insect can be easily selected based on the expression of the marker gene.
• the term "marker gene” refers to a polynucleotide consisting of a base sequence that codes for a marker protein, also called a selection marker.
• the term "marker protein” refers to a protein that can confer a new trait not present in a host lepidopteran insect upon expression of a marker gene, and includes enzymes, fluorescent proteins, pigment synthesis proteins, luminescent proteins, etc. Based on the activity of the marker protein, it becomes possible to easily distinguish a transformant that has an introduced nucleic acid. 5.
• the fifth aspect of the present invention is a method for producing a target protein, a fragment thereof, or a fusion protein containing the target protein or a fragment thereof. According to the production method of this aspect, it is possible to mass-produce a target protein, a fragment thereof, or a fusion protein containing the target protein or a fragment thereof, using the genetically modified lepidopteran insect of the first aspect or the genetically modified lepidopteran insect produced by the production method described in the fourth aspect. 5-2. Production method The production method of the present invention includes a rearing step and a recovery step. Each step will be described below.
• the "rearing step” is a step of rearing the genetically modified lepidopteran insect of the first embodiment or the genetically modified lepidopteran insect produced by the production method described in the fourth embodiment.
• the genetically modified lepidopteran insect may be reared by a technique known in the art for each lepidopteran insect. For example, if the lepidopteran insect is a silkworm, "General Theory of Silkworms; Takami Tsuyoshi, published by the National Silkworm Association" may be referred to.
• the feed may be, for example, natural leaves of food tree species such as leaves of the genus Morus for silkworms and mulberry silkworms, leaves of Ricinus communis or leaves of Ailanthus altissima for Eri silkworms, and leaves of Fagaceae for Scutellaria japonica, or artificial feed such as Silkmate L4M or for 1-3rd instar original silkworm species (Nihon Nosan Kogyo). Artificial feed is preferable because it can suppress the occurrence of diseases, allows feeding of a stable quality and amount, and allows rearing in a sterile environment if necessary. A simple rearing method will be explained below using silkworms as an example.
• Sweeping is performed using eggs laid by an appropriate number of females of the same genetically modified lepidopteran insect (e.g., 4-10 larvae).
• the hatched larvae are transferred from the egg carrier to a container lined with dry-proof paper (paraffin-treated paper) that serves as a silkworm bed, and are fed with artificial feed such as Silkmate arranged on the dry-proof paper.
• the feed is replaced once for the 1st and 2nd instars, and once to three times for the 3rd instar. If there is a lot of leftover old feed, it is removed to prevent decay.
• For rearing the 4th to 5th instar adult silkworm larvae they are transferred to a large container and the number of larvae per container is adjusted appropriately.
• the “recovery step” is a step of recovering the target protein or a fragment thereof, or a fusion protein containing the same, which is expressed in the silk gland cells of the larvae of a genetically modified lepidopteran insect, secreted, and then accumulated in the lumen of the silk gland.
• the genetically modified lepidopteran insect used in this embodiment expresses in silk gland cells a precursor protein in which a signal peptide or a functional fragment thereof is fused to the N-terminus of a fusion protein (hereinafter referred to as "target protein, etc.") containing a mature protein or a C-terminal fragment thereof encoded by an endogenous gene at the C-terminus of the target protein or fragment thereof.
• target protein a fusion protein
• the precursor protein expressed in the silk gland cells is transported to the endoplasmic reticulum by the action of the signal peptide or a functional fragment thereof, and the signal peptide or a functional fragment thereof is cleaved by the action of an enzyme such as a peptidase in the endoplasmic reticulum, and then secreted into the lumen of the silk gland.
• the target protein, etc. after the signal peptide or the functional fragment is cleaved is secreted from the anterior silk gland to the outside of the individual during the pupation stage and spun. Therefore, the method of recovering the target protein, etc. includes a method of recovering it from a cocoon, or a method of directly recovering the target protein, etc.
• the method of recovering it from a cocoon is excellent in that the target protein, etc. can be recovered easily.
• the method for recovering the target protein from the cocoon is as follows: first, the larvae in the final stage are transferred to the cocoon and allowed to spin a cocoon. Next, the target protein is extracted from the cocoon. There is no particular limitation on the extraction method. For example, the target protein can be recovered by simply immersing the cocoon in water or an appropriate neutral extraction buffer that does not contain a protein denaturant (e.g., phosphate-buffered saline, pH 7.2, containing or not containing 1% Tween-20 and 0.05% sodium azide).
• a protein denaturant e.g., phosphate-buffered saline, pH 7.2, containing or not containing 1% Tween-20 and 0.05% sodium azide.
• the cocoon may be cut or crushed before immersion.
• the extraction temperature is low, 0 to 10°C, preferably 0 to 5°C, to prevent thermal denaturation of the target protein.
• the extraction can also be performed at 10 to 40°C.
• the extract may be stirred as necessary.
• the extraction time varies depending on the extraction conditions such as the state of the cocoons (e.g., uncut or powdered), the amount of the extract, the extraction temperature, and the presence or absence of stirring, and may be appropriately determined according to the conditions.
• Insoluble components such as fibroin may be removed from the extract by centrifugation or filtration as necessary.
• the method of extracting the silk gland from the insect body in the late final instar to prognathic stage and recovering the target protein, etc. can be achieved by a method known in the art.
• a silkworm immediately before spinning on the 6th day of the final instar (5th instar) is anesthetized on ice, the dorsal side is incised, and the silk gland is extracted with tweezers without damaging it (see Mori Yasushi, ed., New Biological Experiments with Silkworms, Sanseido, 1970, pp. 249-255).
• the extracted silk gland can be gently shaken in the extraction buffer at a temperature of 0 to 10°C, preferably 0 to 5°C, to elute the target protein, etc.
• the sixth aspect of the present invention is a fusion protein.
• the fusion protein of this aspect contains, in order from the N-terminus, a target protein or a fragment thereof, and any protein constituting silk thread.
• the protein constituting silk thread contained in the fusion protein of this aspect may be fibroin, sericin, or fibrohexamarin.
• the fibroin may be a fibroin H chain and/or a fibroin L chain.
• the protein constituting silk thread contained in the fusion protein of this aspect may be a full-length mature protein.
• the target protein or a fragment thereof contained in the fusion protein of this embodiment is not particularly limited, and may be, for example, a fluorescent protein, an antibody, an antigen polypeptide, an enzyme, a cytokine, or an antibacterial polypeptide. 7.
• Cocoon or silk thread The seventh aspect of the present invention is a cocoon or silk thread.
• the cocoon or silk thread of this aspect contains the fusion protein of the sixth aspect.
• any protein constituting the silk thread of the cocoon or silk thread of this aspect is composed of the fusion protein of the sixth aspect.
• any one or more proteins selected from the group consisting of fibroin H chain, fibroin L chain, sericin 1, sericin 2, sericin 3, and fibrohexamarin contained in the cocoon or silk thread of this aspect are composed of the fusion protein described in the sixth aspect.
• the cocoon or silk thread of this aspect is derived from a genetically modified lepidopteran insect of the first aspect or a genetically modified lepidopteran insect produced by the production method according to the fourth aspect.
• the genetically modified lepidopteran insect may homozygously comprise an exon sequence including a gene sequence of interest.
• the cocoon or silk thread of this embodiment When the fusion protein contained in the cocoon or silk thread of this embodiment is a fluorescent protein fused with fibroin, fibrohexamarin, or the like, the cocoon or silk thread of this embodiment has extremely strong fluorescence, and has an extremely strong color that can be distinguished by the naked eye even under normal white light, for example. This color is overwhelmingly stronger than the color of fluorescent cocoons or fluorescent silk threads that can be produced by conventional techniques, so that a highly useful material can be provided.
• Example 1 Construction of knock-in lines for useful protein production (the purpose)
• the GAL4/UAS system of the prior art is a gene control system that utilizes a combination of yeast-derived transcription factor GAL4 and a control sequence UAS.
• an expression system that expresses a target protein in the silk gland or the like is constructed by crossing a GAL4 line expressing a GAL4 gene under the control of a promoter such as a silk gene with a UAS line expressing a target gene under the control of a control sequence UAS.
• the GAL4/UAS system it is necessary to establish the GAL4 line and the UAS line separately and then cross them, so it takes time to construct the expression system.
• the GAL4 gene and the UAS regulatory sequence are introduced at random positions on the genome, so the expression level of the target protein varies and is difficult to predict. Therefore, after producing a large number of lines, it is necessary to examine the expression level in the individuals obtained by crossing the GAL4 line and the UAS line, and select good parent lines based on the results. Therefore, the present inventors came up with the idea of constructing a new expression system for stable mass production of a target protein by knocking in a target gene sequence into an endogenous gene.
• a target gene encoding a target protein is knocked in so as to be fused with a signal peptide into an exon sequence encoding a signal peptide in an endogenous sericin gene or fibroin gene, it may be possible to highly express the target gene by utilizing the promoter activity and enhancer activity of the endogenous gene as is.
• efficient knock-in to endogenous genes requires cutting the target gene using genome editing enzymes.
• the inventors attempted knock-in by designing a genome cleavage site within an exon sequence, but found that more than 95% of the individuals of the injected generation were unable to produce normal cocoons and more than 98% were unable to develop into mating-capable adults.
• the TAL-PITCh (precise integration into target chromosome) method and homologous recombination method are used to knock in the gene of interest, and the intron sequence adjacent to the 5'-end of the target exon sequence is cleaved with the genome editing enzyme TALEN (transcription activator-like effector nuclease) ( Figure 2).
• TALEN transcription activator-like effector nuclease
• Figure 2 the genome editing enzyme
• this example differs from Examples 5 to 6 described below in that the EGFP gene sequence, which is the gene of interest, has a stop codon and a transcription termination sequence at the 3'-end ( Figure 1).
• Knock-in to the fibroin H (FibH) gene, fibroin L (FibL) gene, and sericin 1 (Ser1) gene was performed by the following method.
• the vector expressing TALEN was constructed with reference to Y. Takasu, S. Sajwan, T. Daimon, M. Osanai-Futahashi, K. Uchino, H. Sezutsu, T. Tamura, M. Zurovec (2013): Efficient TALEN construction for Bombyx mori gene targeting, PLoS One, 8, e73458.
• TALEN mRNA was synthesized using each TALEN expression vector as a template and mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen).
• the SP(FibH)-EGFP donor nucleic acid for TAL-PITCh contains the first recognition sequence, the second spacer sequence, the first spacer sequence, the genome homologous sequence, the target gene sequence, the transcription termination sequence, and the marker gene in this order (FIG. 3B).
• the first spacer sequence is adjacent to the 5'-end side of the genome cleavage position and is a 10-base-long sequence consisting of positions 1945 to 1954 in SEQ ID NO: 3.
• the second spacer sequence is adjacent to the 3'-end side of the genome cleavage position and is a 10-base-long sequence consisting of positions 1955 to 1964 in SEQ ID NO: 3.
• the first recognition sequence is a 20-base-long sequence consisting of positions 1925 to 1944 in SEQ ID NO: 3 that is recognized by Left TALEN on the 5'-end side of the first spacer sequence.
• the genome homologous sequence is a base sequence homologous to the genome sequence from the second recognition sequence to the codon encoding the C-terminal residue of the signal peptide in the second exon sequence, and is a 70-base-long sequence consisting of positions 1965 to 2034 in SEQ ID NO: 3.
• the second recognition sequence is a 20-base-long sequence consisting of positions 1965 to 1984 in SEQ ID NO: 3, which is recognized by Right TALEN at the 3'-end side of the second spacer sequence.
• the target gene sequence is the EGFP gene sequence
• the transcription termination sequence used is the transcription termination sequence of the silkworm-derived sericin 1 gene.
• the base sequence from the first recognition sequence to the transcription termination sequence of the EGFP gene is shown in SEQ ID NO: 13.
• amino acid sequence of a protein in which a signal peptide derived from FibH is fused to the N-terminus of the C-terminal fragment excluding the initiation methionine in EGFP, which is encoded by the gene after knock-in is shown in SEQ ID NO: 14 (hereinafter referred to as "SP(FibH)-EGFP fusion protein").
• SP(FibH)-EGFP donor nucleic acid for TAL-PITCh was injected into silkworm eggs of the white-eyed, white-egg, non-diapause w1-pnd strain maintained by the National Agriculture and Food Research Organization 2 to 8 hours after spawning together with mRNA encoding Left TALEN and Right TALEN synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen).
• the eggs after injection were incubated in a humidified state at 25°C until hatching.
• the silkworms of the current generation after injection were crossed with the parent strain, and the resulting next-generation larvae were selected by the fluorescence of DsRed2 expressed in the whole body or EGFP expressed in the silk gland to obtain knock-in silkworm strains.
• the obtained knock-in line is referred to as the "SP(FibH)-EGFP knock-in line.”
• the FibH gene after this knock-in is referred to as the "SP(FibH)-EGFP knock-in gene.”
• 103 individuals spun cocoons normally, meaning that no cocoon-spinning defects were observed in the injected larvae.
• the larvae were anesthetized on ice just before spinning on the sixth day of the fifth instar, the dorsal side was incised, and the middle and posterior silk glands were removed with tweezers without damaging them, and observed under a fluorescent microscope without fixing. As a result, extremely strong EGFP fluorescence was observed (left side of Figure 5). In addition, the cocoons of this line were clearly yellow-green under normal white light (right side of Figure 5).
• the SP(FibH)-EGFP donor nucleic acid for homologous recombination includes a first genome homologous sequence and a second genome homologous sequence, and an EGFP gene sequence, which is a target gene sequence arranged therebetween, a transcription termination sequence, and a marker gene, and includes a TALEN recognition sequence at the end opposite to the EGFP gene sequence of the first genome homologous sequence and the second genome homologous sequence.
• the first genome homologous sequence is a 1034-base-long sequence that is homologous to the genome sequence from the base located on the 5'-terminal side of the genome cleavage position on the genome (position 1001 in SEQ ID NO: 3) to the codon encoding the C-terminal residue of the signal peptide in the second exon sequence.
• the first genome homologous sequence has a mutation in the vicinity of the above-mentioned genome cleavage position so as not to be recognized by the Left TALEN and Right TALEN described in (1) above (specifically, the base sequence located at positions 1925 to 1984 in SEQ ID NO: 3)
• the second genome homologous sequence is a 377-base-long sequence homologous to the genome sequence located at the 3'-end side of the codon encoding the C-terminal residue of the signal peptide in the second exon sequence on the genome.
• the base sequence from the TALEN recognition sequence and the first genome homologous sequence to the second genome homologous sequence and the TALEN recognition sequence is shown in SEQ ID NO: 15.
• the protein in which the FibH-derived signal peptide is fused to the N-terminus of EGFP, which is encoded by the gene after knock-in (FIG. 4), consists of the amino acid sequence shown in SEQ ID NO: 14 as in (1) above (hereinafter, referred to as "SP(FibH)-EGFP fusion protein" as in (1) above).
• the SP(FibH)-EGFP donor nucleic acid for homologous recombination was injected into silkworm eggs of the w1-pnd strain 2 to 8 hours after spawning together with mRNAs encoding Left TALEN and Right TALEN synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen).
• the eggs after injection were incubated in a humidified state at 25°C until hatching.
• the silkworms of the current generation after injection were crossed with the parent strain, and the resulting next-generation larvae were selected by the fluorescence of DsRed2 expressed in the whole body or EGFP expressed in the silk gland to obtain a knock-in silkworm strain.
• the obtained knock-in strain is referred to as "SP(FibH)-EGFP knock-in strain" as in (1) above.
• SP(FibH)-EGFP knock-in strain In the individuals that developed from the microinjected embryos, no defects in spinning or mating were observed, as in (1) above. Therefore, it was demonstrated that knock-in lines capable of normal spinning and mating can be efficiently produced by cutting the genome within the intron sequence using homologous recombination as well.
• a position in the second intron of the FibL gene (position between positions 8937 and 8954 in SEQ ID NO: 6) was set as the genome cleavage position, and a double-stranded circular DNA was constructed as a donor nucleic acid to be used in homologous recombination (hereinafter referred to as "SP(FibL)-EGFP donor nucleic acid for homologous recombination").
• the SP(FibL)-EGFP donor nucleic acid for homologous recombination includes a first genome homologous sequence and a second genome homologous sequence, and an EGFP gene sequence, which is a target gene sequence arranged therebetween, a transcription termination sequence, and a marker gene, and includes a TALEN recognition sequence at the end opposite to the EGFP gene sequence of the first genome homologous sequence and the second genome homologous sequence.
• the above-mentioned genome cleavage site is cleaved by a Left TALEN that recognizes a 20-base sequence consisting of positions 8917 to 8936 in SEQ ID NO:6, and a Right TALEN that recognizes a 20-base sequence consisting of positions 8955 to 8974 in SEQ ID NO:6.
• the first genome homologous sequence is a 1128-base-long sequence homologous to the genome sequence from the base located on the 5'-terminal side of the genome cleavage position on the genome (position 7864 in SEQ ID NO: 6) to the codon encoding the C-terminal residue of the signal peptide in the third exon sequence.
• the first genome homologous sequence has a mutation in the vicinity of the above-mentioned genome cleavage position so that it is not recognized by the Left TALEN and the Right TALEN (specifically, the base sequence located at positions 8917 to 8974 in SEQ ID NO: 6).
• the second genome homologous sequence is a 1362-base sequence homologous to the genome sequence located at the 3'-end side of the codon encoding the C-terminal residue of the signal peptide in the third exon sequence on the genome.
• the TALEN recognition sequence and the base sequence from the first genome homologous sequence to the second genome homologous sequence and the TALEN recognition sequence are shown in SEQ ID NO: 16.
• the protein in which the FibL signal peptide is fused to the N-terminus of the C-terminal fragment of EGFP excluding the initiation methionine, which is encoded by the gene after knock-in consists of the amino acid sequence shown in SEQ ID NO: 17 (hereinafter referred to as "SP(FibL)-EGFP fusion protein").
• the SP(FibL)-EGFP donor nucleic acid for homologous recombination was injected into silkworm eggs of the w1-pnd strain 2 to 8 hours after egg laying together with mRNAs encoding Left TALEN and Right TALEN synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen).
• the eggs after injection were incubated in a humidified state at 25°C until hatching.
• a knock-in silkworm line was obtained in the same manner as in (2) above.
• the knock-in line obtained is referred to as the "SP(FibL)-EGFP knock-in line" as in (1) above.
• the FibL gene after this knock-in is referred to as the "SP(FibL)-EGFP knock-in gene".
• SP(FibL)-EGFP knock-in gene The FibL gene after this knock-in is referred to as the "SP(FibL)-EGFP knock-in gene”.
• Ser1 The EGFP gene sequence was introduced into the second exon of the sericin 1 (hereinafter referred to as "Ser1”) gene by homologous recombination so that the EGFP protein was fused to the C-terminus of the signal peptide of Ser1.
• a position in the first intron of the Ser1 gene (position between positions 3020 and 3033 in SEQ ID NO: 9) was set as the genome cleavage position, and a double-stranded circular DNA was constructed as a donor nucleic acid to be used in homologous recombination (hereinafter referred to as "SP(Ser1)-EGFP donor nucleic acid for homologous recombination").
• the SP(Ser1)-EGFP donor nucleic acid for homologous recombination includes a first genome homologous sequence and a second genome homologous sequence, and an EGFP gene sequence, which is a target gene sequence arranged therebetween, a transcription termination sequence, and a marker gene, and includes a TALEN recognition sequence at the end opposite to the EGFP gene sequence of the first genome homologous sequence and the second genome homologous sequence.
• the above-mentioned genome cleavage site is cleaved by a Left TALEN that recognizes a 19-base sequence consisting of positions 3001 to 3019 in SEQ ID NO:9, and a Right TALEN that recognizes a 16-base sequence consisting of positions 3034 to 3049 in SEQ ID NO:9.
• the first genome homologous sequence is a 2069-base-long sequence homologous to the genome sequence from the base located on the 5'-terminal side of the genome cleavage position on the genome (position 947 in SEQ ID NO: 9) to the codon encoding the C-terminal residue of the signal peptide in the second exon sequence.
• the first genome homologous sequence has a mutation in the vicinity of the above-mentioned genome cleavage position so that it is not recognized by the Left TALEN and the Right TALEN (specifically, the base sequence located at positions 3000 to 3049 in SEQ ID NO: 9).
• the second genome homologous sequence is a 2000-base-long sequence homologous to a genome sequence located on the 3'-terminal side of the codon encoding the C-terminal residue of the signal peptide in the second exon sequence on the genome.
• the restriction enzyme recognition sequence and the base sequence from the first genome homologous sequence to the second genome homologous sequence and the restriction enzyme recognition sequence are shown in SEQ ID NO: 18.
• the protein in which the signal peptide of Ser1 is fused to the N-terminal side of the C-terminal fragment of EGFP excluding the initiating methionine, which is encoded by the gene after knock-in consists of the amino acid sequence shown in SEQ ID NO: 19 (hereinafter referred to as "SP(Ser1)-EGFP fusion protein").
• the SP(Ser1)-EGFP donor nucleic acid for homologous recombination was injected into silkworm eggs of the w1-pnd strain 2 to 8 hours after spawning together with mRNAs encoding Left TALEN and Right TALEN synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen).
• the eggs after injection were incubated in a humidified state at 25°C until hatching.
• a knock-in silkworm strain was obtained in the same manner as in (2) above.
• the knock-in strain obtained is referred to as the "SP(Ser1)-EGFP knock-in strain" as in (1) above.
• Example 2 Production of EGFP protein (the purpose) The expression level of EGFP protein in the silk gland of each knock-in line prepared in Example 1 is measured. (Methods and Results) (1) Silkworm strains By crossbreeding each of the knock-in strains prepared in (2) to (4) of Example 1, strains having a combination of multiple knock-in genes were prepared.
• a strain having two knock-in genes, an SP(FibH)-EGFP knock-in gene and an SP(FibL)-EGFP knock-in gene is called an SP(FibH)-EGFP/SP(FibL)-EGFP knock-in strain, etc. Note that in the silkworms used in this example, all of the knock-in genes are heterozygous.
• a line obtained by crossing a FibH+Ser1-GAL4 line expressing the GAL4 gene under the control of the FibH gene promoter and the Ser1 gene promoter with a UAS-EGFP line expressing the EGFP gene under the control of the UAS regulatory sequence (hereinafter referred to as the "EGFP-producing GAL4/UAS line") was used as a control group.
• Silkworms were reared as follows. All stages of larvae were reared on artificial diet (Silkmate Original Species 1-3 Stages S, Nippon Nosan Kogyo) in a rearing room at 25-27°C. The artificial diet was changed every 2-3 days (Uchino K.
• the EGFP protein concentration in the water-soluble protein contained in the supernatant was measured by ELISA. Specifically, 100 ⁇ L of the supernatant was added to a 96-well plate coated with an anti-GFP antibody (Aves GFP-1010, Cosmo Bio), and left to stand at room temperature for 1 hour. After washing three times with PBS/0.05% Tween 20, horseradish peroxidase-conjugated anti-GFP antibody (Rockland Immunochemicals) was added and left at room temperature for 1 hour. After washing three times with PBS/0.05% Tween 20, a color reaction was carried out using TMB Peroxidase EIA Substrate Kit (Bio-Rad), and the reaction was stopped by adding 1N sulfuric acid.
• the color development was quantified with a plate reader (SpectraMax iD3; Molecular Devices).
• a standard curve was prepared using serial dilutions (1 to 400 pg/ ⁇ L) of recombinant GFP protein (Takara Bio; Z2373N).
• the results of measuring the amount of EGFP expression per silkworm are shown in FIG.
• the amount of EGFP expression in the SP(Ser1)-EGFP knock-in line (FIG. 6, SP(Ser1)-EGFP) was slightly higher than that of the EGFP-producing GAL4/UAS line (FIG. 6, Control(GAL4/UAS)).
• the GAL4 protein is expressed by two promoters, the Ser1 gene promoter and the FibH gene promoter, and the amount of expression derived from the Ser1 gene promoter out of the 3.3 mg of EGFP expression is estimated to be 1 mg or less. Therefore, the amount of EGFP expression in the SP(Ser1)-EGFP knock-in line is considered to be overwhelmingly large.
• the amount of EGFP expression in the SP(FibH)-EGFP knock-in line (FIG. 6, SP(FibH)-EGFP) was more than twice as high as that in the SP(FibL)-EGFP knock-in line (FIG.
• Example 3 Production of GM-CSF protein (the purpose) A knock-in is performed by homologous recombination so that granulocyte macrophage colony-stimulating factor (GM-CSF) is fused to the C-terminus of the signal peptide of FibH. The amount of GM-CSF produced in the silk gland is evaluated.
• GM-CSF granulocyte macrophage colony-stimulating factor
• a line obtained by crossing the Ser1-GAL4 line expressing the GAL4 gene under the control of the Ser1 gene promoter with the UAS-GM-CSF line expressing the GM-CSF gene under the control of the UAS regulatory sequence (hereinafter referred to as the "middle silk gland GM-CSF-producing GAL4/UAS line"), and a line obtained by crossing the FibH-GAL4 line expressing the GAL4 gene under the control of the FibH gene promoter with the UAS-GM-CSF line (hereinafter referred to as the "posterior silk gland GM-CSF-producing GAL4/UAS line”) were used as controls.
• the membrane was gently shaken for 5 minutes with EZ wash (AE-1480, ATTO), and then reacted with a primary antibody (anti-GM-CSF antibody, 3000-fold dilution, Immundiagnostik, product number AS1021.2) overnight at 4° C.
• the membrane was washed three times for 10 minutes with EZ wash, and then reacted with a secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva, product number NA934-100UL, 50,000-fold dilution) at room temperature for 1 hour.
• Example 4 Antibody production> (the purpose) Knock-in is performed by homologous recombination so that an IgG H chain is fused to the C-terminus of the signal peptide of FibH. Furthermore, knock-in is performed by homologous recombination so that an IgG L chain is fused to the C-terminus of the signal peptide of FibL. The two knock-in strains obtained are crossed to produce an antibody molecule containing an IgG H chain and an IgG L chain.
• FibH-derived signal peptide in which a FibH-derived signal peptide is fused to the N-terminus of the IgG H chain consists of the amino acid sequence shown in SEQ ID NO: 25 (hereinafter referred to as the "SP(FibH)-IgG H chain fusion protein").
• SEQ ID NO: 25 hereinafter referred to as the "SP(FibH)-IgG H chain fusion protein”
• the homologous recombination method described in Example 1(3) was performed by replacing the EGFP gene sequence with an IgG L chain gene sequence.
• the IgG L chain gene sequence consists of the base sequence shown in SEQ ID NO: 26 and encodes an IgG L chain consisting of the amino acid sequence shown in SEQ ID NO: 27.
• the resulting knock-in line is called the "SP(FibL)-IgG L chain knock-in line.”
• the protein encoded by the knock-in gene in which a FibL-derived signal peptide is fused to the N-terminus of the IgG L chain consists of the amino acid sequence shown in SEQ ID NO: 28 (hereinafter referred to as the "SP(FibL)-IgG L chain fusion protein").
• a line obtained by crossing a FibH+Ser1-GAL4 line expressing the GAL4 gene under the control of the FibH gene promoter and the Ser1 gene promoter, a UAS-IgG H chain line expressing the IgG H chain gene under the control of the UAS regulatory sequence, and a UAS-IgG L chain line expressing the IgG L chain gene under the control of the UAS regulatory sequence (hereinafter referred to as "antibody-producing GAL4/UAS line”) was used as a control group.
• (2) Quantification of IgG expression level The middle and posterior silk glands were excised from each line, and proteins in each silk gland were extracted according to the method described in Example 2.
• IgG was purified using Ab SpinTrap (Cytiva), and the amount of IgG in the extract was quantified using a Protein Assay BCA Kit (Nacalai).
• Figure 8C It was shown that the SP(FibH)-IgG H chain/SP(FibL)-IgG L chain knock-in line was able to produce approximately 4.6 times more IgG than the middle and posterior silk glands of the antibody-producing GAL4/UAS line. It was revealed that the silk glands of the SP(FibH)-IgG H chain/SP(FibL)-IgG L chain knock-in line expressed and secreted antibody molecules containing IgG H chain and IgG L chain very efficiently.
• Example 5 Generation of knock-in lines for fusion protein production (the purpose)
• a method of substituting the repeat sequence portion located in the center of FibH with a target protein, or a method of fusing a target protein to the C-terminus of FibL is known.
• the conventional techniques have problems such as low expression levels and loss of activity of the target protein.
• the inventors conceived the idea that by knocking in a gene of interest into an exon sequence encoding a signal peptide in a silk gene such as a sericin gene or a fibroin gene, and fusing the gene of interest between a peptide and a mature protein formed by cleaving the signal peptide from a precursor protein encoded by an endogenous silk gene, it may be possible to highly express a silk protein fused with the gene of interest by utilizing the promoter and enhancer activities of the endogenous silk gene as is.
• a genomic cleavage site is designed within the intron sequence located at the 5' end of the exon sequence encoding the signal peptide, and a homologous recombination method is applied to create a knock-in line expressing a fusion silk protein.
• a gene of interest encoding an EGFP protein is knocked in as a protein of interest fused to the C-terminus of a signal peptide in an exon sequence encoding a signal peptide in an endogenous silk gene.
• a homologous recombination method is used to knock in the gene of interest, and an intron sequence adjacent to the 5'-end of the target exon sequence is cleaved with a genome editing enzyme TALEN (FIG. 10).
• TALEN genome editing enzyme
• the donor nucleic acids used for knock-in into the fibroin L (FibL) gene and the sericin 1 (Ser1) gene were prepared by the following method.
• (1) EGFP-FibL donor nucleic acid In the homologous recombination method described in (3) of Example 1, the transcription termination sequence and marker gene were removed from the SP(FibL)-EGFP donor nucleic acid described in Example 1, and the EGFP gene sequence and the second genome homologous sequence were modified so that the C-terminal amino acid residue of the EGFP protein could be fused with the N-terminal amino acid residue of FibL after the signal peptide was cleaved, to construct a donor nucleic acid (double-stranded circular DNA) for homologous recombination (hereinafter referred to as "EGFP-FibL donor nucleic acid").
• EGFP-FibL donor nucleic acid double-stranded circular DNA
• the configurations of the genome cleavage position in the endogenous FibL gene, the Left TALEN and Right TALEN, the first genome homologous sequence, the TALEN recognition sequence, etc. were in accordance with Example 1.
• the base sequence from the TALEN recognition sequence and the first genome homologous sequence to the second genome homologous sequence and the restriction enzyme recognition sequence is the SP(FibH)-EGFP donor nucleic acid shown in SEQ ID NO: 15 in which the stop codon TAA at positions 1821 to 1823 is deleted.
• the EGFP-FibL precursor protein which is encoded by the knock-in gene and in which the signal peptide of FibL is fused to the N-terminus of EGFP and the mature FibL protein after cleavage of the signal peptide is fused to the C-terminus of EGFP, consists of the amino acid sequence shown in SEQ ID NO: 29.
• the EGFP-FibL mature protein generated by cleavage of the signal peptide from the EGFP-FibL precursor protein consists of the amino acid sequence shown in SEQ ID NO: 30.
• the configurations of the genome cleavage position in the endogenous Ser1 gene, the Left TALEN and Right TALEN, the first genome homologous sequence, the TALEN recognition sequence, etc. were in accordance with Example 1.
• the base sequence from the restriction enzyme recognition sequence and the first genome homologous sequence to the second genome homologous sequence and the restriction enzyme recognition sequence is the SP(Ser1)-EGFP donor nucleic acid shown in SEQ ID NO: 18 in which the stop codon TAA at positions 2841 to 2843 has been deleted.
• the EGFP-Ser1 precursor protein which is encoded by the gene after knock-in and in which the signal peptide of Ser1 is fused to the N-terminus of EGFP and the mature Ser1 protein after cleavage of the signal peptide is fused to the C-terminus of EGFP, consists of the amino acid sequence shown in SEQ ID NO: 31.
• the EGFP-Ser1 mature protein generated by cleavage of the signal peptide from the EGFP-Ser1 precursor protein consists of the amino acid sequence shown in SEQ ID NO: 32.
• the donor nucleic acids prepared in (1) and (2) above were injected into silkworm eggs together with mRNA encoding TALEN according to the method described in (3) and (4) of Example 1 to prepare knock-in lines.
• knock-in lines Hereinafter, they are referred to as "EGFP-FibL knock-in line” and “EGFP-Ser1 knock-in line”, respectively.
• the FibL gene and the Ser1 gene after this knock-in are referred to as “EGFP-FibL knock-in gene” and "EGFP-Ser1 knock-in gene", respectively.
• Example 6 Production of a fusion protein containing EGFP protein and silk protein (the purpose)
• the knock-in line generated in Example 5 is used to produce a fusion protein containing an EGFP protein and a silk protein.
• Methods and Results According to the method described in Example 2, the middle silk gland and the posterior silk gland were excised from each knock-in line prepared in Example 5, and proteins in each silk gland were extracted. Then, Western blotting was performed according to the method described in Example 3 (2). In this example, Peroxidase Conjugate anti-GFP antibody (diluted 4000 times, manufactured by ROCKLAND, product number 600-103-215) was used as the detection antibody.
• the SP(Ser1)-EGFP knock-in line prepared in Example 1 (4) and the w1-pnd line were used as the control group.
• the results of Western blotting are shown in Figure 11.
• EGFP-FibL knock-in line EGFP-FibL mature protein was detected ( Figure 11A).
• EGFP-Ser1 knock-in line EGFP-Ser1 mature protein was detected ( Figure 11B).
• Example 7 Homozygote of knock-in gene (the purpose) Individuals homozygously carrying the EGFP-FibL knock-in gene are produced and allowed to spin cocoons.
• the wild-type silkworm cocoons, FibL-fused EGFP cocoons produced using the piggyBac system, and cocoons produced by the heterozygotes and homozygotes were lined up and observed under white light or fluorescence, as shown in FIG. 13.
• the fluorescence was detected using an EGFP filter after excitation with blue light. It was revealed that the heterozygote of the EGFP-FibL knock-in line produced by the method of the present invention exhibited strong fluorescence, and the homozygote exhibited overwhelmingly strong fluorescence, compared to the FibL-fused EGFP cocoons produced using the piggyBac system.
• Comparative Example 1 Preparation of knock-in line when genome cleavage site is designed in exon sequence (the purpose) The genome cleavage site is provided within the exon sequence, not within the intron sequence, and the EGFP gene is knocked into the fibroin H gene by homologous recombination. The efficiency of generating knock-in lines is compared with that in the case of cleaving the genome within the intron sequence. (Methods and Results) In the homologous recombination method described in (2) of Example 1, the method was modified so that the genome cleavage position was set within the second exon of the FibH gene, and the EGFP gene sequence was knocked into the fibroin H gene.
• a mutation was introduced into the donor nucleic acid for homologous recombination near the genome cleavage position in the second exon so that it would not be recognized by TALEN.
• the donor nucleic acid for homologous recombination was injected into 384 eggs together with mRNA encoding TALEN synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen) into silkworm eggs of the w1-pnd strain 2 to 8 hours after spawning.
• the eggs after injection were incubated in a humidified state at 25°C until hatching.
• the silkworms of the current generation after injection were raised to adulthood and crossed with the parent strain to determine whether they had mating ability. The results are shown in FIG.
• Example 1(2) where almost no cocoon spinning or mating defects were observed when the genome was cut within the intron sequence ( FIG. 14A ), and indicates that the method for producing knock-in silkworm lines according to the method described in Example 1 can produce knock-in lines with overwhelmingly high efficiency. This is thought to be because even if some mutations are introduced into the intron sequence, the effect on normal protein expression is negligible.
• a target protein can be stably and mass-produced in a lepidopteran insect such as a silkworm.

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