WO2022080234A1 - ガルデリア属に属する藻類のゲノム改変方法 - Google Patents

ガルデリア属に属する藻類のゲノム改変方法 Download PDF

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WO2022080234A1
WO2022080234A1 PCT/JP2021/037195 JP2021037195W WO2022080234A1 WO 2022080234 A1 WO2022080234 A1 WO 2022080234A1 JP 2021037195 W JP2021037195 W JP 2021037195W WO 2022080234 A1 WO2022080234 A1 WO 2022080234A1
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genome
genus
garderia
desired substance
algae belonging
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French (fr)
Japanese (ja)
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夢 國分
広顕 山崎
進也 宮城島
俊亮 廣岡
崇之 藤原
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DIC Corp
Inter University Research Institute Corp Research Organization of Information and Systems
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DIC Corp
Inter University Research Institute Corp Research Organization of Information and Systems
Dainippon Ink and Chemicals Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

  • the present invention relates to a method for modifying the genome of algae belonging to the genus Garderia. It also contains a method for producing algae belonging to the genus Garderia with a genome modification, a method for producing a desired substance using the algae belonging to the genus Garderia with a genome modification produced by the production method, and the desired substance. Regarding the manufacturing method of food. It also relates to algae belonging to the genus Garderia, which have a requirement for nutritional components. This application claims priority based on Japanese Patent Application No. 2020-172163 filed in Japan on October 12, 2020, the contents of which are incorporated herein by reference.
  • microalgae Since microalgae have a high carbon dioxide fixation capacity compared to land plants, and because their habitat does not compete with agricultural products, some species are mass-cultured to feed, functional foods, and cosmetic materials. It is used industrially as such. When microalgae are used industrially, it is desirable that they are microalgaes that can be mass-cultured outdoors from the viewpoint of cost. However, in order to be a microalgae that can be mass-cultured outdoors, it must be resistant to environmental changes (light, temperature, etc.), can be cultivated under conditions where other organisms cannot survive, and can grow to high densities. Conditions such as that are required.
  • the genus Galdia is a unicellular red alga that preferentially grows in sulfuric acid-acidic hot springs.
  • the genus Garderia is characterized in that it can be cultivated in an environment where other organisms such as high salt concentration, high temperature, and low pH are difficult to grow. Therefore, it is considered to be suitable for industrial use. Further, if a desired trait can be imparted to a unicellular red alga by a gene modification technique or the like, a cell line more suitable for industrial use can be produced. Furthermore, in addition to the ability to grow by photosynthesis, the genus Garderia also has the ability to assimilate various organic substances and grow heterotrophically. Therefore, it can be efficiently propagated even in a dark place.
  • the genus Garderia belongs to the Cyanidiophyceae class.
  • Cyanidioschyzon genus the entire genome sequence has been decoded in Cyanidioschyzon merolae, which belongs to the genus Cyanidioschyzon, and the development of gene modification technology is underway (non-patent). Documents 1 and 2).
  • the genus Garderia is considered to be a promising alga for industrial use because it can grow in an environment where it is difficult for other organisms to grow and high-density culture is possible.
  • a transformation method capable of stably expressing a foreign gene has not been established. Therefore, it is difficult to produce the genus Garderia having a desired trait, which is a barrier to industrial use.
  • a method for modifying the genome of an alga belonging to the genus Garderia which comprises a step of modifying the genome of a haploid of the alga belonging to the genus Gardenia.
  • [4] The method for modifying the genome of algae belonging to the genus Garderia according to [3], wherein the genome editing system is selected from the group consisting of CRISPR / Cas, ZNF, and TALEN.
  • the genomic modification is at least one genomic modification selected from the group consisting of the following (a) to (c). Genome modification methods for algae belonging to: (a) genome modification to produce a desired substance; (b) genome modification to improve the production amount of the desired substance; and (c) genome modification to promote or reduce cell proliferation.
  • Production method. [7] The method for producing algae belonging to the genome-modified genus Garderia according to [6], further comprising a step (B) of diploidizing the algae after the step (A).
  • a method for producing a desired substance which comprises a step of recovering the desired substance.
  • a method for producing a desired substance including a step of recovering the substance of the substance.
  • a method for modifying the genome of algae belonging to the genus Garderia which can stably impart a desired trait to the algae belonging to the genus Garderia. It also contains a method for producing algae belonging to the genus Galderia whose genome has been modified using the genome modification method, a method for producing a desired substance using the algae belonging to the genus Garderia whose genome has been modified, and the desired substance.
  • a method for producing food is provided. Further provided are algae belonging to the genus Garderia, which have a nutritional requirement and can be used for genome modification.
  • Micrographs of monoploids and diploids of algae belonging to the genus Garderia are shown.
  • the results of culturing a haploid of Galdia partita NBRC102759 (hereinafter, also referred to as “Garderia (polyploid)”) using MA medium adjusted to pH 0.1 to 2.0 are shown.
  • the target sequence of gRNA used for the preparation of the uracil demanding strain is shown.
  • the construct of the plasmid for genome editing used for the preparation of the uracil-requiring strain is shown.
  • the construct of the plasmid for genome editing before the insertion of the target sequence is shown.
  • the primer sequence of the primer designed for inserting the target sequence by the In-Fusion reaction and the design position of the primer are shown.
  • the results of evaluation of the susceptibility of adaptia (polyploid) to Blasticidin S (BS) are shown.
  • the construct of the donor DNA used for the preparation of the BS resistant strain is shown.
  • the NS1 region, which is a neutral site of Galdia partita NBRC102759, and the base sequences of 200 bp upstream and downstream thereof are shown.
  • the result of confirming the BS resistance of the transformant (BSD) prepared by introducing the donor DNA containing the BSD marker set into adaptia (diploid) is shown.
  • WT indicates untransformed gardenia (diploid).
  • the result of confirming the BS resistance of the transformant (BSD) prepared by introducing the donor DNA containing the BSD marker set into adaptia is shown.
  • WT indicates untransformed gardenia (polyploid).
  • the results of PCR amplification of the target region in a transformant (BSD) of Garderia (polyploid) in which BS resistance was confirmed are shown.
  • WT indicates untransformed gardenia (polyploid).
  • the construct of the donor DNA used for the preparation of the mVenus expression strain is shown.
  • FIG. 3 shows a fluorescence microscope image of a transformant (TF) prepared by introducing donor DNA containing the mVenus gene set into adaptia (polyploid).
  • DIC is a differential interference microscope image
  • Chl is a fluorescence microscope image in which autofluorescence of chlorophyll is detected
  • mVenus is a fluorescence microscope image in which mVenus fluorescence is detected
  • merged is a merged image of Chl and mVenus fluorescence microscope images.
  • the proteins, peptides, polynucleotides, vectors, and cells described herein can be isolated.
  • isolated means the native state or the state separated from other components. What is “isolated” can be substantially free of other components. "Substantially free of other components” means that the content of other components contained in the isolated component is negligible.
  • the content of other components contained in the isolated component is, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0. It can be 5% by mass or less, or 0.1% by mass or less.
  • the proteins, peptides, polynucleotides, vectors, and cells described herein are isolated proteins, isolated peptides, isolated polynucleotides, isolated vectors, and isolated cells. Can be a cell.
  • the first aspect of the present invention is a method for modifying the genome of algae belonging to the genus Garderia, which comprises a step of modifying the genome (genome modification step) of a primal algae belonging to the genus Garderia.
  • the algae belonging to the genus Garderia are algae belonging to the genus Cyanidiophyceae, the genus Cyanidiophyceae, and the genus Cyanidiophyceae.
  • Examples of algae belonging to the genus Garderia include G.I. Partita, G.M. sulphuraria, G.M. phlegrea, G.M. daedala, G.M. Examples include, but are not limited to, maxima and the like.
  • the algae belonging to the genus Garderia are unicellular red algae that preferentially grow in sulfuric acid acidic hot springs, and can preferably grow under conditions of high salt concentration, high temperature, and low pH.
  • Examples of the algae strain of the genus Garderia include G.I. Partita NBRC102759, G.M. sulphuraria CCCryo127-00, G.M. sulphuraria 074W, G.M. sulphuraria MS1, G.M. sulphuraria RT22, G.M. sulphuraria SAG21, G.M. sulphuraria SAG21, G.M. sulphuraria Azora, G.M. sulphuraria YNP, G.M. sulphuraria 5571, G.M. sulphuraria 002, G.M. phlegrea DBV009, G.M. Examples include, but are not limited to, phlegrea Soos, those shown in FIG. 10 of International Publication No. 2019/107385, and mutants thereof.
  • genome modification is performed on a haploid alga belonging to the genus Garderia.
  • the genus Garderia has diploid and ploid cell morphology.
  • FIG. 1 shows micrographs of haploids and diploids of the genus Garderia.
  • the haploid has an irregular or spherical cell morphology and does not have a strong cell wall.
  • the diploid has a spherical cell morphology and has a strong cell wall. Diploid cells form 4-32 endoplasmic spores during cell division, but the method of cell division of diploid cells is not yet known.
  • Whether the algae belonging to the genus Garderia are haploid or diploid can be determined by confirming the number of copies of the same locus. That is, if the number of copies of the same locus is 1, it is determined to be haploid.
  • a next-generation sequencer can also be used to determine that it is haploid. For example, sequence reads of the entire genome are acquired by a next-generation sequencer, the sequence reads are assembled, and then the sequence reads are mapped to the sequence obtained by assembling. In diploidy, differences in bases for each allele can be found in various regions on the genome, but in diploidy, only one allele exists, so such regions cannot be found.
  • the cell is homodiploid, it can be determined whether it is monoploid or diploid by measuring the DNA content of the cell. The DNA content of haploid cells is 1 ⁇ 2 of the DNA content of diploid cells.
  • the haploid does not have a strong cell wall and the diploid has a strong cell wall, it is possible to distinguish between a haploid cell and a diploid cell by observing the cell morphology. For example, in haploid cells, the cell wall is usually not observed when observed with an optical microscope (for example, at a magnification of 600 times). Therefore, if the cell wall is not observed by an optical microscope, it can be determined that the cell is a haploid cell. Since haploid cells do not have a strong cell wall, they can be destroyed by relatively mild treatment (neutralization treatment, hypotonic treatment, freeze-thaw treatment, surfactant treatment, etc.).
  • the cells are suspended in a medium containing 2% by mass of the detergent and the cells are disintegrated within 5 minutes after the addition of the detergent, it is determined that the cells are haploid.
  • the surfactant include sodium dodecyl sulfate. More specifically, sodium dodecyl sulfate is added to the cell suspension of algae belonging to the genus Garderia so as to be 2% by mass, and if the cells are disrupted within 5 minutes after the addition, the polyploid is used. It can be determined that there is. Whether or not the cells have collapsed can be confirmed by observing the cells with an optical microscope.
  • haploid cells When algae belonging to the genus Garderia are cultured in a solid medium, it is also possible to determine whether the cells are haploid cells based on the shape of the colonies. Since haploid cells do not have a strong cell wall, they are flatter and have a shape that spreads on the surface of a solid medium as compared with a colony of diploid cells. When a colony having such a shape appears on a solid medium, it can be determined to be a haploid colony.
  • the diploid of the algae belonging to the genus Garderia can be obtained by culturing the diploid of the algae belonging to the genus Garderia until the quiescent phase, and continuing the culturing in the quiescent phase for an arbitrary period.
  • Examples of the period for culturing in the stationary period include half a day or more, one day or more, two days or more, three days or more, five days or more, and the like.
  • the upper limit of the culture period is not particularly limited, and examples thereof include 60 days or less, 40 days or less, 30 days or less, 20 days or less, or 10 days or less.
  • cells may be recovered from the culture solution in the stationary phase, subcultured, and further cultured for about 1 to 5 days.
  • a diploid of algae belonging to the genus Garderia can be obtained by culturing a diploid of algae belonging to the genus Garderia in a medium containing an osmoregulator of 80 mM or more.
  • concentration of the osmotic pressure adjusting agent is preferably 100 mM or more, more preferably 150 mM or more, further preferably 200 mM or more, and even more preferably 300 mM or more, 350 mM or more, or 400 mM or more.
  • the upper limit concentration of the osmotic pressure adjusting agent is not particularly limited and may be a limit value that can be dissolved in the medium.
  • the upper limit concentration of the osmotic pressure regulator is, for example, 2M or less, 1.5M or less, 1.4M or less, 1.3M or less, 1.2M or less, 1.1M or less, or 1M. It can be as follows.
  • a diploid of algae belonging to the genus Garderia can be obtained by culturing a diploid of algae belonging to the genus Garderia in a medium having an osmotic pressure of 150 mOsm / kg or more.
  • the osmotic pressure is preferably 200 mOsm / kg or more, more preferably 250 mOsm / kg or more, further preferably 300 mOsm / kg or more, still more preferably 350 mOsm / kg or more, or even more preferably 400 mOsm / kg or more.
  • the upper limit of the osmotic pressure is not particularly limited, and may be a limit value at which the osmotic pressure adjusting agent can be dissolved in the medium. From the viewpoint of cell proliferation rate, the upper limit of osmotic pressure can be, for example, 2000 mOsm / kg or less, 1500 mOsm / kg or less, or 1400 mOsm / kg or less.
  • the osmotic pressure adjusting agent is not particularly limited as long as it is a chemical substance whose osmotic pressure can be adjusted by adding it to the medium.
  • examples of the osmotic pressure adjusting agent include sugars (glucose, sucrose, etc.), sugar alcohols (mannitol, sorbitol, etc.), amino acids (glycine, proline, arginine, etc.), metal salts (alkali metal salts, alkaline earth metal salts), and the like. Examples include urea, protein, betaine, inositol, and polysaccharides.
  • the medium used for culturing algae belonging to the genus Garderia is not particularly limited, and a known medium for culturing microalgae can be used.
  • the medium is not particularly limited, and examples thereof include an inorganic salt medium containing a nitrogen source, a phosphorus source, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, etc.) and the like.
  • examples of the nitrogen source include ammonium salts, nitrates, nitrites and the like
  • examples of the phosphorus source include phosphates and the like. Examples of such a medium include Gross medium, 2 ⁇ Allen medium (Allen MB. Arch. Microbiol.
  • M-Alllen medium Minoda A et al. Plant Cell Physiol. 2004 45: 667-71.
  • MA2 medium Ohnuma M et al. Plant Cell Physiol. 2008 Jan; 49 (1): 117-20.
  • Modified M-Alllen medium etc., but are not limited thereto.
  • Algae belonging to the genus Garderia may be autotrophically cultured under light irradiation, or may be heterotrophically cultured in the dark.
  • a carbon source (glucose or the like) may be added to the above-mentioned inorganic salt medium.
  • the culture conditions are not particularly limited, and the conditions normally used for culturing algae belonging to the genus Garderia can be used.
  • Examples of the pH condition include pH 0.25 to 8.0, preferably pH 0.5 to 6.0, more preferably pH 0.5 to 4.0, further preferably pH 0.5 to 3.0, and pH 0. 5 to 2.0 is particularly preferable.
  • Examples of the temperature condition include 15 to 50 ° C, preferably 30 to 50 ° C, and more preferably 35 to 50 ° C.
  • the light intensity includes 5 to 2000 ⁇ mol / m2s, preferably 5 to 1500 ⁇ mol / m2s. It may be cultured with continuous light, or a light-dark cycle (10L: 14D, etc.) may be provided. When culturing heterotrophically, it can also be cultivated in a dark place.
  • Algae belonging to the genus Garderia may be cultivated in a liquid medium or a solid medium.
  • haploid cells appearing in the culture medium can be collected while observing under a microscope. Since haploid cells do not have a strong cell wall, cells in which no cell wall is observed may be collected.
  • monosomatic cells can be obtained by collecting colonies of cells characteristic of monosomatic cells (for example, colonies that are flat and have a shape that spreads on the surface of the solid medium). ..
  • Genome modification means inducing a mutation at an arbitrary position on the genome.
  • the genome modification method is not particularly limited, and any modification method can be used. Genome modification may be performed sequence-specifically or non-sequence-specifically for the genomic DNA sequence.
  • sequence-specific genome modification method include a method using a genome editing system containing a sequence-specific endonuclease, a homologous recombination method, and the like.
  • sequence non-specific genome modification method include introduction of DNA fragments by microinjection method, particle gun method, transposon method, etc .; induction of mutation by ultraviolet irradiation, irradiation, chemical treatment with nitrite, etc. ..
  • the genome modification method according to this embodiment is preferably a sequence-specific genome modification method. By performing sequence-specific genome modification, a variant having the desired properties can be rapidly obtained.
  • the "genome editing system containing a sequence-specific endonuclease” means a system capable of sequence-specific cleavage of genomic DNA by a sequence-specific endonuclease and inducing mutation in the cleavage region.
  • Genomic DNA cleaved by a sequence-specific endonuclease is subsequently endogenous to the cell, such as homologous directed repair (HDR) or non-homologous end-joining repair (NHEJ).
  • HDR homologous directed repair
  • NHEJ non-homologous end-joining repair
  • HDR is a repair mechanism using donor DNA, and it is also possible to introduce a desired mutation into a target region.
  • a targeting vector used in the homologous recombination method described later can be used.
  • a sequence-specific endonuclease is an enzyme that can cleave a nucleic acid with a predetermined sequence.
  • the sequence-specific endonuclease is preferably a sequence-specific endodeoxyribonuclease capable of cleaving double-stranded DNA at a predetermined sequence.
  • the sequence-specific endonuclease is not particularly limited, and examples thereof include, but are not limited to, zinc finger nucleases (Zinc finger nucleoses (ZFNs)), TALENs (Transaction activator-like effector nucleoses), and Cas proteins. .. In the present specification, the genome editing system containing these sequence-specific endonucleases is described as ZFN, TALEN, and CRISPR (Crustered Regularly Interspaced Short Palindromic Repeat) / Cas, respectively.
  • ZFN means a genome editing system using an artificial nuclease containing a nucleic acid cleavage domain conjugated to a binding domain containing a zinc finger array.
  • cleavage domain of ZFN include the cleavage domain of the type II restriction enzyme FokI.
  • FokI restriction enzyme
  • TALEN means a genome editing system using an artificial nuclease containing a DNA binding domain of a transcriptional activator-like (TAL) effector in addition to a DNA cleavage domain (eg, FokI domain).
  • TAL transcriptional activator-like
  • the design of the TALE construct capable of cleaving the target sequence can be performed by a known method (for example, Zhang, Feng et. Al. (2011) Nature Biotechnology 29 (2)).
  • CRISPR / Cas means a genome editing system using Cas protein and guide RNA.
  • Cas protein is a general term for sequence-specific endonucleases used for genome editing by the CRISPR / Cas system, and refers to CRISPR-associated proteins.
  • the Cas protein preferably forms a complex with a guide RNA and exhibits endonuclease activity or nickase activity.
  • the Cas protein is not particularly limited, and examples thereof include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein.
  • the Cas protein is not particularly limited as long as it exhibits endonuclease activity or nickase activity in cooperation with the guide RNA.
  • Cas protein includes wild-type Cas protein and its homologs (paralogs and orthologs), as well as variants thereof.
  • the Cas protein is preferably involved in a class 2 CRISPR / Cas system, more preferably a type II CRISPR / Cas system.
  • Preferred examples of Cas protein include Cas9 protein.
  • the Cas9 protein is a Cas protein involved in the type II CRISPR / Cas system, which forms a complex with a guide RNA and exhibits an activity of cleaving DNA in a target region in cooperation with the guide RNA.
  • the Cas9 protein is not particularly limited as long as it has the above-mentioned activity.
  • Cas9 protein includes wild-type Cas9 protein and its homologs (paralogs and orthologs), as well as variants thereof.
  • the wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein herein may be one in which either the RuvC domain or the HNH domain is inactivated.
  • the species from which the Cas9 protein is derived is not particularly limited, but bacteria belonging to the genus Streptococcus, Staphylococcus, Neisseria, Treponema and the like are preferably exemplified. More specifically, S. pyogenes, S. streptococcus. thermophilus, S.A. aureus, N. et al. Meningitidis, or T.I. Cas9 protein derived from dentalcola and the like is preferably exemplified. A preferred example of the Cas9 protein is S. cerevisiae. Cas9 protein derived from pyogenes can be mentioned.
  • Guide RNA means RNA that can form a complex with Cas protein and induce Cas protein to a target region.
  • Guide RNAs include, for example, CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA).
  • the crRNA is involved in binding to a target region on the genome, and the tracrRNA is involved in binding to the Cas protein.
  • the crRNA comprises a spacer sequence and a repeat sequence, in which the spacer sequence binds to the complementary strand of the target sequence in the target region.
  • the tracrRNA comprises an anti-repeat sequence and a 3'tail sequence.
  • the anti-repeat sequence has a sequence complementary to the repeat sequence of crRNA and forms a base pair with the repeat sequence, and the 3'tail sequence usually forms three stem loops.
  • the guide RNA may be a single guide RNA (sgRNA) in which the 3'end of the crRNA is linked to the 5'end of the tracrRNA, and the crRNA and the tracrRNA are separate RNA molecules, and base pairs are used in repeat sequences and anti-repeat sequences. May be formed.
  • sgRNA single guide RNA
  • the repeat sequence of crRNA and the sequence of tracrRNA can be appropriately selected according to the type of Cas protein, and those derived from the same bacterial species as Cas protein can be used.
  • the length of sgRNA can be about 50 to 220 nucleotides (nt), preferably about 60 to 180 nt, and more preferably about 80 to 120 nt.
  • the length of crRNA can be about 25 to 70 bases including the spacer sequence, and is preferably about 25 to 50 nt.
  • the length of the tracrRNA can be about 10 to 130 nt, preferably about 30 to 80 nt.
  • the repeat sequence of crRNA may be the same as that in the bacterial species from which the Cas protein is derived, or may be the one in which a part of the 3'end is deleted.
  • the tracrRNA may have the same sequence as the mature tracrRNA in the bacterial species from which the Cas protein is derived, or may be a terminal-cleaving type in which the 5'end and / or the 3'end of the mature tracrRNA is cleaved.
  • the tracrRNA can be a terminal-cleaving type in which about 1 to 40 nucleotide residues are removed from the 3'end of the mature tracrRNA.
  • the tracrRNA can be a terminal-cleaving type in which about 1 to 80 nucleotide residues are removed from the 5'end of the mature tracrRNA. Further, the tracrRNA can be, for example, a terminal-cleaving type in which about 1 to 20 nucleotide residues are removed from the 5'end and about 1 to 40 nucleotide residues are removed from the 3'end.
  • Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNAs based on known techniques (eg, Jinek et al. (2012) Science, 337, 337, 816-21; Mali et al.
  • PAM Proto-spacer Adjacent Motif
  • the sequence and position of PAM depends on the type of Cas protein. For example, in the case of Cas9 protein, the PAM needs to be adjacent immediately after the 3'side of the target sequence.
  • the sequence of PAM corresponding to the Cas9 protein depends on the bacterial species from which the Cas9 protein is derived. For example, S.
  • the PAM corresponding to the Cas9 protein of pyogenes is "NGG” and S. streptococcus.
  • the PAM corresponding to the Cas9 protein of thermophilus is "NNAGAA", and S.I.
  • the PAM corresponding to the Cas9 protein of aureus is "NNGRRT” or “NNGRR (N)".
  • the PAM corresponding to the Cas9 protein of meningitidis is "NNNNGATT", which is T.I. It is "NAAAAC” corresponding to the Cas9 protein of detentola ("R” is A or G; “N” is A, T, G or C).
  • the target sequence targeted for cleavage by the Cas protein can be designed using a known method.
  • the PAM can be searched in the target region, and the sequence adjacent to the 5'side of the PAM can be used as the target sequence.
  • the target sequence For example, G.
  • the nucleotide sequence set forth in SEQ ID NO: 33 can be used as the target sequence.
  • amino acid sequence information and gene sequence information of the above sequence-specific endonuclease can be obtained from various databases such as GenBank, UniProt, and DDBJ.
  • sequence-specific endonuclease may be introduced into cells as a protein or may be introduced into cells as a polynucleotide encoding a sequence-specific endonuclease.
  • a sequence-specific endonuclease mRNA may be introduced into a cell, or a sequence-specific endonuclease expression vector may be introduced into a cell.
  • the "expression vector” refers to a vector containing a target polynucleotide, which is provided with a system for making the target polynucleotide expressible in the cell into which the vector has been introduced.
  • the "expressible state” means that the polynucleotide is in a state in which it can be transcribed in the cell into which the polynucleotide has been introduced.
  • sequence-specific endonuclease sequence-specific endonuclease gene
  • “Functionally linked” means that the first base sequence is located sufficiently close to the second base sequence and the first base sequence is the second base sequence or a region under the control of the second base sequence. Means that it can affect.
  • functionally ligating a polynucleotide to a promoter means that the polynucleotide is ligated to be expressed under the control of the promoter.
  • the promoter is not particularly limited, and for example, various pol II promoters can be used.
  • the pol II promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a ⁇ -actin promoter, a CAG promoter, and a CBh promoter.
  • the promoter of the sequence-specific endonuclease the EF1 promoter of algae belonging to the genus Garderia can be used.
  • Examples of the EF1 promoter for algae belonging to the genus Garderia include G.I. Partita's EF1 ⁇ promoter (SEQ ID NO: 18) can be mentioned.
  • the expression vector a known one can be used without particular limitation.
  • the expression vector include a plasmid vector, a viral vector, a linear DNA fragment and the like.
  • the sequence-specific endonuclease is a Cas protein
  • the expression vector may contain a guide RNA coding sequence (guide RNA gene) in addition to the Cas protein coding sequence (Cas protein gene).
  • the guide RNA coding sequence (guide RNA gene) is preferably functionalized by the pol III promoter.
  • the pol III promoter include U6-snRNA promoters of algae belonging to the genus Garderia, H1-RNase P RNA promoters, valine-tRNA promoters and the like.
  • the U6-snRNA promoter of algae belonging to the genus Garderia include G.I. Partita's U6 promoter (SEQ ID NO: 16) can be mentioned.
  • the homologous recombination method is a genome modification method utilizing a phenomenon in which recombination occurs between two DNA double strands having a homologous sequence.
  • a targeting vector can be used, and the targeting vector usually contains a sequence homologous to a region adjacent to a target region of interest for genomic modification.
  • the targeting vector can include a 5'homology arm adjacent to the 5'side of the target region and a 3'homology arm adjacent to the 3'side of the target region.
  • the targeting vector can include between the 5'homology arm and the 3'homology arm any sequence intended to be introduced into the target region (hereinafter referred to as "introduction sequence").
  • the sizes of the 5'homology arm and the 3'homology arm are not particularly limited as long as they are large enough to cause homologous recombination with genomic DNA.
  • the 5'homology arm and the 3'homology arm can be, for example, about 500 to 3000 bp.
  • the targeting vector for example, a plasmid vector, a linear DNA fragment, or the like can be used.
  • the introduction sequence is not particularly limited and can be any sequence.
  • Examples of the introduction sequence include (A) a sequence involved in the production of a desired substance, (B) a sequence involved in improving the production amount of the desired substance, (C) a sequence involved in cell proliferation, and the like. ..
  • the desired substances in the above (A) and (B) include, for example, various physiologically active substances such as various nutritional components (amino acids, vitamins, proteins, fatty acids, dietary fiber, etc.), enzymes, hormones, active ingredients of pharmaceutical products, and the like. , Hydrocarbons and the like, but are not limited thereto.
  • sequences related to the production of a desired substance include gene sequences of proteins involved in the synthesis of a desired substance.
  • proteins involved in the synthesis of a desired substance include, but are limited to, a synthase of a desired substance, a synthase of a precursor of a desired substance, a degrading enzyme of a substance that inhibits the synthesis of a desired substance, and the like. Not done.
  • sequences involved in improving the production amount of a desired substance include gene sequences of proteins involved in improving the production amount of a desired substance.
  • the proteins involved in improving the production of the desired substance include proteins involved in improving the expression of the synthase of the desired substance and synthase of the precursor of the desired substance. Examples include, but are not limited to, proteins involved in improving the expression of a desired substance, proteins involved in suppressing the expression of a degrading enzyme of a desired substance, and proteins involved in suppressing the expression of a degrading enzyme of a precursor of a desired substance.
  • Examples of the protein involved in improving the expression of the synthase of a desired substance include, but are not limited to, a protein that inhibits the binding of a transcriptional repressor to the promoter of the synthase gene, a transcriptional promoter of the synthase, and the like. ..
  • Examples of the protein involved in improving the expression of the precursor synthase of a desired substance include a protein that inhibits the binding of a transcriptional repressor to the promoter of the precursor synthase gene, a transcription promoting factor of the precursor synthase, and the like. However, it is not limited to these.
  • Examples of the protein involved in suppressing the expression of the degrading enzyme of a desired substance include, but are not limited to, a transcription inhibitor of the degrading enzyme, a protein that inhibits the binding of the transcription promoting factor to the promoter of the degrading enzyme gene, and the like. ..
  • Examples of proteins involved in suppressing the expression of a precursor-degrading enzyme of a desired substance include a transcription-suppressing factor of the precursor-degrading enzyme, a protein that inhibits the binding of a transcription-promoting factor to the promoter of the precursor-degrading enzyme gene, and the like. However, it is not limited to these.
  • the sequence involved in improving the production of the desired substance may be a highly expressed promoter sequence functionally linked to the endogenous synthase gene of the desired substance or the endogenous synthase gene of the precursor of the desired substance. good.
  • the sequence involved in improving the production of the desired substance may be a low expression promoter sequence that functionally links to the endogenous degrading enzyme gene of the desired substance or the endogenous degrading enzyme gene of the precursor of the desired substance. good.
  • sequences involved in cell proliferation include gene sequences of proteins that control cell division.
  • the protein may be a protein that promotes cell proliferation or a protein that suppresses cell proliferation.
  • the introduction sequence may contain, in addition to the gene sequence, a sequence that controls the expression of the gene sequence.
  • the expression control sequence include, but are not limited to, promoters, enhancers, polyA addition signals, terminators, and the like.
  • the gene sequence is preferably functionally linked to any promoter.
  • the promoter is not particularly limited as long as it can function in the cells of algae belonging to the genus Garderia.
  • the promoter may be a promoter of the gene or a promoter of another gene. Examples of promoters of other genes include, but are not limited to, promoters of APCC, promoters of CPCC, promoters of Catalase, and the like.
  • the promoter may be a promoter of an alga belonging to the genus Garderia, or may be a promoter of a gene of another organism (for example, another alga). Suitable promoters include, for example, G.I. Examples include, but are not limited to, the Partita EF1 ⁇ promoter (SEQ ID NO: 18), the Catalase promoter (SEQ ID NO: 19), and the like. It is preferable that an arbitrary terminator is linked to the 3'end of the gene sequence. The terminator is not particularly limited as long as it can function in the cells of algae belonging to the genus Garderia. The terminator may be the terminator of the gene, or may be the terminator of another gene.
  • Examples of the terminator of other genes include a ⁇ -tubulin terminator, a ubiquitin terminator, and the like, in addition to the gene terminator exemplified as the above promoter.
  • Suitable terminators include, for example, G.I. Examples thereof include, but are not limited to, a partita ubiquitin terminator (SEQ ID NO: 20) and a ⁇ -tubulin terminator (SEQ ID NO: 21).
  • the introduction sequence may contain a selectable marker gene in addition to the gene sequence of any protein.
  • the selectable marker include an antibiotic resistance gene, a gene related to auxotrophy, a fluorescent protein gene, and the like.
  • antibiotic resistance gene include a resistance gene to an antibiotic to which algae belonging to the genus Garderia are susceptible.
  • Blasticidin S resistance gene for example, Blasticidin S deaminase (BSD) gene; SEQ ID NO: 25
  • BSD Blasticidin S deaminase
  • chloramphenicol resistance gene and the like.
  • the gene related to auxotrophy include the URA5.3 gene and the like.
  • the fluorescent protein gene examples include the mVenus gene (SEQ ID NO: 27), the GFP gene, the mCherry gene and the like. These selectable marker genes may also have expression control sequences such as promoters, enhancers, polyA addition signals, and terminators.
  • the selectable marker gene is preferably functionally linked to a promoter that functions in algae belonging to the genus Garderia. Examples of the promoter and terminator include the same as above.
  • the protein coding sequence may be codon-optimized based on the codon usage frequency of algae belonging to the genus Garderia. By codon optimization, the translation efficiency of the protein coding sequence can be improved.
  • the region targeted for genome modification can be appropriately set according to the purpose of genome modification.
  • the target region can be set within the coding region of the particular gene or in the promoter region of the particular gene.
  • the purpose of genome modification is to introduce a gene of an arbitrary protein, it is preferable to set the target region to a region other than the gene endogenously present in the genome and the peripheral region thereof.
  • the target area can be set to, for example, a safe harbor area (neutral area).
  • Safe harbor region (neutral region) means a genomic region that has been demonstrated to be capable of inserting foreign DNA without exerting any detrimental effects on cells.
  • Examples of the safe harbor region of algae belonging to the genus Garderia include a region consisting of the base sequence set forth in SEQ ID NO: 15 (NS1 region) and the like.
  • the 5'homology arm may include, for example, part or all of the nucleotide sequence set forth in SEQ ID NO: 29.
  • the 3'homology arm may include, for example, part or all of the nucleotide sequence set forth in SEQ ID NO: 30.
  • the purpose of the genome modification is not particularly limited, and examples thereof include any of the following genome modifications (a) to (c).
  • Genome modification to produce a desired substance can be performed, for example, using a targeting vector containing the sequence involved in the production of the desired substance (A).
  • Genome modification may be performed by a genome editing system containing a sequence-specific endonuclease, or by a homologous recombination method.
  • the target region is preferably set in a genomic region other than the endogenous gene and its peripheral region, and more preferably set in a safe harbor region.
  • Genome modification to improve the production amount of the desired substance is, for example, a targeting vector containing the sequence involved in the improvement of the production amount of the desired substance (B). Can be done using. Genome modification may be performed by a genome editing system containing a sequence-specific endonuclease, or by a homologous recombination method.
  • the target region is preferably set in a genomic region other than the endogenous gene and its peripheral region, and more preferably set in a safe harbor region.
  • a genomic modification that improves the production of a desired substance may be a genomic modification that changes the endogenous promoter of the endogenous synthase gene of the desired substance or its precursor or its endogenous expression-promoting factor to a highly expressed promoter. good.
  • the target region can be set within the endogenous promoter region of the endogenous synthase gene.
  • a genomic modification that improves the production of a desired substance may be a genomic modification that changes the endogenous promoter of the endogenous degrading enzyme gene of the desired substance or its precursor or its endogenous expression-promoting factor to a low-expression promoter. good.
  • the target region can be set within the endogenous promoter region of the endogenous degrading enzyme gene.
  • the genomic modification that improves the production of the desired substance may be a genomic modification that disrupts the endogenous degrading enzyme gene of the desired substance or its precursor or an endogenous expression-promoting factor thereof.
  • the target region can be set in the coding region of the endogenous degrading enzyme gene or in the endogenous promoter region thereof.
  • Genome modification that promotes or decreases cell proliferation can be performed using, for example, a targeting vector containing the sequence involved in (C) cell proliferation.
  • Genome modification may be performed by a genome editing system containing a sequence-specific endonuclease, or by a homologous recombination method.
  • the target region is preferably set in a genomic region other than the endogenous gene and its peripheral region, and more preferably set in a safe harbor region.
  • Genome modification that promotes or reduces cell proliferation changes the endogenous gene of the protein that controls cell division or the endogenous promoter of the endogenous expression regulator (expression promoter or expression suppressor) into a high expression promoter or a low expression promoter. It may be a genomic modification to be modified.
  • the target region can be set within the endogenous promoter region of the endogenous gene or its endogenous expression regulator.
  • the genomic modification that promotes or reduces cell proliferation may be a genomic modification that disrupts an endogenous gene of a protein that controls cell division or an endogenous expression regulator (expression promoter or expression suppressor) thereof.
  • the target region can be set within the coding region of the endogenous gene or within the endogenous promoter region thereof.
  • the sequence-specific endonuclease may be introduced as a protein, may be introduced as an mRNA encoding the protein, or encodes the protein. It may be introduced as DNA to be used.
  • a Cas protein or an mRNA or DNA encoding a Cas protein and a guide RNA containing a sequence homologous to the target region can be used.
  • donor DNA can be used if desired.
  • the targeting vector as described above can be used.
  • the method for introducing a component necessary for genome modification into a haploid alga belonging to the genus Garderia is not particularly limited, and a known method can be used.
  • the genome-modifying component is nucleic acid
  • examples of the nucleic acid introduction method include a polyethylene glycol method, a lipofection method, a microinjection method, a DEAE dextran method, a particle gun method, an electroporation method, and a calcium phosphate method.
  • the genome-modifying component is a protein, for example, a method using a protein-introducing reagent, a method using a protein-introduced domain (PTD) fusion protein, a microinjection method, and the like can be mentioned.
  • PTD protein-introduced domain
  • the sequence-specific endonuclease is preferably introduced into cells as mRNA or DNA from the viewpoint of introduction efficiency.
  • the guide RNA in the CRISPR / Cas system may be introduced into the cell as RNA or may be introduced into the cell as DNA to express RNA in the cell.
  • the Cas protein is introduced into cells as DNA, the Cas protein and the guide RNA may be cloned on the same expression vector.
  • the genome modification method according to this embodiment may include an arbitrary step in addition to the above-mentioned genome modification step.
  • the optional step include a step of selecting genome-modified cells (selection step), a step of culturing genome-modified cells (culture step), and the like.
  • the genome modification method may include a step of selecting genome-modified cells after the genome modification step.
  • a selectable marker is introduced in the genome modification step
  • genome-modified cells can be selected using the expression of the selectable marker as an index.
  • genome-modified cells can be selected by culturing the genome-modified cells in a medium containing the antibiotic.
  • the genome-modified cells can be selected by culturing the genome-modified cells in a medium containing Blasticidin S.
  • the genome-modified cells are selected by culturing the cells after the genome modification in a medium containing no such nutritional component. can do.
  • genome-modified cells can be selected by culturing the cells after the genome modification in a medium containing the specific component.
  • the genome is modified using the URA5.3 gene as a target region and the URA5.3 gene is disrupted, the cells after the genome modification are cultured in a medium containing uracil and 5-fluoroorotic acid (5-FOA).
  • Genome-modified cells can be selected. This is because in cells that normally express the URA5.3 gene, the gene product of the URA5.3 gene converts uracil and 5-FOA to the toxic 5-fluorouracil. When genome modification is performed on monoploid cells that do not have the normal URA5.3 gene using the URA5.3 gene as a selection marker, the cells after the genome modification are cultured in a medium containing no uracil. Genome-modified cells can be selected.
  • genome-modified cells can be selected by selecting cells after genome modification by flow cytometry or the like based on the fluorescence of the fluorescent protein. ..
  • the medium and culture conditions used in the selection step the same media and culture conditions as those mentioned above (algae belonging to the genus Garderia) can be used.
  • the culture time in the selection step is not particularly limited as long as the cells whose genome has not been modified die. Examples of the culture time in the selection step include 1 to 5 days or more, 2 to 5 days or more, 3 to 5 days or more, and the like.
  • the genome modification method may include a step of culturing genome-modified cells after the genome modification step.
  • the culturing step may be performed before the selection step or after the selection step.
  • the medium and culture conditions used in the culture step the same culture medium and culture conditions as those mentioned above (algae belonging to the genus Garderia) can be used.
  • the culture step By performing the culture step, the number of genome-modified cells can be increased to an arbitrary amount.
  • the culturing step is performed after the selection step, the same medium as the selection step may be used as the medium.
  • the genome modification method since the genome is modified for the haploid of algae belonging to the genus Garderia, it is not necessary to modify the two alleles. Therefore, genome-modified cells having a desired trait can be easily obtained. Since the haploid does not have a strong cell wall, the efficiency of introducing the genome-modifying component is improved as compared with the diploid. Therefore, it is possible to efficiently modify the genome.
  • the second aspect of the present invention belongs to the genome-modified genus Garderia, which comprises a step (A; genome modification step) of modifying the genome of algae belonging to the genus Garderia by the genome modification method according to the first aspect. It is a method for producing algae.
  • the genome modification step can be performed in the same manner as the genome modification method according to the first aspect.
  • the production method according to this embodiment may include an arbitrary step in addition to the genome modification step.
  • the optional steps include, for example, a step of diploidizing genome-modified cells (B; diploidization step), a step of culturing the diploid (C; diploid culture step), and the diploid. (D; re-haploidization step) and the like.
  • the production method according to this embodiment may include a step of diploidizing the genome-modified cells after the genome-modifying step (step (A)).
  • step (A) genome modification is performed on the haploids of algae belonging to the genus Garderia. Therefore, the obtained genome-modified cells are haploid.
  • Polyploid cells do not have a strong cell wall and are more fragile and more susceptible to culture conditions than diploid cells. Therefore, it is considered that by making the genome-modified cells diploid, the genome-modified cells can be efficiently proliferated without being affected by the culture conditions.
  • Examples of the method of diploidizing the genome-modified cells include a method of culturing the monoploid genome-modified cells for an arbitrary period. By continuing the culture while substituting in a timely manner, diploid cells appear. By collecting the diploid cells that have appeared, diploid cells can be obtained.
  • Examples of the medium used for culturing include the same medium as the medium mentioned above (algae belonging to the genus Garderia).
  • the content of the osmotic pressure adjusting agent is preferably less than 80 mM, more preferably 70 mM or less, and even more preferably 60 mM or less.
  • the osmotic pressure of the medium is preferably less than 150 mOsm / kg, preferably 140 mOsm / kg or less, and even more preferably 120 mOsm / kg or less.
  • the culture conditions are not particularly limited, and the same culture conditions as those mentioned above (algae belonging to the genus Garderia) can be used.
  • Examples of the pH condition include pH 0.25 to 8.0, preferably pH 0.5 to 6.0, more preferably pH 0.5 to 4.0, further preferably pH 0.5 to 3.0, and pH 0. 5 to 2.0 is particularly preferable.
  • Examples of the temperature condition include 15 to 50 ° C, preferably 30 to 50 ° C, and more preferably 35 to 50 ° C.
  • the light intensity includes 5 to 2000 ⁇ mol / m2s, preferably 5 to 1500 ⁇ mol / m2s. It may be cultured with continuous light, or a light-dark cycle (10L: 14D, etc.) may be provided. When culturing heterotrophically, it can also be cultivated in a dark place.
  • the cells may be cultured in a liquid medium or a solid medium.
  • diploid cells appearing in the culture medium can be collected while observing under a microscope. Since diploid cells have a strong cell wall, cells in which the cell wall is observed may be collected. Further, when cultured in a solid medium, the colonies of diploid cells do not spread and have a raised shape as compared with the colonies of diploid cells. Therefore, diploid cells can be obtained by collecting colonies of cells characteristic of diploid cells.
  • the acquired cells are diploid by the same method as the method mentioned above (algae belonging to the genus Garderia).
  • the production method according to this embodiment may further include a step of culturing the diploid after the diploidization step (step (B)).
  • step (B) By culturing diploid genome-modified cells, it is considered that the genome-modified cells can be efficiently proliferated without being affected by the culture conditions.
  • Examples of the medium and culture conditions used in the diploid culture step include the same medium and culture conditions as those mentioned in the diploidization step. It is preferable that the cultured cells are appropriately subcultured before the quiescent phase.
  • the subculture interval can be appropriately adjusted according to the growth state of the diploid, and examples thereof include 3 to 10 days, 4 to 8 days, or 5 to 6 days.
  • Culturing is preferably performed in a liquid medium because it is easy to grow.
  • the production method according to this embodiment may further include a step of forming a haploid after the diploid culture step (step (C)).
  • the genome may be modified again, or the substance produced by the genome variant may be recovered. Since haploids do not have a strong cell wall, they can be destroyed by relatively mild treatment. Therefore, the intracellular components can be easily and efficiently recovered. Further, the haploid may be contained in foods and the like. Since the monoploid does not have a strong cell wall, it is considered that the digestion and absorption rate is higher than that of the diploid.
  • the production method it is possible to efficiently produce a genome-modified product having a desired trait by modifying the genome of a haploid cell.
  • the diploid is formed and the diploid is cultured, so that the genome variant having a desired trait can be efficiently propagated.
  • the genome can be efficiently modified again.
  • the substance produced by the genome variant can be efficiently recovered.
  • a third aspect of the present invention is a step of obtaining an alga belonging to the genome-modified Garderia genus (genome-modified product manufacturing step) by the production method according to the second aspect, and belonging to the genome-modified Garderia genus.
  • a method for producing a desired substance (hereinafter, “manufacturing a desired substance”, which comprises a step of causing algae to produce a desired substance (desired substance production step) and a step of recovering the desired substance (desired substance recovery step). Method ").
  • the desired substance produced by the method for producing a desired substance according to this embodiment is not particularly limited and may be any substance.
  • the desired substance may be a substance produced endogenously by algae belonging to the genus Garderia, or may be a substance produced by introducing a foreign gene. Examples of the desired substance include substances similar to those mentioned above (genome modification method).
  • the genome variant production step can be performed in the same manner as the production method according to the second aspect.
  • the genome modification performed in this step may be any of the genome modifications (a) to (c) mentioned in the above (genome modification method), and two or more genome modifications (a) to (c) may be combined. You may.
  • the genome modification of (c) is preferably a genome modification that promotes cell proliferation.
  • the genome variant By performing a genome modification that produces a desired substance, the genome variant can be made to produce a substance that is not produced by a wild strain.
  • B By performing genome modification to improve the production amount of a desired substance, it is possible to improve the production amount of the substance endogenously produced by algae belonging to the genus Garderia in the genome modification.
  • C Genome modification that promotes or reduces cell proliferation By performing genome modification that promotes cell proliferation, a genomic variant that produces a desired substance can be efficiently propagated.
  • the desired substance production step is a step of causing the genome variant obtained in the genome variant manufacturing step to produce a desired substance. This step can be performed by culturing the genomic variant.
  • the genomic variant to be cultured in this step may be diploid or diploid.
  • the haploid cells after genome modification may be cultured as they are. Alternatively, the haploid cells after the genome modification may be made into diploids and proliferated, and then returned to the haploids and cultured.
  • the medium and culture conditions the same medium and culture conditions as those mentioned above (algae belonging to the genus Garderia) can be used.
  • the genomic variant can be made diploid by the method described in the above (diploidization step).
  • the diploid culture can be carried out in the same manner as described above (diploid culture step).
  • This step is preferably performed by culturing diploid cells.
  • the genome variant By culturing the genome variant as a diploid, the genome variant can be efficiently propagated and a desired substance can be efficiently produced.
  • the desired substance recovery step is a step of recovering the desired substance produced by the genomic variant in the desired substance production step.
  • This step may be carried out using a haploid of a genomic variant, or may be carried out using a diploid, but it is preferable to use a haploid. Since haploids do not have a strong cell wall, they can destroy cells under relatively mild conditions. Therefore, the desired substance can be recovered easily and efficiently.
  • the haploid may be one obtained by culturing the haploid after genome modification as it is to produce a desired substance. Alternatively, the haploid may be cultivated as a diploid after the genome is modified to produce a desired substance, and then made into a haploid again.
  • a method of forming a haploid the same method as described above (algae belonging to the genus Garderia) can be mentioned.
  • the desired substance recovered in this step does not have to be a completely purified product of the desired substance, and may contain other components other than the desired substance.
  • the desired substance recovered in this step may be a haploid cell or a diploid cell containing the desired substance as a cell content, or may be a cell disruptor of these cells, or may be a cell disruption. It may be the one obtained by removing the solid content from the substance.
  • the desired substance can be recovered, for example, by recovering these cells from a haploid or diploid culture medium.
  • the cells can be recovered from the culture solution by, for example, centrifugation, filtration, or the like.
  • the recovered cells may be used as they are, or the cells may be destroyed and used.
  • Cell destruction can be performed using known methods.
  • Cells can be destroyed, for example, by physical treatment. Examples of the physical treatment method include cell destruction by glass beads, a mortar, ultrasonic treatment, a French press, a homogenizer, and the like.
  • Cells can be destroyed, for example, by chemical treatment. Examples of the method of chemical treatment include cell destruction by neutralization treatment, hypotonic treatment, freeze-thaw treatment, dry swelling treatment, enzyme treatment, surfactant treatment and the like. Since diploid cells have a strong cell wall, it is preferable to destroy the cells by physical treatment or a combination of physical treatment and chemical treatment. Since haploid cells do not have a strong cell wall, they can be destroyed by relatively mild chemical treatment.
  • Examples of the neutralization treatment method include a method of immersing monoploid cells in a neutralizing solution having a pH of about 7 to 10.
  • the composition of the neutralizing solution is not particularly limited, but for example, a buffer solution such as a phosphate buffer solution or a Tris buffer solution can be used.
  • the time for immersing the cells in the neutralizing solution may be such that the cells are destroyed, and examples thereof include about one week.
  • Examples of the hypotonic treatment method include a method of immersing monoploid cells in a hypotonic solution such as water.
  • the composition of the hypotonic liquid is not particularly limited as long as it is a hypotonic liquid to the extent that monoploid cells rupture.
  • hypotonic solution examples include water, a buffer solution having a low salt concentration, and the like.
  • the time for immersing the cells in the hypotonic solution may be such that the cells rupture, and examples thereof include about 1 to 30 minutes.
  • the algae cells may be collected by centrifugation or the like and resuspended in the hypotonic solution, which may be repeated.
  • the number of resuspensions is not particularly limited, and examples thereof include 1 to 5 times.
  • the method of freeze-thaw treatment include a method in which a cycle of freezing and thawing is performed once or more for haploid cells. The number of freezing and thawing cycles may be, for example, about 1 to 5 times.
  • Each time of freezing and thawing is not particularly limited, and for example, about 10 to 30 minutes each is exemplified.
  • Examples of the dry swelling treatment method include a method in which haploid cells are subjected to one or more cycles of drying and resuspension in a buffer solution. The number of drying and resuspending cycles may be, for example, about 1 to 5 times.
  • Examples of the enzyme treatment method include a method using an enzyme such as cellulase, pectinase, and lysozyme.
  • Examples of the method using a surfactant include a method using a surfactant such as sodium dodecyl sulfate.
  • the solid content may be removed by centrifugation, filtration, or the like.
  • the crude extract after removing the solid content may be subjected to an appropriate combination of methods generally used for separation / purification of biochemical substances, and further separation / purification of desired substances may be carried out.
  • Separation / purification includes, for example, salting, dialysis, recrystallization, reprecipitation, solvent extraction, adsorption, concentration, filtration, gel filtration, ultrafiltration, various types of chromatography (thin layer chromatography, column chromatography, ion exchange chromatography, high speed). (Liquid chromatography, adsorption chromatography, etc.), etc., but are not limited thereto.
  • the desired substance when the desired substance is a substance secreted extracellularly, the desired substance can be recovered by recovering the culture supernatant from the haploid or diploid culture solution.
  • the culture supernatant can be collected by centrifugation, filtration, or the like of the culture solution.
  • a method generally used for separation / purification of biochemical substances may be appropriately combined with the collected culture supernatant to further separate / purify the desired substance.
  • the desired substance production method in order to obtain a genome variant by the production method according to the second aspect and produce a desired substance, the production of the desired substance or the recovery of the desired substance is efficient. You can do it well.
  • the desired substance obtained by the method for producing a desired substance according to this embodiment can be appropriately used in foods, cosmetics, feeds or pet foods, feeds, industrial products, etc., depending on the type of desired substance.
  • a fourth aspect of the present invention is a step of producing a desired substance (desired substance manufacturing step) and a step of producing a food containing the desired substance (a step of producing a desired substance by the desired substance manufacturing method according to the third aspect).
  • a food manufacturing process) and a food manufacturing method (hereinafter referred to as “food manufacturing method”) including.
  • the food produced by the food production method according to this embodiment is not particularly limited and may be any food.
  • Foods include, for example, various noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles, cup noodles; carbohydrates such as bread, wheat flour, rice flour, hot cakes, mashed potatoes; green juice, soft drinks, carbonated drinks, etc.
  • Beverages such as nutritional drinks, fruit drinks, vegetable drinks, lactic acid drinks, milk drinks, sports drinks, tea and coffee; bean products such as tofu, okara and natto; various soups such as curry roux, stew roux and instant soup; ice cream Cold confectionery such as cream, ice sherbet, shaved ice; confectionery such as candy, cookies, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and other baked confectionery; Fisheries and processed livestock foods such as; processed milk, fermented milk, butter, cheese, yogurt and other dairy products; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils and fat processed foods; sauces, dressings , Miso, soy sauce, seasonings such as sauce; various retort foods, other processed foods such as sprinkles, pickles, etc., but are not limited thereto.
  • the food may be a functional food or a dietary supplement.
  • the functional food or dietary supplement may be in the form of a general food as described above, or may be in the form of a dry powder, granules, tablets, jelly or drink.
  • the desired substance production step can be performed by the desired substance production method according to the third aspect.
  • the food manufacturing process is a step of manufacturing a food containing a desired substance.
  • the food can be produced according to a known method according to the type of food by adding a desired substance to the food raw material and appropriately adding other food additives.
  • a nutritionally fortified food can be obtained by adding a desired substance.
  • a fifth aspect of the present invention is an alga belonging to the genus Garderia, which has a mutation in a gene involved in the synthesis of a nutritional component and has a requirement for the nutritional component.
  • Genes involved in the synthesis of nutritional components means genes encoding proteins involved in the synthesis of arbitrary nutritional components.
  • Examples of the gene involved in the synthesis of the nutritional component include a synthase gene of the nutritional component, a synthase gene of a precursor of the nutritional component, a gene encoding an activated protein of the synthase gene, and a transcription of the synthase gene. Examples include, but are not limited to, genes that regulate.
  • the nutritional component is not particularly limited and may be any nutritional component. Examples of nutritional components include, but are not limited to, bases, amino acids, vitamins and the like. Specific examples of nutritional components include uracil. Specific examples of genes involved in the synthesis of nutritional components include the URA5.3 gene.
  • Having a mutation in a gene involved in the synthesis of nutritional components means at least one selected from the group consisting of substitutions, deletions, insertions, and additions in the base sequence of genes involved in the synthesis of nutritional components. It means that a mutation has occurred.
  • the number of bases substituted, deleted, inserted, and / or added is not particularly limited. Whether or not a gene involved in the synthesis of a nutritional component has a mutation is evaluated based on, for example, the base sequence of the gene possessed by a wild strain (WT) that does not show the requirement of any nutritional component.
  • WT wild strain
  • the target algae strain is involved in the synthesis of the nutritional component. It is evaluated as having a mutation in the gene.
  • Wild strains of algae belonging to the genus Garderia can be obtained from, for example, ATCC, NIES collection and other algae culture collections.
  • the mutation is preferably not a silent mutation, and is preferably a mutation that impairs the function of the protein expressed from the gene.
  • the mutation can be, for example, a mutation that causes a frameshift. Mutations in genes involved in the synthesis of nutrient components are preferably introduced by genomic modification.
  • the algae belonging to the genus Garderia according to this embodiment are preferably genomic variants.
  • all of the two or more copies of the genes have mutations. It is more preferred that all of the two or more copies of the gene are mutated and all of the two or more copies of the gene are unable to express a functional protein.
  • Having the requirement of nutritional components means that it cannot grow in the absence of any nutritional components. Algae having the requirement of any nutritional component cannot grow in the absence of the nutritional component, but can grow in the presence of the nutritional component. It is preferable that the algae belonging to the genus Garderia according to this embodiment have a mutation in a gene involved in the synthesis of the nutritional component and cannot express a functional protein from the gene, and thus have a requirement for the nutritional component.
  • the algae belonging to the genus Garderia according to this embodiment may be diploid or diploid.
  • the genes involved in the synthesis of nutritional components have mutations in both alleles. If the algae belonging to the genus Garderia are diploid and there are two or more copies of the gene involved in the synthesis of nutritional components, then all of the genes in both alleles have mutations and all of the genes in both alleles are functional. It is more preferable that a typical protein cannot be expressed.
  • the mutation of the gene involved in the synthesis of the nutritional component may be the same mutation in both alleles or may be a different mutation, but it is preferable that the mutation is the same.
  • homodiploid having a homozygous mutation in a gene involved in the synthesis of nutritional components.
  • Such homodiploid induces diploid cells after introducing a mutation into a gene involved in the synthesis of nutritional components in monoploid cells by the genome modification method according to the first aspect. By doing so, it can be produced.
  • Examples of the algae belonging to the genus Garderia according to this embodiment include those having a mutation in the URA5.3 gene and being uracil-requiring (hereinafter, also referred to as "uracil-requiring strain").
  • the URA5.3 gene is a gene encoding orotidine 5'-phosphate decarboxylase.
  • Orotidine 5'-phosphate decarboxylase is an enzyme that catalyzes the reaction of converting orotidine 5'-phosphate to uridine 5'-phosphate (UMP; uracil precursor).
  • the URA5.3 gene is mutated and the functional orotidin-5'-decarboxylase is not expressed, the cells will not be able to synthesize UMP and uracil, resulting in uracil requirement.
  • Oroticin 5'-phosphate decarboxylase also catalyzes the reaction of converting 5-fluoroorotic acid (5-FOA) to the cytotoxic 5-fluorouridine (5-FU). Therefore, if the URA5.3 gene is not mutated and functional orotidin 5'-phosphate decarboxylase is expressed, the cells cannot survive in the presence of 5-FOA. On the other hand, if the URA5.3 gene is mutated and the functional orotidine 5'-phosphate decarboxylase is not expressed, the cells can survive in the presence of 5-FOA and uracil.
  • the uracil-requiring strain may be diploid or diploid. Since algae belonging to the genus Garderia usually have two copies of the URA5.3 gene, they have mutations in any of the two copies of the URA5.3 gene and cannot express the functional orotidine 5'-phosphate decarboxylase. Is preferable.
  • the uracil-requiring strain is diploid, it is preferably a homodiploid having a mutation in the URA5.3 gene.
  • Such a uracil-requiring strain can be obtained by introducing a mutation into the URA5.3 gene in a haploid cell by the genome modification method according to the first aspect, and then inducing the diploid cell. Can be made.
  • a mutation can be introduced into the URA5.3 gene by designing a target sequence in the coding region of URA5.3 and performing genome editing by the CRISPR / Cas system using a gRNA containing the target sequence. .. G.
  • the target sequence in the URA5.3 coding region includes the target sequence shown in FIG. 3 (SEQ ID NO: 33).
  • the mutation of the uracil-requiring strain in the URA5.3 gene is not particularly limited as long as it cannot express the functional orotidine 5'-phosphate decarboxylase.
  • the type of mutated base and the number of mutated bases are also not particularly limited.
  • specific examples of mutations in the URA5.3 gene include mutations (deletion, insertion, and / or substitution) in the range of positions 194 to 195 of the URA5.3 gene shown in SEQ ID NO: 22. Those having the above are mentioned.
  • Examples of the mutation include a deletion of the thymine residue (T) at position 194, a deletion of the adenine residue (A) at position 195, and a thymine residue at position 194 in the URA5.3 gene represented by SEQ ID NO: 22. Insertion of one to several (for example, 2, 3, 4 or 5) nucleotide residues between the group and the adenine residue (A) at position 195, a combination thereof and the like can be mentioned. Specific examples include mutations of # 1_1, # 1_2, # 2_1, # 2_2, # 3_1, and # 3_2 in FIG. That is, G.
  • Examples of the uracil-requiring strain of partita include those containing a base sequence selected from the group consisting of SEQ ID NOs: 7 to 12 at the URA5.3 locus. Mutations in the URA5.3 gene are not limited to these and may have other mutations in place of or in addition to these mutations.
  • the auxotrophic algae belonging to the genus Garderia make it possible to use the auxotrophic algae as a selectable marker for genome modification in the algae belonging to the genus Garderia. This provides a technique for realizing self-cloning in algae belonging to the genus Garderia.
  • the present invention provides algae belonging to the genus Garderia, which has been genomically modified, produced by the production method according to the second aspect.
  • the algae may be haploid or diploid. When it is diploid, it is preferably homodiploid having a genome-modified sequence homozygous.
  • the present invention provides algae belonging to the genus Garderia containing an antibiotic resistance gene.
  • the algae may be haploid or diploid. When it is diploid, it is preferably homodiploid having an antibiotic resistance gene homozygous. Examples of the antibiotic resistance gene include the BSD gene.
  • the present invention provides a desired substance produced by the method for producing a desired substance according to the third aspect.
  • the desired substance include the same substances as those exemplified above.
  • the present invention provides a food containing algae belonging to the genus Garderia, which has been genomically modified, produced by the production method according to the second aspect.
  • the algae may be haploid or diploid. When it is diploid, it is preferably homodiploid having a genome-modified sequence homozygous. From the viewpoint of digestion and absorption, it is preferable that the food according to this embodiment contains haploids of algae belonging to the genus Garderia whose genome has been modified.
  • Table 4 shows the composition of the MA2 medium (Ohnuma M et al. Plant Cell Physiol. 2008 Jan; 49 (1): 117-20.) Used in the test.
  • Garderia (polyploid) was cultured in MA medium adjusted to pH 0.1-2.0. Seven days after the start of the culture, the OD 750 of the culture solution was measured to confirm the growth status.
  • gRNA guide RNA
  • dsDNA double-stranded DNA
  • FIG. 3 shows the target sequence of gRNA (SEQ ID NO: 33) used in the preparation of the uracil auxotrophic strain.
  • a target sequence was designed within the coding sequence of the URA5.3 gene (SEQ ID NO: 22).
  • P URA 5.3 represents the promoter of the URA 5.3 gene and T URA 5.3 represents the terminator of the URA 5.3 gene.
  • a DNA fragment having a target sequence was commissioned to Eurofins and synthesized by the phosphoramidite method.
  • the CDS of the URA5.3 gene is shown in SEQ ID NO: 23.
  • FIG. 4 shows the construct of the plasmid for genome editing used in the preparation of the uracil auxotrophy strain.
  • Pu6 is the U6 promoter (SEQ ID NO: 16)
  • PEF1 -a is the promoter of the EF1-a gene (SEQ ID NO: 18)
  • TUBQ is the ubiquitin gene terminator (SEQ ID NO: 20)
  • NLS is the nuclear localization signal. (Nuclear localization signal) is shown.
  • the gRNA scaffold (SEQ ID NO: 17) indicates an sgRNA that does not contain a target sequence.
  • a 20 bp target sequence is inserted between Pu6 and the gRNA scaffold.
  • a plasmid containing the construct shown in FIG. 5 was prepared.
  • PCR was performed using the plasmid as a template using the primers shown in FIG.
  • the obtained PCR product was subjected to an In-Fusion reaction using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio).
  • Escherichia coli was transformed with the obtained plasmid, propagated, and then the plasmid was extracted.
  • PCR amplification was performed using the obtained plasmid as a template using the following primers (puc19_F, puc19_R), and the obtained DNA fragment was used as a genome editing DNA for producing a uracil-requiring strain.
  • the DNA for genome editing was introduced into Garderia (polyploid) by the PEG method.
  • Incubation was performed in a light-dark cycle (12 L / 12D) at 42 ° C. for 4 to 5 days (aeration culture, 2% CO 2 , 300 mL / min).
  • the transformant prepared as described above can be used as an MA medium (pH 1.0), a uracil-containing MA medium (pH 1.0, uracil 0.5 mg / mL), or a 5-FOA / uracil-containing MA medium (pH 1.0, Uracil (0.5 mg / mL, 5-FOA 0.4 mg / mL) was planted and cultured for 10 days under the same conditions as above.
  • untransformed gardenia (polyploid) (WT) was also cultured in the same manner.
  • Genome analysis Three strains of uracil-requiring strains were cloned (# 1, # 2, # 3), and the target region targeted for genome editing was analyzed. PCR was performed using DNA extracted from untransformed WT or uracil-requiring strains (# 1, # 2, # 3) as a template to amplify the target region. Sequence analysis was performed using the amplified DNA fragment, and the sequence of the target region was confirmed. The sequences of the primers used for PCR are shown below.
  • BS is converted to non-toxic deaminhohydroxy-blastidin (d-BS) by Blasticidin S deaminase (EC 3.5.4.23; BSD). Therefore, we introduced BSD into Garderia (polyploid) and tried to establish a drug selection system by BS.
  • FIG. 10 shows the construct of the donor DNA.
  • Neutral site (NS1) was selected as the area for introducing the BSD.
  • P catalase indicates a promoter of a catalase gene
  • TUBQ indicates a terminator of a ubiquitin gene.
  • the obtained DNA fragment was cloned and the BSD marker set of P- catalase (SEQ ID NO: 19) -BSD (SEQ ID NO: 25) -TUBQ (SEQ ID NO: 20) was inserted.
  • Escherichia coli was transformed with the obtained plasmid, propagated, and then the plasmid was extracted.
  • PCR amplification was performed using the obtained plasmid as a template using puc19_F and puc19_R, and the obtained DNA fragment was used as a donor DNA for a BSD marker.
  • the sequence of the NS1 region and its upstream and downstream 200 bp is shown in FIG.
  • the sequence of the NS1 region is shown in SEQ ID NO: 15.
  • the sequence of the NS1 region used as the 5'homology arm in the donor DNA is shown in SEQ ID NO: 29.
  • the sequence of the NS1 region used as the 3'homology arm in the donor DNA is shown in SEQ ID NO: 30.
  • NS1_F cggtacccggggatcTTTATGGAGAGCATCGTGAATAACGGC (SEQ ID NO: 13)
  • NS1_R cgactctagaggatcTGCAGAATAACCGGTGAAATTTATGAAC (SEQ ID NO: 14)
  • the transformant (BSD) prepared as described above is planted in MA medium (pH 1.0) or BS-containing MA medium (pH 1.0, BS 100 ⁇ g / mL) and cultured for 21 days under the same conditions as above. bottom.
  • MA medium pH 1.0
  • BS-containing MA medium pH 1.0, BS 100 ⁇ g / mL
  • WT gardenia
  • FIG. 12 is the result of Garderia (diploid)
  • FIG. 13 is the result of Garderia (polyploid).
  • Neither the untransformed WT nor the transformant (BSD) could grow Garderia (diploid) in BS-containing MA medium.
  • WT could not grow in BS-containing MA medium
  • transformant (BSD) could grow in BS-containing MA medium. From this result, it was confirmed that in Garderia (polyploid), a BS-resistant transformant can be obtained by homologous recombination.
  • Garderia (diploid) BS-resistant transformants could not be obtained. It was considered that this is because gardenia (diploid) has a strong cell wall, so that donor DNA is difficult to be introduced into cells.
  • PS1_F TCCCAAGATAATAGACAGTGCTCGG (SEQ ID NO: 31)
  • PS1_R TTGTTACCTACTCATACCCCTACTCC (SEQ ID NO: 32)
  • FIG. 15 shows the construct of the donor DNA for introducing mVenus.
  • the mVenus gene set (PEF1 - ⁇ -mVenus-T ⁇ -tubulin ) was inserted upstream of the BSD marker set.
  • the obtained plasmid was used as a donor DNA for introducing mVenus.
  • P EF1- ⁇ indicates the promoter of the EF1- ⁇ gene
  • T ⁇ -tubulin indicates the terminator of the ⁇ -tubulin gene.
  • the transformant (TF) or WT obtained above was immunobloted using Anti-GFP antibody (JL-8, Clontech). The result is shown in FIG. In the transformant (TF), a band of mVenus was confirmed. From this result, it was confirmed that the transformant (TF) expressed the mVenus protein.
  • DIC is a differential interference microscope image
  • Chl is a fluorescence microscope image in which autofluorescence of chlorophyll is detected
  • mVenus is a fluorescence microscope image in which mVenus fluorescence is detected
  • merged is Chl and mVenus. It is a merged fluorescence microscope image.
  • fluorescence of mVens could be confirmed in the transformant (TF), and it was confirmed that functional mVenus was produced.

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