JP7587838B2 - Methods for performing genome editing in microalgae - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applied. Published in the 83rd Annual Meeting of the Botanical Society of Japan, published on September 1, 2019.

Application of Article 30, Paragraph 2 of the Patent Act Presented at the 83rd Annual Meeting of the Botanical Society of Japan held at Tohoku University on September 16, 2019

The present disclosure provides a nucleic acid construct for performing genome editing in Nannochloropsis microalgae, or a method for performing genome editing using the same. The present disclosure also provides a Nannochloropsis microalgae that includes the nucleic acid construct. Furthermore, the present disclosure provides a nucleic acid for constructing the nucleic acid construct, or a method for constructing the same.

Nannochloropsis microalgae have the property of accumulating oils and fats, and are expected to be used as a means of producing biofuel and as feed for fish and other animals. When applying and utilizing Nannochloropsis species that have such properties, technology for artificially modifying their genome sequence is useful. Genome editing technology is one of the technologies for artificially modifying the genome of an organism. The present invention can be used to precisely modify the genome of Nannochloropsis species with high efficiency using genome editing technology, and to create varieties (strains) of Nannochloropsis species that are endowed with new properties.

To perform genome editing in eukaryotes, methods such as Crispr/Cas9 and TALEN can be used (e.g., International Publication No. 2014/199358). In the Crispr/Cas9 method, two elements, the Cas9 protein and gRNA, are introduced into target cells. Once an experimental system has been successfully constructed, Crispr/Cas9 can perform genome editing on almost all genes contained in the genome of the organism by simply modifying a portion (about 20 bp) of the base sequence of the gRNA. Therefore, it is desirable to establish a Crispr/Cas9 method that is useful in target organisms.

International Publication No. 2014/199358

The present inventors have found a structure of a nucleic acid construct that can perform genome editing with high efficiency in Nannochloropsis microalgae. Based on this finding, the present disclosure also provides a nucleic acid construct for performing genome editing in Nannochloropsis microalgae, a method for performing genome editing using the same, and Nannochloropsis microalgae containing the nucleic acid construct. Furthermore, the present disclosure provides a nucleic acid for constructing the nucleic acid construct, or a method for constructing the same.

Thus, the present invention provides the following:
(Item 1)
A promoter sequence that recruits Type III RNA polymerase from Nannochloropsis microalgae;
a sequence encoding a guide RNA (gRNA), operably linked to the promoter sequence; and a terminator sequence operably linked to the sequence encoding the gRNA, which terminates transcription by the Type III RNA polymerase of Nannochloropsis microalgae.
A nucleic acid construct comprising:
(Item 2)
2. The nucleic acid construct according to claim 1, wherein the promoter sequence is a promoter sequence of the U6 RNA gene of Nannochloropsis microalgae.
(Item 3)
The promoter sequence is the sequence of SEQ ID NO: 1 (CCAAATGTCTTTGACGGTCGTCACGGTTTTGCGTCGTAAAACCTACGCAAGGAAAATTACCATCATCGACCCGACACAAACAAGCGGTCCTTAGAAATCAGGACCTTTTATTTTTTAAGAAGGGAGTGAATGGGTAACATTTCGTGGTGAGACTTCAACGAAAGTTTGGAGAAAAATTCCACATGTTGTACGCTCCCGGAAAAGAGAAGGAATATAAGGGCGTCACGAGTCATGACTTTCTAAACAGCAAGGTTCGCGTGAAACTCGTTTACGGGAAACC).
The nucleic acid construct according to claim 1 or 2, comprising a sequence having at least 85% identity with
4. The nucleic acid construct according to any one of items 1 to 3, wherein the terminator sequence comprises at least 4 consecutive thymidine bases.
(Item 5)
5. The nucleic acid construct according to any one of items 1 to 4, wherein the terminator sequence is a terminator sequence of the U6 RNA gene of Nannochloropsis microalgae.
(Item 6)
The terminator sequence is the sequence of SEQ ID NO: 2 (TTTTTTTCTTTTGATCAAATTACATTCTGATTCTTAGATACTTCACATGTTGATACTCCAC).
6. The nucleic acid construct according to any one of items 1 to 5, comprising a sequence having at least 85% identity with
(Item 7)
7. The nucleic acid construct of any one of items 1 to 6, wherein the sequence encoding the gRNA comprises a protospacer adjacent motif (PAM).
(Item 8)
The sequence encoding the gRNA is the sequence of SEQ ID NO: 3 (GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC).
8. The nucleic acid construct according to any one of items 1 to 7, comprising a sequence having at least 85% identity with
(Item 9)
The nucleic acid construct according to any one of items 1 to 8, wherein the sequence encoding the gRNA comprises a sequence of 15 to 25 bases in length of a sequence encoding a target gene.
(Item 10)
10. The nucleic acid construct according to any one of items 1 to 9, wherein the sequence encoding the gRNA comprises a PAM and a 15 to 25 base sequence present on the 5' side of the PAM in the sequence encoding the target gene.
(Item 11)
11. The nucleic acid construct according to item 9 or 10, wherein the target gene is green fluorescent protein (GFP).
(Item 12)
12. The nucleic acid construct of any one of items 1 to 11, wherein the gRNA is a single-stranded guide RNA.
(Item 13)
13. The nucleic acid construct of any one of items 1 to 12, further comprising a sequence encoding a Cas9 protein.
(Item 14)
16. The nucleic acid construct of any one of items 1 to 15, further comprising a sequence encoding a drug selection marker.
(Item 15)
15. The nucleic acid construct of any one of claims 1 to 14, wherein the Nannochloropsis microalgae is N. oceanica.
(Item 16)
16. The nucleic acid construct of any one of items 1 to 15, which is a plasmid.
(Item 17)
17. A Nannochloropsis microalga comprising the nucleic acid construct according to any one of items 1 to 16.
(Item 18)
A kit for preparing a nucleic acid construct for performing genome editing in Nannochloropsis microalgae,
A nucleic acid comprising a promoter sequence that recruits Type III RNA polymerase from Nannochloropsis microalgae;
and a nucleic acid comprising a terminator sequence that terminates transcription by Type III RNA polymerase of Nannochloropsis microalgae.
(Item 19)
1. A method for performing genome editing in Nannochloropsis microalgae, comprising:
A method comprising the step of introducing the nucleic acid construct according to any one of items 1 to 16, or the nucleic acid in the kit according to item 18, into Nannochloropsis microalgae.

It is contemplated that one or more of the above features may be provided in combinations in addition to those combinations explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present disclosure may enable highly efficient genome editing in Nannochloropsis microalgae, thereby providing easy genetic manipulation of Nannochloropsis microalgae.

The results of electrophoresis confirming the production of the 1006 bp α-tubulin promoter are shown. 1 shows a schematic diagram of the construction of pNAN0006. A) Schematic diagram of the structure of pNAN0006. B) SDS-PAGE of proteins extracted from wild-type strain (1) and pNAN0006 transformed strains (2, 3, 4). C) Western blot with anti-Cas9 antibody. A schematic diagram of the construction of pNAN1001, pNAN1002, and pNAN1003 is shown. The U6RNA-like gene and its surrounding sequences obtained from N. oceanica NIES 2145 strain are shown. The bold (black) part is presumed to code for the promoter, the sequence between (red) for the U6RNA, and the italics (green) for the terminator. The underlined part indicates the sequence used for the primer. The 3' and 5' parts (light blue) shown separately represent part of the sequence of the vector used for cloning. A) Schematic diagram of the structure of pNAN1001, pNAN1002, and pNAN1003. B) Schematic diagram of the structure of the expression cassette of pNAN1001, pNAN1002, and pNAN1003. A) pNAN1001 transformant, pNAN1002 transformant, pNAN1003 transformant, wild type (WT) RNA extracted from the reverse transcriptase (+) or without (-) conditions is shown electrophoresis of the sample (110 bp gRNA cDNA). B) pNAN1001 transformant, pNAN1002 transformant, pNAN1003 transformant, wild type (WT) RNA extracted from the reverse transcriptase (+) or without (-) conditions is shown electrophoresis of the sample (230 bp β-tubulin mRNA cDNA). A schematic diagram of the structure of pNAN0005 is shown. A) Fluorescent stereomicroscope images of colonies formed on an agar medium of a pNAN0005 transformed strain. B) to D) Fluorescent stereomicroscope images of colonies formed from a strain further transformed with pNAN1002. This shows the analysis of the GFP gene in cells in which genome editing was performed by transformation with pNAN1002. After PCR amplification of the GFP gene (a 201 bp or 756 bp fragment appears), the fragment was digested with AluI. 1 to 10 show the experimental results of independent strains. In strains in which genome editing has occurred, a 105 bp band appears. Genome editing has occurred in 1, 2, 5, 6, and 7. 1 shows changes in the partial base sequence of the GFP gene caused by transformation with pNAN1002. 1 shows a schematic diagram of the construction of pNAN1006 based on pNAN1002. The results of electrophoresis of a portion of the PCR amplified product of the CA2 gene obtained from each colony with or without EcoRV treatment are shown. 1: Wild type, not treated with EcoRV. 2: Wild type, treated with EcoRV. 3 to 10: Zeocin-resistant colonies generated after transformation with pNAN1006, treated with EcoRV, respectively. The 516 bp band indicates the occurrence of genome editing. Shown are fluorescent micrographs (A-C) of colonies of the NIES-2146, NIES-2587, and NIES-2588 strains into which pNAN0005 was introduced, as well as photographs (D, E) of colonies of each strain into which genome editing was performed by introducing pNAN1002, and photographs (F-H) of colonies that became entirely red after single colony isolation from colonies containing red parts. The figure shows the change in the base sequence at the target site of the GFP gene of the colony strains that started to show red fluorescence. The top is the sequence of the wild-type GFP gene (16-80). 1 and 2 are the base sequences of the GFP gene of the strains derived from NIES-2146, 3 and 4 are the strains derived from NIES-2587, and 5 and 6 are the base sequences of the GFP gene of the strains derived from NIES-2588.

Hereinafter, the present disclosure will be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified. Therefore, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.

The following provides definitions of terms and/or basic technical content that are particularly used in this specification.

As used herein, "genome editing" refers to the artificial modification of genes within a cell. The genes modified by genome editing may be endogenous genes of a species, or may be present on a nucleic acid construct (e.g., a plasmid) that has been exogenously introduced into the organism.

As used herein, "protein," "polypeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term also encompasses naturally or artificially modified polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety).

As used herein, "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. Examples of nucleic acids include DNA, RNA, cDNA, mRNA, rRNA, tRNA, microRNA (miRNA), and lncRNA. The term also includes "polynucleotide derivatives." "Polynucleotide derivatives" refers to polynucleotides that contain nucleotide derivatives or have unusual internucleotide linkages. "Nucleotide derivative" refers to a nucleotide having a structure different from that of the normal nucleotide used in natural DNA or RNA, such as locked nucleic acid (LNA), ethylene nucleic acid such as 2'-O,4'-C-ethylene bridged nucleic acid (ENA), other bridged nucleic acid (BNA), hexitol nucleic acid (HNA), amido-bridged nucleic acid (AmNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), polyether nucleic acid (see, for example, U.S. Patent No. 5,908,845), cyclohexene nucleic acid (CeNA), etc. Examples of internucleotide bonds that are different from the normal ones include interoligonucleotide bonds in which the phosphodiester bond is converted to a phosphorothioate bond, interoligonucleotide bonds in which the phosphodiester bond is converted to an N3'-P5' phosphoramidate bond, and interoligonucleotide bonds in which the ribose and phosphodiester bond are converted to a peptide nucleic acid bond.

Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass the sequence explicitly indicated, as well as conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.

As used herein, the term "nucleic acid construct" refers to an artificially created nucleic acid molecule. Nucleic acid constructs can be created using any known method, such as a method using an enzyme (such as a restriction enzyme), chemical synthesis, or a method using an organism (such as E. coli). Nucleic acid constructs may be RNA or DNA, may contain modified nucleotides, and are typically plasmids.

As used herein, a "gene" refers to a nucleic acid portion that performs a certain biological function. Examples of such biological functions include encoding a polypeptide or protein, encoding a non-protein-coding functional RNA (such as guide RNA, rRNA, tRNA, microRNA (miRNA), lncRNA, etc.), controlling the production of a polypeptide, protein, or non-protein-coding functional RNA, specifically binding to a specific protein, and controlling the cleavage or replication of a nucleic acid. Thus, in this specification, a gene includes not only a nucleic acid portion that encodes a protein or a non-protein-coding functional RNA, but also transcription/translation regulatory sequences such as promoters, terminators, enhancers, silencers, and replication origins. As used herein, a "gene product" may refer to a polypeptide, protein, or non-protein-coding functional RNA encoded by a gene.

As used herein, "operably linked" refers to a sequence in which the expression (operation) of a desired sequence is placed under the control of a transcriptional/translational regulatory sequence (e.g., a promoter, enhancer, etc.) or a translational regulatory sequence. For a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but is not necessarily placed adjacent to the gene.

As used herein, "transcriptional/translational regulatory sequences" refers collectively to promoters, polyadenylation signals, transcriptional termination sequences, upstream regulatory domains, replication origins, enhancers, and the like, which cooperate to allow the replication, transcription, and translation of a coding sequence in a cell. Not all of these transcriptional/translational regulatory sequences need necessarily be present, so long as the replication, transcription, and translation of a selected coding sequence is possible in an appropriate host cell. Those skilled in the art can deduce regions containing regulatory nucleic acid sequences from published information. Furthermore, those skilled in the art can identify transcriptional/translational regulatory sequences that are applicable to the intended use, e.g., in vivo, ex vivo, or in vitro.

As used herein, a "promoter" refers to a segment of a nucleic acid sequence that controls transcription of an operably linked nucleic acid sequence. A promoter contains specific sequences that are sufficient for recognition, binding, and initiation of transcription by RNA polymerase. A promoter may contain sequences that regulate RNA polymerase recognition, binding, or initiation of transcription.

As used herein, "enhancer" refers to a segment of a nucleic acid sequence that functions to increase the efficiency of expression of a gene of interest.

As used herein, "silencer" refers to a segment of a nucleic acid sequence that, in contrast to an enhancer, functions to reduce the expression efficiency of a gene of interest.

As used herein, "terminator" refers to a segment of a nucleic acid sequence that is located downstream of a region encoding a protein or functional RNA and is involved in terminating transcription when a nucleic acid is transcribed into RNA.

As used herein, "origin of replication" refers to a segment of a nucleic acid sequence where replication begins when a protein that recognizes that nucleic acid sequence binds, partially unwinding the DNA double helix.

In this specification, the "homology" of a nucleic acid refers to the degree of identity between two or more nucleic acid sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two nucleic acids, the higher the identity or similarity of their sequences. "Similarity" is a value that takes into account similar bases in addition to identity, and similar bases here refer to cases where a portion of the mixed bases (e.g., R=A+G, M=A+C, W=A+T, S=C+G, Y=C+T, K=G+T, H=A+T+C, B=G+T+C, D=G+A+T, V=A+C+G, N=A+C+G+T) match. Whether two types of nucleic acids have homology can be examined by direct comparison of the sequences or by hybridization methods under stringent conditions. When two nucleic acid sequences are compared directly, the genes have homology if the nucleic acid sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.

Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one-letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.10.1+ (published 2020.6.18). The identity value in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them shall be regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

In this specification, unless otherwise specified, a reference to a biological substance (e.g., a protein, a nucleic acid, a gene) is understood to also refer to a variant of that biological substance (e.g., a variant having modifications in the amino acid sequence) that exerts a biological function similar to, but not necessarily to, the same degree as, the biological function of the biological substance. Such variants can include fragments of the original molecule, molecules that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical across the amino acid or nucleic acid sequence of the original biological substance of the same size, or compared to the sequence of the original molecule as aligned by computer homology programs known in the art. Variants can include molecules with modified amino acids (e.g., modified by disulfide bond formation, glycosylation, lipidation, acetylation or phosphorylation) or modified nucleotides (e.g., modified by methylation).

As used herein, "stringent conditions" refers to conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, stringent conditions include hybridization in 6xSSC (sodium chloride/sodium citrate) at about 45°C, followed by one or more washes in 0.2xSSC, 0.1% SDS at 50-65°C. See Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has, or is predicted to have, the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide that is used as a reference for comparison. A corresponding amino acid may be, for example, a specific amino acid that is cysteinized, glutathionylated, S-S bond formed, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Such a "corresponding" amino acid or nucleic acid may be a region or domain spanning a certain range. In such cases, it is referred to as a "corresponding" region or domain in this specification.

As used herein, a "corresponding" gene (e.g., a polynucleotide sequence or molecule) refers to a gene (e.g., a polynucleotide sequence or molecule) in a given species that has or is predicted to have the same function as a given gene in a species that is used as a reference for comparison, and in the case where there are multiple genes with such function, refers to genes that have the same evolutionary origin. For example, a corresponding gene in a given microalgae species can be found by searching a sequence database for that microalgae species using the gene sequence of the reference microalgae species for the corresponding gene as a query sequence.

In accordance with the present invention, the term "activity" as used herein refers to the function of a molecule in its broadest sense. Activity generally includes, but is not intended to be limiting, the biological, biochemical, physical or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress or destabilize the function of other molecules, stability, ability to localize to a particular subcellular location. Where applicable, the term also relates to the function of protein complexes in their broadest sense.

As used herein, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, and includes, but is not limited to, a specific cell surface structure recognition ability, an enzyme activity, and a binding ability to a specific protein. In the present disclosure, for example, a function that a promoter is recognized in a specific host cell may be included, but is not limited to these. As used herein, a biological function may be exerted by "biological activity." As used herein, "biological activity" refers to an activity that a certain factor (e.g., a polynucleotide, a protein, etc.) may have in a living body, and includes an activity that exerts various functions (e.g., a transcription promoting activity), and also includes, for example, an activity that activates or inactivates another molecule by interacting with another molecule. For example, when a certain factor is an enzyme, its biological activity includes its enzymatic activity. In another example, when a certain factor is a ligand, it includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as a measure of the amount of an upstream or downstream protein or other similar function in a host cell.

As used herein, "plasmid" refers to circular DNA that exists separately from chromosomes in bacterial cells for construction and amplification, and that, when introduced into algal cells, is either integrated into the chromosome or exists as circular DNA separately from the chromosome. When introducing into algal cells, all or part of the plasmid can be linearized by means of restriction enzyme treatment or PCR amplification, etc., before use.

As used herein, the term "transformation" refers to the introduction of nucleic acid into a host cell. Any method for introducing nucleic acid into a host cell can be used as a transformation method, including various well-known techniques such as the use of competent cells, electroporation, a method using a particle gun (gene gun), and the calcium phosphate method.

As used herein, a "purified" substance or biological factor (e.g., a nucleic acid or protein, etc.) refers to a biological factor from which at least a portion of the factors naturally associated with the biological factor have been removed. Thus, typically, the purity of the biological factor in a purified biological factor is higher (i.e., concentrated) than the state in which the biological factor is normally present. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor is present. The substance used in the present invention is preferably a "purified" substance.

In this specification, the terms "drug", "agent" or "factor" (all of which correspond to the English word agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), composite molecules thereof, and mixtures thereof.

As used herein, a "complex" or a "complex molecule" refers to any construct that includes two or more parts. For example, if one part is a polypeptide, the other part may be a polypeptide or another substance (e.g., a substrate, a sugar, a lipid, a nucleic acid, another carbohydrate, etc.). As used herein, the two or more parts that make up a complex may be bonded by a covalent bond or by another bond (e.g., hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.).

In this specification, the term "label" refers to an entity (e.g., substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When a target protein or a factor or means for capturing it is labeled by a fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the fluorescence emission maximum wavelength is preferably 10 nm or more. Any label that does not affect the function can be used, and an example of a fluorescent substance is Alexa ^™ Fluor. Alexa ^™ Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes with a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa ^™ 555 and Alexa ^™ 633, and a combination of Alexa ^™ 488 and Alexa ^™ 555. Other fluorescent labels include cyanine dyes (e.g., Cy3, Cy5, etc. of the CyDye ^™ series), rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc. In the present disclosure, such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.

As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., elements constituting a nucleic acid construct, instructions, etc.) are provided, usually divided into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit is advantageously provided with instructions or instructions describing how to use the parts provided or how to handle the reagents. When the kit is used herein as a reagent kit, the kit usually includes instructions describing how to use each element to constitute the nucleic acid construct.

The term "about" refers to the indicated value plus or minus 10%. When "about" is used in reference to temperature, it refers to the indicated temperature plus or minus 5° C., and when "about" is used in reference to pH, it refers to the indicated pH plus or minus 0.5.

Preferred Embodiments
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used alone or in combination.

In one aspect, the present disclosure provides nucleic acid constructs for performing genome editing in Nannochloropsis microalgae. Any means for accomplishing this is contemplated as being within the scope of the present disclosure. For example, even if not explicitly stated, a description of a nucleic acid construct also contemplates embodiments reflecting other means, such as compositions including the nucleic acid construct, methods of using the nucleic acid construct, and methods or materials for making the nucleic acid construct.

Nucleic acid constructs for genome editing of Nannochloropsis microalgae
In one aspect, the present disclosure provides a nucleic acid construct for performing genome editing in Nannochloropsis microalgae. Nannochloropsis microalgae have a small (about 1.5 to 5 micrometers) spherical cell body and are not motile. Nannochloropsis microalgae are known to be characterized by having chlorophyll a but lacking chlorophyll b and chlorophyll c. Nannochloropsis microalgae are also known to have pigments such as astaxanthin, zeaxanthin, and canthaxanthin. Nannochloropsis microalgae are known to have the ability to accumulate many polyunsaturated fatty acids, and are expected to be used as feed for fish, food additives for humans, a source of biofuel, and a source for the biological production of various useful compounds. Nannochloropsis microalgae species include australis, gaditana, granulata, limnetica, oceanica, oculata, and salina. These species were previously classified as Nannochloropsis, but recent studies have led to the classification of some as Microchloropsis (Marvin W. Fawley et al., Phycologia Volume 54 (5), 545-552). There are also cases where microalgae previously classified as Marine Chlorella have been reclassified as N. oceanica. Microalgae classified into the genus Nannochloropsis or Microchloropsis according to the latest classification can be used as suitable Nannochloropsis microalgae in the present disclosure. In a specific embodiment, the nucleic acid construct of the present disclosure can be used for genome editing of Nannochloropsis microalgae of N. oceanica, N. oculata, N. gaditana (Microchloropsis gaditana) or N. granulata.

In one embodiment, the nucleic acid construct comprises a promoter sequence that recruits the Type III RNA polymerase of Nannochloropsis microalgae. In one embodiment, the nucleic acid construct comprises a sequence encoding a guide RNA (gRNA) operably linked to the promoter sequence. In one embodiment, the nucleic acid construct comprises a terminator sequence that terminates transcription by the Type III RNA polymerase of Nannochloropsis microalgae operably linked to the sequence encoding the gRNA. In one embodiment, the nucleic acid construct comprises these promoter sequence, gRNA encoding sequence, and terminator sequence. In one embodiment, the nucleic acid construct of the present disclosure causes genome editing in microalgae by the Crispr/Cas9 system. In one embodiment, the nucleic acid construct is a plasmid.

(Promoter)
In one embodiment, the nucleic acid construct of the present disclosure comprises a promoter sequence that recruits Type III RNA polymerase of Nannochloropsis microalgae to express a guide RNA. Type III RNA polymerase is believed not to add additional structures such as 5'-end modification and 3'-end modification to the transcript, so gRNA produced by transcription with Type III RNA polymerase can enable highly efficient genome editing without activity reduction due to the addition of modified structures. The inventors have found a promoter sequence in Nannochloropsis microalgae that is expected to recruit Type III RNA polymerase, and have made highly efficient genome editing possible by utilizing this promoter sequence. Whether a nucleic acid having a specific sequence recruits Type III RNA polymerase can be examined, for example, by contacting the nucleic acid with Type III RNA polymerase derived from Nannochloropsis microalgae in vivo or in vitro and observing binding.

、配列番号８または９の配列と少なくとも７０％、７５％、８０％、８５％、９０％、９５％、または９９％の同一性を有する配列を含む。本願実施例において、配列番号１の配列の下線部の２４塩基を除去してもゲノム編集が達成されることが確認された。そのため、一つの実施形態において、プロモータ配列は、配列番号１の配列の３’末端から１～５０塩基（例えば、約１、約２、約５、約１０、約１５、約２０、約２５、約３０、約３５、約４０、約４５または約５０塩基など）だけ短縮された配列と少なくとも７０％、７５％、８０％、８５％、９０％、９５％、または９９％の同一性を有する配列を含んでもよい。 In one embodiment, the promoter sequence that recruits the Type III RNA polymerase of Nannochloropsis microalgae can include the promoter sequence of the U6 RNA gene of Nannochloropsis microalgae. In one embodiment, the promoter sequence has the sequence of SEQ ID NO:1.

, SEQ ID NO: 8 or 9. In the present examples, it was confirmed that genome editing can be achieved even if the underlined 24 bases of the sequence of SEQ ID NO: 1 are removed. Therefore, in one embodiment, the promoter sequence may include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a sequence shortened by 1 to 50 bases (e.g., about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 bases, etc.) from the 3' end of the sequence of SEQ ID NO: 1.

の５’末端から１～１０塩基（例えば、１、２、３、４、５、６、７、８、９または１０塩基）の配列をさらに含んでもよい。配列番号１の配列は、Ｎ．ｏｃｅａｎｉｃａから取得された配列であるが、他の種のナンノクロロプシス微細藻類にも適用され得る。特に、Ｎ．ｏｃｅａｎｉｃａは、Ｎ．ｇｒａｎｕｌａｔａ、およびＮ．ｏｃｕｌａｔａと近縁であるため（ＰＬｏＳ Ｇｅｎｅｔ． ２０１４ Ｊａｎ；１０（１）：ｅ１００４０９４）、これらの種において有利に使用され得る。また、本明細書の記載を参考にして、Ｎ．ｏｃｅａｎｉｃａ以外のナンノクロロプシス微細藻類のＵ６プロモータ配列も、マウスなどの公知の生物のまたは本明細書に記載のＮ．ｏｃｅａｎｉｃａのＵ６ ＲＮＡ様遺伝子配列および／またはＵ６プロモータ配列との相同性に基づいて当業者は決定することができ、Ｎ．ｏｃｅａｎｉｃａのＵ６プロモータ配列と同様に本開示のゲノム編集において使用することができる。 In one embodiment, the promoter sequence is the U6 RNA-like gene sequence of SEQ ID NO: 4, which is located immediately following the sequence of SEQ ID NO: 1 at the 3' end in N. oceanica.

The sequence of SEQ ID NO: 1 may further include a sequence of 1 to 10 bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) from the 5' end of N. oceanica. The sequence of SEQ ID NO: 1 is a sequence obtained from N. oceanica, but may also be applied to other species of Nannochloropsis microalgae. In particular, since N. oceanica is closely related to N. granulata and N. oculata (PLoS Genet. 2014 Jan; 10(1): e1004094), it may be advantageously used in these species. In addition, with reference to the description in this specification, a person skilled in the art can determine the U6 promoter sequence of Nannochloropsis microalgae other than N. oceanica based on the homology with the U6 RNA-like gene sequence and/or U6 promoter sequence of known organisms such as mice or N. oceanica described herein, and the U6 promoter sequence of N. The U6 promoter sequence of N. oceanica can be used in the genome editing of the present disclosure.

(Guide RNA)
In one embodiment, the nucleic acid construct of the present disclosure may include a sequence encoding a guide RNA. As used herein, "guide RNA" (gRNA) refers to an RNA molecule that is used in cleaving a target gene by Cas9 protein in a CRISPR/Cas9 system, and includes a portion that targets the target gene and a portion that assists the cleavage of nucleic acid by Cas9 protein. The guide RNA may include CRISPR RNA (crRNA). In one embodiment, the guide RNA may not include a transactivating CRISPR RNA (tracrRNA), and in such an embodiment, tracrRNA may be provided by some means in the target cell of genome editing. For example, the tracrRNA may be encoded in a portion of the nucleic acid construct of the present disclosure that is separate from the portion that encodes the guide RNA, or may be supplied by introducing a nucleic acid construct (e.g., a plasmid encoding the tracrRNA, the tracrRNA itself, etc.) separate from the nucleic acid construct of the present disclosure into the target cell, or may be endogenously supplied in a target cell that has been modified to express the tracrRNA. In one embodiment, the guide RNA may be a single-stranded guide RNA that induces nucleic acid cleavage by the Cas9 protein as a single molecule, and in this embodiment, the supply of the tracrRNA may not be necessary.

In one embodiment, the guide RNA may include a portion having a sequence (which may be referred to herein as a homing sequence) that hybridizes with a portion of the target gene (e.g., a continuous base sequence having a length of about 10 to 30 bases, e.g., about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, or about 30 bases). In the present specification, the target gene refers to a gene to be cut in the genome editing described herein, and when the gene is on a double strand, even if the portion that exerts its function (e.g., a coding sequence) is only on one strand, it refers to both the portion that exerts its function and the complementary portion present on the other strand. In one embodiment, the homing sequence in the guide RNA may have a sequence that is completely complementary to a portion of the target gene, but even if it does not have a sequence that is completely complementary to a portion of the target gene, the desired function can be exerted as long as it can hybridize with a portion of the target gene. In one embodiment, the homing sequence in the guide RNA has a sequence that hybridizes under stringent conditions with a nucleic acid molecule having a partial base sequence of the target gene. In one embodiment, the homing sequence in the guide RNA may include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with a base sequence complementary to a partial base sequence of the target gene.

In one embodiment, the portion of the target gene targeted by the guide RNA can be determined taking into account the protospacer adjacent motif (PAM). The PAM differs for each Cas9 protein used. For example, the Cas9 protein of S. pyogenes recognizes the PAM in the base sequence NGG and is known to cause cleavage in the target sequence adjacent to the 5' side of the PAM.

In a preferred embodiment, the homing sequence is located at the 5' end of the guide RNA. In one embodiment, the 5' end of the homing sequence is within 1, 2, 3, 4, or 5 bases from the 5' end of the guide RNA. A common sequence can be used for the partial sequence of the guide RNA other than the homing sequence (e.g., depending on the Cas9 protein used), and such sequences are known to those skilled in the art or can be easily designed.

Examples of target genes include genes encoding or regulating proteins (e.g., lipid metabolism-related enzymes, photosynthesis-related genes, growth-related genes, proteins that regulate protein expression), genes encoding or regulating functional RNAs (e.g., microRNAs), and marker genes for evaluating the occurrence of genome editing (e.g., genes encoding luminescent proteins such as green fluorescent protein (GFP)). More specific examples of target genes include, for example, the acetyl-CoA carboxylase gene, the ADP-glucose pyrophosphorylase gene, the ribulose 1,5-bisphosphate carboxylase/oxygenase gene, the Lhc gene, the carbonic anhydrase gene, the cyclin gene, and the MAP kinase gene. By appropriately selecting the target gene, modifications of the growth ability, metabolic products, photosynthetic efficiency, and the like of the Nannochloropsis microalgae can be achieved. Those skilled in the art can appropriately design a homing sequence that targets a suitable site of the target gene, taking into account the Cas9 protein, PAM, and the like to be used.

と少なくとも７０％、７５％、８０％、８５％、９０％、９５％、または９９％の同一性を有する配列を含み得る。 Those skilled in the art can design a sequence encoding a guide RNA based on the structure of the guide RNA of interest, and can prepare a nucleic acid construct containing a sequence encoding a guide RNA. The guide RNA may be complementary to a partial sequence and/or a protospacer adjacent motif (PAM) of a target gene, so that the sequence encoding a guide RNA may have a partial sequence and/or a PAM of a target gene. In one embodiment, the sequence encoding a gRNA may include a PAM and a 15-25 base-long sequence present on the 5' side of the PAM in the sequence encoding the target gene. In one embodiment, the sequence encoding a guide RNA may be operably linked to a promoter sequence downstream of the promoter. In one embodiment, the sequence encoding a guide RNA may be a sequence of SEQ ID NO: 3.

The sequence may include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to

と少なくとも７０％、７５％、８０％、８５％、９０％、９５％、または９９％の同一性を有する配列を含み得る。 (terminator)
In one embodiment, the nucleic acid construct of the present disclosure comprises a terminator sequence that terminates the transcription by Type III RNA polymerase of Nannochloropsis microalgae, operably linked to a sequence encoding gRNA. For example, whether the terminator sequence terminates the transcription by Type III RNA polymerase can be easily confirmed by those skilled in the art, for example, by determining the length (size) of the transcript of the sequence operably linked to the promoter sequence that recruits Type III RNA polymerase as described herein. In one embodiment, the terminator sequence can include at least 3, 4, 5, 6, 7 or 8 consecutive thymine bases.
In one embodiment, the terminator sequence is the sequence of SEQ ID NO:2.

The sequence may include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to

(Other elements)
In one embodiment, the nucleic acid construct of the present disclosure may include a sequence encoding Cas9. In this embodiment, it may be possible to express the Cas9 protein and the guide RNA in the target cell by simply introducing a single nucleic acid construct (e.g., a plasmid), which may simplify the operation and improve the efficiency of genome editing. In another embodiment, the Cas9 protein may be introduced into the target cell separately from the nucleic acid construct of the present disclosure (e.g., by transforming the target cell with another plasmid encoding the Cas9 protein, modifying the target cell to endogenously express the Cas9 protein, or by introducing the Cas9 protein into the target cell). In one embodiment, the nucleic acid construct of the present disclosure may include a sequence encoding Cas9 and a promoter sequence upstream thereof, which may be the same or different from the promoter sequence operably linked to the guide RNA. In one embodiment, the promoter sequence upstream of the sequence encoding Cas9 may be a promoter of a constitutively expressed gene, such as the promoter of the tubulin gene of the Nannochloropsis microalgae, and such promoters can be easily determined by one skilled in the art.

The Cas9 protein used in the present disclosure may be any known Cas9 protein, and may be not only a Cas9 protein naturally obtained from a microorganism, but also a modified version thereof. Such modified versions are known to those skilled in the art, and include, for example, Cas9 proteins modified to have a nuclear localization signal. Cas9 from any known biological species can be used. The sequence of the PAM recognized by Cas9 and the mode of cleavage of the target nucleic acid by Cas9 are known, and a person skilled in the art can select an appropriate Cas9 depending on the purpose. For example, S. Cas9 from P. pyogenes recognizes the PAM sequence 5'-XGG-3', Cas9 from Neisseria meningitidis recognizes the PAM sequence 5'-XXXXGATT-3', Cas9 from Streptococcus thermophilus recognizes the PAM sequence 5'-XXAGAA-3', and Cas9 from Treponema denticola recognizes the PAM sequence 5'-XAAAAC-3'. Those skilled in the art can select an appropriate Cas9 protein taking into consideration the target gene, etc.

In one embodiment, the nucleic acid construct of the present disclosure may include a sequence encoding one or more drug selection markers. The drug selection marker may confer resistance to any drug (e.g., zeocin, hygromycin) that inhibits colony formation (growth) of Nannochloropsis microalgae, so that successfully transformed colonies can be formed by culturing cells transformed with a nucleic acid construct containing the drug selection marker in the presence of the drug.

In one embodiment, the nucleic acid construct (e.g., a plasmid) of the present disclosure may have a replication origin that operates in Nannochloropsis microalgae. In one embodiment, the nucleic acid construct (e.g., a plasmid) of the present disclosure may have an enhancer that operates in Nannochloropsis microalgae. Any known replication origin and/or enhancer may be used. Nucleic acid elements that operate in Nannochloropsis microalgae do not need to be derived from Nannochloropsis microalgae. The nucleic acid construct of the present disclosure may be produced in cells of organisms other than Nannochloropsis microalgae, and may therefore include transcriptional/translational regulatory sequences, such as replication origins, promoters, and transcription termination sequences, that operate in those organisms. Transcriptional/translational regulatory sequences for each organism are known and can be selected appropriately by those skilled in the art.

In certain embodiments, the present disclosure provides a nucleic acid construct comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the sequence of pNAN1001 (Accession Number: NITE AP-03261), pNAN1002 (Accession Number: NITE AP-03262), or pNAN1003 (Accession Number: NITE AP-03263).

(Note)
As described above, the preferred embodiment of the present disclosure, the nucleic acid construct (e.g., a plasmid) of the present disclosure, may have a replication origin that operates in Nannochloropsis microalgae. In one embodiment, the nucleic acid construct (e.g., a plasmid) of the present disclosure may have an enhancer that operates in Nannochloropsis microalgae. Any known replication origin and/or enhancer may be used. The nucleic acid element that operates in Nannochloropsis microalgae does not need to be derived from Nannochloropsis microalgae. The nucleic acid construct of the present disclosure may be produced in cells of organisms other than Nannochloropsis microalgae, and may therefore include a replication origin, a promoter, a transcription termination sequence, and other transcriptional translation regulatory sequences that operate in those organisms. The transcriptional translation regulatory sequences for each organism are known and can be appropriately selected by those skilled in the art.

(Preparation of Nucleic Acid Construct)
In one aspect, the present disclosure provides a method for producing the nucleic acid construct of the present disclosure and a nucleic acid for producing the same. The nucleic acid construct of the present disclosure can be produced by combining any suitable method (such as ligation using a restriction enzyme) that includes any element of the nucleic acid construct of the present disclosure (e.g., a promoter sequence, a sequence encoding a gRNA, a terminator sequence) alone or collectively. In one embodiment, the nucleic acid for producing the nucleic acid construct of the present disclosure can be provided as a kit. In one embodiment, the kit can include one or more containers filled with at least one of the nucleic acids for producing the nucleic acid construct of the present disclosure. The kit may include instructions showing the method for producing the nucleic acid construct. Since a common system can be used regardless of the target gene of genome editing other than the homing sequence of the guide RNA, in one embodiment, the kit can include a nucleic acid that includes the nucleic acid construct of the present disclosure alone or collectively other than the base sequence encoding the homing sequence.

(Introduction of Nucleic Acid Construct)
The method of introducing the nucleic acid construct of the present disclosure into a target cell may be any method, including, for example, the use of competent target cells, electroporation, a method using a particle gun (gene gun), calcium phosphate method, and the like. Details of each method are known, and those skilled in the art can carry out the methods appropriately. The nucleic acid construct may be introduced into a target cell in the form of a circular plasmid, or may be introduced into a target cell in a linearized state by restriction enzyme treatment or PCR. In addition, only a portion containing a necessary element among the elements constituting the nucleic acid construct may be extracted by restriction enzyme treatment or PCR, and then introduced into the target cell. It is contemplated that the nucleic acid construct of the present disclosure includes both a nucleic acid construct to be introduced into a target cell and a nucleic acid construct (e.g., a plasmid) that is the source for preparing the nucleic acid construct to be introduced into the target cell. In one embodiment, the present disclosure provides a cell (such as Nannochloropsis microalgae) transformed with the nucleic acid construct of the present disclosure. Whether or not a cell has been transformed can be confirmed by any marker (such as expression of a color-producing protein, drug resistance, etc.).

(Composition)
In one embodiment, the present disclosure provides a composition for performing genome editing, comprising the nucleic acid construct of the present disclosure. The composition can be provided in any suitable form (e.g., a fluid containing the nucleic acid construct). The composition can include any suitable component, such as a component for stabilizing the nucleic acid construct of the present disclosure, a component that does not damage target cells, etc.

In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "alternative." In this specification, when it is stated that "within the range of" "two values," the range includes the two values themselves.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth herein.

Specific reagents used were those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

(Construction of plasmids for expressing cas9 in Nannochloropsis microalgae)
In order to stably express cas9 in Nannochloropsis microalgae, a plasmid pNAN0002 expressing a Zeocin resistance gene was first constructed using the β-tubulin promoter of Nannochloropsis oceanica NIES 2145 strain (obtained from the National Institute for Environmental Studies) and the Nos terminator derived from pCAMBIA1300, and then a cassette expressing the Cas9 gene with the promoter of the α-tubulin gene of N. oceanica NIES 2145 strain was introduced into pNAN0002 to obtain pNAN0006. The outline is shown in FIG.

pNAN0002 was constructed as follows.
The downstream of the Zeocin resistance gene of pEM7/Zeo (Invitrogen, V50020) was cleaved by PCR using primers P1 and P2 to obtain a DNA fragment of about 300 bp of Nos terminator obtained by PCR using primers P3 and P4 from pCAMBIA1300 (purchased from Marker Gene Technologies, Inc., USA) by in-fusion method, and pEM7/Zeo-NosT was obtained. Next, the sequence encoding β-tubulin was searched for in the database of N. oceanica CCMP1779 v2.0 on the JOINT GENOME INSTITUTE website (https://jgi.doe.gov/), and the upstream sequence was identified as the promoter sequence.
The upstream portion of pEM7/Zeo-NosT was cleaved by PCR using P5 and P6, and a DNA fragment of about 550 bp of β-tubulin promoter obtained from N. oceanica NIES 2145 strain by PCR using P7 and P8 was ligated by in-fusion method to obtain pNAN0002 of about 3.6 Kbp.

A cassette expressing the Cas9 gene using the promoter of the α-tubulin gene of the 2145 strain was constructed as follows.
The sequence (below) containing the Cas9 gene and terminator sequence (a part (2780 bp-7347 bp) described in Supplementary Note 1 of Nature Biotechnology VOLUME 35 NUMBER 7 2017 647-655) modified by Imad Ajjawi et al. based on SpCas9 was totally synthesized to obtain the plasmid pNAN-cas9-t.
SEQ ID NO: 5: >2780-7347

The sequence encoding α-tubulin was searched for in the database of N. oceanica CCMP1779 v2.0 on the JOINT GENOME INSTITUTE website (https://jgi.doe.gov/), and the upstream sequence was identified as the promoter sequence (SEQ ID NO: 6). A DNA fragment of about 1000 bp of the promoter region of the α-tubulin gene was obtained from N. oceanica NIES 2145 strain by PCR using P29 and P30, and confirmed by electrophoresis (Figure 1).
SEQ ID NO: 6: α-tubulin promoter

The upstream of the Cas9 gene in pNAN-cas9-t was cleaved by PCR using primers P26 and P27, and the DNA encoding the approximately 1000 bp α-tubulin promoter was connected by in-fusion to obtain pNAN aTubP-Cas9-t. The DNA fragment encoding the expression cassette of pNAN aTubP-Cas9-T (α-tubulin promoter: Cas9 gene: terminator) was obtained by PCR using primers P17 and P28, and pNAN0002 was ligated by blunt ligation to the fragment cleaved by PCR using primers P31 and P32 to obtain pNAN0006 (Figure 2).

pNAN0006 was introduced into N. oceanica NIES 2145 by electroporation, and colonies were selected based on Zeocin resistance to generate transformants (2, 3, and 4 each represent a different colony). The transformants were dissolved, and proteins were extracted and subjected to SDS-PAGE (Figure 3B). Western blotting was then performed using an anti-Cas9 antibody (Guide-it Cas9 Polyclonal Antibody, 632607, Takara Bio) to compare the production of Cas9 protein with that of the wild-type strain (1) (Figure 3C). Cas9 protein was not detected in the wild-type strain, but was detected only in the transformants.

配列番号７：Ｕ６プロモータ

配列番号２：Ｕ６ターミネータ

(Construction of plasmids for expressing sgRNA in Nannochloropsis microalgae)
The outline of the construction is shown in FIG. 4. A promoter for expressing sgRNA in Nannochloropsis microalgae was examined. A Nannochloropsis gene corresponding to the promoter of the U6 gene, which is known to mobilize TypeIII RNA polymerase in humans, etc., was searched for. A sequence homologous to the mouse U6 gene was searched for in the N. oceanica CCMP1779 v2.0 database, and a region containing a base sequence that may be the U6-like gene of N. oceanica was identified. The U6-like gene region has low conservation at the site thought to be the transcription start point, and may be a pseudogene, and it was not easy to identify the transcription start point in particular. The upstream region of GTATAA of the N. oceanica U6-like gene (SEQ ID NO: 4) was identified as the U6 promoter (SEQ ID NO: 7), and the downstream region was identified as the terminator (SEQ ID NO: 2).
SEQ ID NO: 4: U6-like gene

SEQ ID NO: 7: U6 promoter

SEQ ID NO:2: U6 terminator

Based on this, a U6 gene-like sequence and its surrounding sequence were obtained from N. oceanica NIES 2145 strain by PCR using primers P33 and P34, and inserted into pGEM-T Easy Vector (Promega) to obtain 2145-U6-Tvec (a partial sequence is shown in Figure 5).

Using 2145-U6-Tvec as a template and three primer pairs (P37 and P38, P37 and P39, and P37 and P40), inverse PCR was performed to obtain a DNA fragment lacking the U6-like gene but containing the U6 terminator and three types of U6 promoter sequences of different lengths.

A fragment of sgRNA containing a sequence complementary to sGFP was inserted into these three fragments by the in-fusion method. The sgRNA containing a sequence complementary to sGFP was prepared as follows. A plasmid (pGuide-it-sGFP36-58) containing a sequence corresponding to the sgRNA was obtained using oligo DNAs P35 and P36 and Guide-it CRISPR/Cas9 System (Green) (Clontech, Cat. No. 632601).

Using pGuide-it-sGFP36-58 as a template and three primer pairs (P41 and P42, P41 and P43, and P41 and P44), three types of DNA fragments were obtained. The fragments derived from P37 and P38 were linked to the fragments derived from P41 and P42, the fragments derived from P37 and P39 were linked to the fragments derived from P41 and P43, and the fragments derived from P37 and P40 were linked to the fragments derived from P41 and P44 by in-fusion, to obtain pU6-1 sGFP36-55, pU6-2 sGFP36-55, and pU6-3 sGFP36-55, respectively.

pU6-1 sGFP36-55, pU6-2 sGFP36-55, and pU6-3 sGFP36-55 were each digested with NotI to obtain DNA fragments encoding the U6 promoter (three different lengths; SEQ ID NO: 8, 9, and 1, respectively), gRNA (SEQ ID NO: 10), and U6 terminator (SEQ ID NO: 2).

・配列番号９：Ｕ６プロモータ（ｐＮＡＮ１００２）

・配列番号７３：Ｕ６プロモータ（ｐＮＡＮ１００３）

・配列番号１０：ｇＲＮＡ

（下線は、ＧＦＰをコードする配列の一部（ターゲット配列）；太字は、ｃａｓ９のＰＡＭ）
・配列番号２：Ｕ６ターミネータ

The three types of U6 promoters, gRNA, and U6 terminator coding sequences described in the previous section were introduced into the NotI site of a plasmid (pNAN0006) containing a cas9 coding sequence downstream of an α-tubulin promoter to produce pNAN1001, pNAN1002, and pNAN1003.
SEQ ID NO: 8: U6 promoter (pNAN1001)

SEQ ID NO: 9: U6 promoter (pNAN1002)

SEQ ID NO: 73 : U6 promoter (pNAN1003)

SEQ ID NO: 10: gRNA

(The underlined part of the sequence encoding GFP (target sequence); the bolded part is the PAM of cas9.)
SEQ ID NO: 2: U6 terminator

pNAN1001, pNAN1002, and pNAN1003 were deposited at the National Institute of Technology and Evaluation's Patent Microorganism Depositary Center and received on August 19, 2020. The accession numbers are NITE AP-03261, NITE AP-03262, and NITE AP-03263, respectively.

(Confirmation of sgRNA expression)
These plasmids were used to transform N. oceanica NIES 2145. RNA was extracted from pNAN1001 transformants, pNAN1002 transformants, pNAN1003 transformants, and wild-type strains, and the contaminating genomic DNA was digested with DNase, and then cDNA was synthesized using reverse transcriptase. Then, sgRNA was detected by PCR using cDNA as a template. The size of the target sgRNA was 110 bp. Since the 110 bp band was not detected in the sample to which reverse transcriptase was not added, it was confirmed that the 110 bp band was derived from the sgRNA transcribed in the cell (Figure 7A). In addition, using an equivalent amount of RNA to the above, the mRNA of β-tubulin constitutively expressed in N. oceanica was detected by RT-PCR, and it was confirmed that the same amount of sample was used in the above experiment (Figure 7B).

(Detection of genome editing)
We tested whether genome editing occurred by introducing the constructed plasmid.
A plasmid pNAN0005 encoding GFP was constructed. The structure of pNAN0005 is shown in FIG. 8. The T-BORDER(L) to T-BORDER(R) of pNAN0005 was extracted by PCR using P13 and P14, and this was used to transform N. oceanica NIES 2145 to create a strain that emits green fluorescence of GFP as a model experimental system (FIG. 9A). A genome editing vector (pNAN1001, pNAN1002 or pNAN1003) was introduced into this GFP-expressing strain. When genome editing is performed in the GFP gene, the GFP gene becomes nonfunctional and the fluorescence of GFP is lost. After the introduction of the plasmid, colonies of either GREEN (FIG. 9B), MIX (FIG. 9C) or RED (FIG. 9D) appear. GREEN refers to a colony that has not been genome-edited, MIX refers to a colony in which genome editing has occurred during the colony formation process, and RED refers to a colony formed from a single genome-edited cell. Note that a RED colony can be produced by obtaining a single cell from a MIX colony.

It was found that both plasmids induced genome editing with high efficiency. In particular, the genome editing efficiency with pNAN1003 was high.

ｐＮＡＮ１００２から生産されるｓｇＲＮＡはこのうちの第２のＡｌｕＩ認識部分に改変を導入し得るので、第２のＡｌｕＩ認識部分が失われることによって第１および第３のＡｌｕＩ認識部分の間の塩基配列を有する約１０５ｂｐの断片が生じ得る。 (Analysis of GFP gene in pNAN1002 transformants)
A single cell was obtained from the MIX colony of the pNAN1002 transformant, and a RED colony was formed and analyzed (1 to 10 each indicate a different colony). A plasmid was extracted from the colony, and a nucleic acid fragment (201 bp or 756 bp) containing the GFP gene of pNAN0005 was amplified by PCR, and the amplified fragment was digested with AluI (Figure 10). The 5'-side portion of the amplified GFP gene contains four AluI recognition sites, as underlined below.

The sgRNA produced from pNAN1002 may be modified in the second AluI recognition site, resulting in the loss of the second AluI recognition site and the generation of an approximately 105 bp fragment having the base sequence between the first and third AluI recognition sites.

Samples 1, 2, 5, and 6, which amplified a 201 bp fragment, were confirmed to produce a 105 bp band upon AluI treatment, while sample 7, which amplified a 756 bp fragment, was confirmed to produce a 105 bp band upon AluI treatment. Therefore, genome editing was confirmed in these samples.

Next, the base sequence of the GFP gene was determined for the samples in which genome editing was confirmed (Figure 11). As a result, a 5-bp deletion in the GFP gene of samples 1, 2, 5, and 6, and a 1-bp substitution in the GFP gene of sample 7 were confirmed.

（下線は、標的配列の相補配列を示す。）
・配列番号１３：ｓｇＲＮＡの５’末端側配列

（下線は、ｇＲＮＡ配列を示し、太字は、ＰＡＭを示す。） (Design of sgRNA for CA2 gene)
An sgRNA sequence (SEQ ID NO: 13) was designed that targets a sequence in the CA2 gene (SEQ ID NO: 12) of N. oceanica.
SEQ ID NO: 12: CA2 gene

(The underlined portion indicates the complementary sequence of the target sequence.)
SEQ ID NO: 13: 5'-end sequence of sgRNA

(Underlining indicates gRNA sequence, bold indicates PAM.)

A plasmid containing this sgRNA sequence was constructed based on pNAN1002. Specifically, PCR was performed using pNAN1002 as a template and primers P39 and P51. The PCR product was self-ligated to produce pNAN1006 (Figure 12).

(Detection of genome editing in the CA2 gene)
N. oceanica NIES 2145 was transformed with pNAN1006, and Zeocin-resistant colonies (colonies 3 to 10) were formed by incubation in the presence of Zeocin. For the wild type and each colony, PCR was performed to amplify a part of the CA2 gene (516 bp), and the PCR product was digested with EcoRV and analyzed (FIG. 13). When genome editing occurs, the EcoRV recognition sequence present in the 516 bp (present in the targeted sequence in the CA2 gene) changes, and a variant that is not cleaved by EcoRV appears. The presence of a 516 bp fragment was confirmed in the electrophoretic pattern, indicating that genome editing occurred in the CA2 gene.

(Genome editing using different strains)
pNAN0005 is introduced into various strains of Nannochloropsis microalgae to produce strains that emit GFP fluorescence. Then, pNAN1001, 1002, or 1003 is introduced into the strains that emit GFP fluorescence. Strains that may have undergone genome editing due to the disappearance of GFP fluorescence are selected, and finally, the base sequence of the relevant portion is determined to confirm genome editing.

Specifically, it was confirmed that genome editing is possible in N. oculata, N. gaditana (Microchloropsis gaditana), and N. granulata as follows.

1. Construction of strains for genome editing monitoring N. oculata NIES-2146, N. gaditana NIES-2587, and N. granulata NIES-2588 obtained from the National Institute for Environmental Studies were used to monitor genome editing in the strains. Plasmid pNAN0005 expressing GFP was introduced by electroporation.

2. Introduction of pNAN1002 into strains for monitoring pNAN1002 is an all-in-one vector that performs genome editing on the GFP gene. Strains into which pNAN1002 has been introduced acquire Zeocin resistance. Furthermore, if the introduced strain contains the GFP gene, genome editing is performed on the GFP gene. In cells in which genome editing has occurred, the sequence of the GFP gene changes and GFP fluorescence is lost. Cells that have lost GFP fluorescence begin to emit chlorophyll autofluorescence (red). pNAN1002 was further introduced into the above pNAN0005-introduced strain. After that, colonies that showed an overall red color were obtained by single colony isolation from colonies containing red parts that appeared on the selective medium containing Zeocin.

The results are shown in Figure 14. Introduction of pNAN0005 caused each strain to exhibit green fluorescence (Figures 14A-C), but in colonies where genome editing was further performed by introducing pNAN1002, cells that had lost the fluorescence of the target GFP appeared, and chlorophyll autofluorescence (red) was partially observed (Figures 14D, E). Furthermore, single colony isolation from colonies containing red parts yielded colonies that were entirely red (Figures 14F-H).

3. Confirmation of genome editing by base sequence analysis The structural changes of the GFP gene were analyzed for the above cell lines that lost GFP fluorescence and showed red color. From each line that lost fluorescence after genome editing, a fragment of the GFP gene was obtained by PCR method, and the sequence of the target site was determined by Sanger analysis.

The results are shown in FIG. 15. It was observed that the base sequence of the target site (upstream of the PAM sequence (TGG)) of the GFP gene was mutated in all strains derived from NIES-2146 (2146-7c, 2146-8C), NIES-2587 (2587-9C, 2587-12c), and NIES-2588 (2588-3c, 2588-9c). It was demonstrated that the nucleic acid construct of the present disclosure is capable of genome editing of various Nannochloropsis microalgae.

The primers used in this example are listed below.

(Note)
Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure should be interpreted in scope only by the claims. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference in their entirety as if the contents themselves were specifically set forth in this specification.

The present disclosure may enable highly efficient genome editing in Nannochloropsis microalgae, and may provide advantages in various applications using Nannochloropsis microalgae (biofuel production and other bioconversion, feed, etc.).

pNAN1001 (Accession number: NITE AP-03261)
pNAN1002 (Accession number: NITE AP-03262)
pNAN1003 (Accession number: NITE AP-03263)

Claims (17)

A promoter sequence that recruits Type III RNA polymerase from Nannochloropsis microalgae;
a sequence encoding a guide RNA (gRNA), operably linked to the promoter sequence; and a terminator sequence operably linked to the sequence encoding the gRNA, which terminates transcription by the Type III RNA polymerase of Nannochloropsis microalgae.
A nucleic acid construct comprising:
The promoter sequence is the sequence of SEQ ID NO:1
(CCAAATGTCTTTGACGGTCGTCACGGTTTTGCGTCGTAAAACCTACGCAAGGAAAATTACCATCATCGACCCGACACAAACAAGCGGTCCTTAGAAATCAGGACCTTTTATTTTTTAAGAAGGGAGTGAATGGGTAACAT TTCGTGGTGAGACTTCAACGAAAGTTTGGAGAAAAATTCCACATGTTGTACGCTCCCGGAAAAGAGAAGGAATATAAGGGCGTCACGAGTCATGACTTTCTAAACAGCAAGGTTCGCGTGAAACTCGTTTACGGGAAACC)
or a promoter sequence of the U6 RNA gene of Nannochloropsis microalgae, comprising a sequence shortened by 1 to 24 bases from the 3' end of the sequence of SEQ ID NO: 1;
Nucleic acid constructs . The nucleic acid construct of claim 1, wherein the terminator sequence comprises at least four consecutive thymine bases. 3. The nucleic acid construct of claim 1 or 2 , wherein the terminator sequence is the terminator sequence of the U6 RNA gene of Nannochloropsis microalgae. The terminator sequence is the sequence of SEQ ID NO: 2 (TTTTTTTCTTTTGATCAAATTACATTCTGATTCTTAGATACTTCACATGTTGATACTCCAC).
The nucleic acid construct according to any one of claims 1 to 3 , comprising a sequence having at least 85% identity with The nucleic acid construct of claim 1 , wherein the sequence encoding the gRNA comprises a protospacer adjacent motif (PAM). The sequence encoding the gRNA is the sequence of SEQ ID NO: 3 (GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC).
The nucleic acid construct according to any one of claims 1 to 5 , comprising a sequence having at least 85% identity with The nucleic acid construct according to any one of claims 1 to 6 , wherein the sequence encoding the gRNA comprises a sequence of 15 to 25 bases in length from a sequence encoding a target gene. The nucleic acid construct according to any one of claims 1 to 7 , wherein the sequence encoding the gRNA comprises a PAM and a 15 to 25 base sequence located on the 5' side of the PAM in the sequence encoding the target gene. The nucleic acid construct of claim 7 or 8 , wherein the target gene is green fluorescent protein (GFP). The nucleic acid construct according to any one of claims 1 to 9 , wherein the gRNA is a single-stranded guide RNA. The nucleic acid construct of any one of claims 1 to 10 , further comprising a sequence encoding a Cas9 protein. The nucleic acid construct according to any one of claims 1 to 11 , further comprising a sequence encoding a drug selection marker. The nucleic acid construct according to any one of claims 1 to 12 , wherein the Nannochloropsis microalgae is Nannochloropsis oceanica ( N. oceanica ) . The nucleic acid construct according to any one of claims 1 to 13 , which is a plasmid. A Nannochloropsis microalga comprising the nucleic acid construct according to any one of claims 1 to 14 . A kit for preparing a nucleic acid construct for performing genome editing in Nannochloropsis microalgae,
A nucleic acid comprising a promoter sequence that recruits the Type III RNA polymerase of the Nannochloropsis microalga, said promoter sequence being the sequence of SEQ ID NO:1.
(CCAAATGTCTTTGACGGTCGTCACGGTTTTGCGTCGTAAAACCTACGCAAGGAAAATTACCATCATCGACCCGACACAAACAAGCGGTCCTTAGAAATCAGGACCTTTTATTTTTTAAGAAGGGAGTGAATGGGTAACAT TTCGTGGTGAGACTTCAACGAAAGTTTGGAGAAAAATTCCACATGTTGTACGCTCCCGGAAAAGAGAAGGAATATAAGGGCGTCACGAGTCATGACTTTCTAAACAGCAAGGTTCGCGTGAAACTCGTTTACGGGAAACC)
or a nucleic acid comprising a sequence shortened by 1 to 24 bases from the 3' end of the sequence of SEQ ID NO: 1 ;
and a nucleic acid comprising a terminator sequence that terminates transcription by Type III RNA polymerase of Nannochloropsis microalgae. 1. A method for performing genome editing in Nannochloropsis microalgae, comprising:
A method comprising the step of introducing the nucleic acid construct according to any one of claims 1 to 14 , or the nucleic acid in the kit according to claim 16 , into Nannochloropsis microalgae.