Vector, method for producing recombinant in which DNA fragment is inserted in target genome region of subject cell, and method for producing recombinant in which DNA fragment is inserted in plurality of target genome regions of subject cell

Abstract

The present invention provides, for example, a vector that makes it possible to insert, via genome editing, a DNA fragment into a target genome region of a subject cell with higher efficiency. Employed is a vector for inserting a DNA fragment into a target genome region of a subject cell. The vector comprises an expression cassette of a programmable endonuclease and a donor DNA cassette including the DNA fragment, wherein the programmable endonuclease recognizes the target genome region.

Description

Vector, method for producing recombinant in which DNA fragment is inserted into target genome region of target cell, and method for producing recombinant in which DNA fragment is inserted into multiple target genome regions of target cell

[0001] The present invention relates to a vector, a method for producing a recombinant in which a DNA fragment is inserted into a target genomic region of a target cell, and a method for producing a recombinant in which a DNA fragment is inserted into multiple target genomic regions of a target cell. This application claims priority to U.S. provisional patent application Ser. No. 63 / 559,819, filed Feb. 29, 2024, the contents of which are incorporated herein by reference.

[0002] Filamentous fungi of the genus Aspergillus are less expensive to cultivate than animals and plants and secrete many hydrolases. Because the glycosylation system of Aspergillus is similar to that of higher animals, Aspergillus is expected to be used for producing proteins derived from higher organisms. For example, Non-Patent Document 1 describes a method for improving the expression level of a recombinant protein by using a plasmid with a highly active promoter.

[0003] Minetoki T, Tsuboi H, Koda A, Ozeki K (2003) Development of high expression system with the improved promoter using the cis-acting element in Aspergillus species. J. Biol. Macromol., 3(3) 89-96

[0004] In the method described in Non-Patent Document 1, plasmid DNA is inserted in tandem into the host genome. The tandemly inserted sequences are likely to undergo homologous recombination during cell division of the host and be lost from the host genome.

[0005] By using genome editing techniques such as the CRISPR Cas9 system, donor DNA such as an expression cassette for a recombinant protein can be inserted into a target genome region of a host cell. However, there is a problem in that genome editing efficiency is low.

[0006] Therefore, an objective of the present invention is to provide a vector that can insert a DNA fragment into a target genomic region of a target cell with higher efficiency through genome editing, a method for producing a recombinant in which a DNA fragment has been inserted into a target genomic region of a target cell, and a method for producing a recombinant in which DNA fragments have been inserted into multiple target genomic regions of a target cell.

[0007] The present invention includes the following aspects. [1] A vector for inserting a DNA fragment into a target genomic region of a target cell, the vector comprising an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragment, wherein the programmable endonuclease recognizes the target genomic region. [2] The vector according to [1], wherein the programmable endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease, or a Cas protein. [3] The vector according to [2], wherein the programmable endonuclease is a Cas protein, and the vector further comprises an expression cassette for a guide RNA. [4] The vector according to [1], wherein the target cell has a plurality of target genomic regions, and the plurality of target genomic regions are dispersed throughout the genome of the target cell. [5] The vector according to [4], wherein the plurality of target genomic regions share 95% or more sequence identity with each other. [6] The vector according to [1], wherein the target genome region is present within a transposon-like sequence. [7] The vector according to [1], wherein the vector is a plasmid, phagemid, cosmid, artificial chromosome, bacteriophage, or viral vector. [8] The vector according to [1], wherein the target cell is a cell of a filamentous fungus. [9] The vector according to [8], wherein the filamentous fungus belongs to the genus Aspergillus, Emericella, Penicillium, Trichoderma, Cephalosporium, or Acremonium.

[10] The vector according to [9], wherein the filamentous fungus belongs to the genus Aspergillus or Emericella.

[11] The vector according to

[10] , wherein the filamentous fungus is Aspergillus oryzae, Aspergillus nidulans, or Emericella nidulans.

[0008]

[12] A method for producing a recombinant in which a DNA fragment has been inserted into a target genomic region of a target cell, the method comprising the step of introducing the vector according to any one of [1] to

[11] into the target cell.

[0009]

[13] A method for producing a recombinant in which DNA fragments are inserted into multiple target genomic regions of a target cell, the method comprising the step of introducing into the target cell an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragments, wherein the programmable endonuclease recognizes the target genomic regions.

[14] The method according to

[13] , wherein the multiple target genomic regions have 95% or more sequence identity with each other.

[15] The method according to

[13] , wherein the target genomic regions are present within a transposon-like sequence.

[16] The method according to

[13] , wherein the target cell is a filamentous fungal cell.

[0010] The present invention can also be said to include the following aspects: [P1] A vector for inserting a DNA fragment into a target genomic region of a target cell, the vector comprising an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragment, wherein the programmable endonuclease recognizes the target genomic region. [P2] The vector according to [P1], further comprising an autonomous replication sequence. [P3] The vector according to [P1] or [P2], further comprising an expression cassette for a selectable marker gene.

[0011] [P4] The vector according to any one of [P1] to [P3], wherein the programmable endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease, or a Cas protein. [P5] The vector according to [P4], wherein the programmable endonuclease is a Cas protein, and the vector further comprises an expression cassette for a guide RNA. [P6] The vector according to [P5], wherein the expression cassette for the programmable endonuclease comprises a promoter, a DNA region encoding the Cas protein, and a terminator. [P7] The vector according to [P5] or [P6], wherein the Cas protein is a Cas9 protein. [P8] The vector according to any one of [P1] to [P7], wherein the promoter of the expression cassette for the programmable endonuclease is an inducible promoter.

[0012] [P9] The vector according to [P5], wherein the guide RNA expression cassette comprises a promoter, a DNA region encoding the guide RNA, and a terminator. [P10] The vector according to [P9], wherein the promoter of the guide RNA expression cassette is an RNA polymerase III-dependent promoter. [P11] The vector according to [P9], wherein the terminator of the guide RNA expression cassette is a terminator of a U6 snRNA gene.

[0013] [P12] The vector according to any one of [P1] to [P11], wherein the donor DNA cassette comprises a 5' homology arm and a 3' homology arm, wherein the 5' homology arm comprises a base sequence having 95% or more sequence identity with a base sequence near the 5' upstream of one DNA strand of the target genomic region in the genome of the target cell, and the 3' homology arm comprises a base sequence having 95% or more sequence identity with a base sequence near the 3' downstream of one DNA strand of the target genomic region in the genome of the target cell. [P13] The vector according to any one of [P1] to [P12], wherein the DNA fragment of the donor DNA cassette comprises a promoter, a DNA fragment encoding a target protein, and a terminator.

[0014] [P14] The vector according to any one of [P1] to [P13], wherein the target cell has multiple target genomic regions, and the multiple target genomic regions are dispersed throughout the genome of the target cell. [P15] The vector according to [P14], wherein the multiple target genomic regions share 95% or more sequence identity with each other. [P16] The vector according to any one of [P1] to [P15], wherein the target genomic region is present within a transposon-like sequence. [P17] The vector according to any one of [P1] to [P16], wherein the vector is a plasmid, phagemid, cosmid, artificial chromosome, bacteriophage, or viral vector.

[0015] [P18] The vector according to any one of [P1] to [P17], wherein the target cell is a cell of a filamentous fungus. [P19] The vector according to [P18], wherein the filamentous fungus belongs to the genus Aspergillus, Emericella, Penicillium, Trichoderma, Cephalosporium, or Acremonium. [P20] The vector according to [P19], wherein the filamentous fungus belongs to the genus Aspergillus or Emericella. [P21] The vector according to [P20], wherein the filamentous fungus is Aspergillus oryzae, Aspergillus nidulans, or Emericella nidulans.

[0016] [P22] The vector according to any one of [P18] to [P21], wherein the target genomic region is present within a transposon-like sequence, and the transposon-like sequence comprises a base sequence having 95% or more sequence identity with a base sequence represented by any one of SEQ ID NOs: 1 to 13, 17 to 32. [P23] The vector according to any one of [P18] to [P22], wherein the sequence of the guide RNA comprises the base sequence represented by SEQ ID NO: 16.

[0017] [P24] The vector according to any one of [P18] to [P23], wherein the donor DNA cassette comprises a 5' homology arm and a 3' homology arm, and the 5' homology arm and the 3' homology arm comprise sequences capable of homologous recombination with a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 13, 17 to 32. [P25] The vector according to [P24], wherein the 5' homology arm comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 14, and the 3' homology arm comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 15.

[0018] [P26] A method for producing a recombinant in which a DNA fragment has been inserted into a target genomic region of a target cell, the method comprising the step of introducing into the target cell a vector described in any one of [P1] to [P25].

[0019] [P27] A method for producing a recombinant in which DNA fragments are inserted into multiple target genomic regions of a target cell, the method comprising the step of introducing into the target cell an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragments, wherein the programmable endonuclease recognizes the target genomic regions.

[0020] [P28] The method according to [P27], wherein the plurality of target genomic regions have 95% or more sequence identity with each other. The method according to [P27] or [P28], wherein the target genomic region is present within a transposon-like sequence.

[0021] [P30] The method according to any one of [P27] to [P29], wherein the target cell is a cell of a filamentous fungus. [P31] The method according to [P30], wherein the filamentous fungus belongs to the genus Aspergillus, Emericella, Penicillium, Trichoderma, Cephalosporium, or Acremonium. [P32] The method according to [P31], wherein the filamentous fungus belongs to the genus Aspergillus or Emericella. [P33] The method according to [P32], wherein the filamentous fungus is Aspergillus oryzae, Aspergillus nidulans, or Emericella nidulans.

[0022] [P34] The method according to any one of [P30] to [P33], wherein the target genomic region is present within a transposon-like sequence, and the transposon-like sequence comprises a base sequence having 95% or more sequence identity with a base sequence represented by any one of SEQ ID NOs: 1 to 13 and 17 to 32.

[0023] According to the present invention, it is possible to provide a vector that can insert a DNA fragment into a target genomic region of a target cell with higher efficiency by genome editing, a method for producing a recombinant in which a DNA fragment has been inserted into a target genomic region of a target cell, and a method for producing a recombinant in which DNA fragments have been inserted into multiple target genomic regions of a target cell.

[0024] Figure 1A is a diagram schematically showing the location of a transposon-like sequence in A. oryzae. Figure 1B is a diagram schematically showing an example of a process for introducing a DNA fragment into the Aspergillus oryzae genome using a vector according to the first aspect. Figure 1C is a diagram schematically showing a process for detecting by PCR whether or not an expression cassette for a xylanase gene has been introduced into a transposon-like sequence in the Aspergillus oryzae genome, and the location of primers used for PCR. Figure 1D is a diagram showing the results of PCR amplification and electrophoresis of a transposon-like sequence from A. oryzae before vector introduction in Experimental Example 1. Figure 1E is a diagram showing the results of PCR amplification and electrophoresis of a transposon-like sequence from A. oryzae introduced with a vector and cultured in a glucose-containing medium in Experimental Example 1. Figure 1F is a diagram showing the results of PCR amplification and electrophoresis of a transposon-like sequence from A. oryzae introduced with a vector and cultured in a xylose-containing medium to induce Cas9 expression.

[0025] Figure 2A shows the results of SDS-PAGE analysis of the xylanase secreted into the medium after culturing in a glucose-containing medium a strain in which a xylanase gene expression cassette was introduced into 13 transposon-like sequences and a strain in which a xylanase gene expression cassette was introduced into a single site in the genome in Experimental Example 2. Figure 2B shows the results of SDS-PAGE analysis of the xylanase secreted into the medium after culturing in a maltose-containing medium a strain in which a xylanase gene expression cassette was introduced into 13 transposon-like sequences and a strain in which a xylanase gene expression cassette was introduced into a single site in the genome in Experimental Example 2. Figure 2C shows the results of RT-PCR analysis of the expression level of xylanase mRNA after culturing in a glucose-containing medium a strain in which a xylanase gene expression cassette was introduced into 13 transposon-like sequences and a strain in which a xylanase gene expression cassette was introduced into a single site in the genome in Experimental Example 2. Figure 2D shows the results of analyzing the expression level of xylanase mRNA by RT-PCR after culturing a strain in which a xylanase gene expression cassette was introduced into 13 transposon-like sequences and a strain in which a xylanase gene expression cassette was introduced into one location in the genome in Experimental Example 2 in a maltose-containing medium.

[0026] 3A shows the results of PCR amplification and electrophoresis of a transposon-like sequence from E. nidulans before the introduction of a vector in Experimental Example 3. FIG. 3B shows the results of PCR amplification and electrophoresis of a transposon-like sequence from E. nidulans into which a vector was introduced and which was cultured in a xylose-containing medium to induce Cas9 expression in Experimental Example 3.

[0027] Figure 4A shows the results of PCR amplification and electrophoresis of transposon-like sequences from A. oryzae into which a vector was introduced and which was cultured in a xylose-containing medium to induce Cas9 expression in Experimental Example 4. Figure 4B shows the results of quantification of kojic acid secreted into the medium after culturing a strain in which a kojA gene expression cassette was introduced into 13 transposon-like sequences and a strain in which a kojA gene expression cassette was introduced into one site in the genome in Experimental Example 4.

[0028] (First Aspect: Vector) The vector according to this embodiment is a vector for inserting a DNA fragment into a target genomic region of a target cell. The vector includes an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragment. The programmable endonuclease recognizes the target genomic region.

[0029] By introducing the vector according to this embodiment into a target cell, a desired DNA fragment can be inserted into a target genomic region. During this process, known as genome editing, a programmable endonuclease recognizes and cleaves the target genomic region. Then, the DNA fragment of the donor DNA cassette is inserted into the target genomic region by homologous recombination.

[0030] In conventional genome editing, the programmable endonuclease expression cassette and the donor DNA cassette are carried on separate vectors, making it unlikely that these two cassettes will be introduced into the target cell at the same time, and as a result, it is difficult for the DNA fragment to be inserted into the target genome region.

[0031] The vector according to this embodiment contains both an endonuclease expression cassette and a donor DNA cassette, which increases the probability that these two cassettes are introduced into the target cell, thereby improving the efficiency of genome editing. That is, cells in which a DNA fragment has been inserted into a target genome region can be obtained with higher efficiency than conventional methods.

[0032] <Target Cells, Target Genomic Regions> The biological species of the target cells is not particularly limited as long as the effects of the present invention are achieved, and may be any of eukaryotes, eubacteria, and archaea. The eukaryotes may be either multicellular or unicellular organisms. The multicellular organisms may be any of animals, plants, and fungi. Examples of the animals include vertebrates such as mammals, reptiles, birds, amphibians, and fish, as well as insects. Examples of the mammals include rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; Carnivora such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees.

[0033] When the target cell is an animal, plant, or fungal cell, the target cell may be either a somatic cell or a germ cell, preferably a somatic cell. The target cell may be a cell cultured in vitro or an in vivo cell. The target cell may also be a primary culture cell or an established cell line. The established cell line is generally an immortalized cell, and may be a cancer cell.

[0034] The target cell is preferably a mutant lacking a human DNA ligase IV homolog. Human DNA ligase IV functions in double-strand repair by non-homologous end joining. Mutants lacking a human DNA ligase IV homolog are unable to perform double-strand repair by non-homologous end joining, and homologous recombination is promoted. The human DNA ligase IV homolog of the target cell can be identified using a protein database, a BLAST program, or the like.

[0035] The target cell species is preferably a filamentous fungus, more preferably a filamentous fungus belonging to the genus Aspergillus, Emericella, Penicillium, Trichoderma, Cephalosporium, or Acremonium, even more preferably a filamentous fungus belonging to the genus Aspergillus or Emericella, and particularly preferably Aspergillus oryzae, Aspergillus nidulans, or Emericella nidulans.

[0036] <Target Genomic Region> A target genomic region is a genomic sequence recognized by a programmable endonuclease. The programmable endonuclease recognizes a target sequence in the target genomic region and cleaves the target genomic region at a specific position. As used herein, the term "target genomic region" refers to a genomic region that includes a target sequence and its complementary strand.

[0037] The number of target genomic regions in the genome of the target cell may be one or two or more (i.e., a plurality of regions). By having two or more target genomic regions in the genome of the target cell, DNA fragments can be inserted into multiple target genomic regions simultaneously.

[0038] When a target cell has multiple target genomic regions, the number of target genomic regions in the genome of the target cell is 2 or more, or may be 3 or more, 5 or more, or 10 or more. The upper limit of the number is not particularly limited, and may be 1000 or less, 500 or less, 300 or less, 100 or less, 50 or less, or 30 or less.

[0039] Preferably, the target cell has multiple target genomic regions. When the target cell has multiple target genomic regions, the DNA fragments contained in the donor DNA cassette can be inserted into multiple target genomic regions simultaneously.

[0040] When a target cell has multiple target genomic regions, the multiple target genomic regions are preferably scattered throughout the genome of the target cell. When multiple target genomic regions are scattered throughout the genome of the target cell, loss of inserted DNA fragments due to homologous recombination or the like, which may occur between DNA fragments inserted into the target genomic regions, can be suppressed, making it easier to maintain the DNA fragments inserted into the target genomic regions.

[0041] It is preferable that the multiple target genomic regions have a sequence identity of 95% or more with each other. When the multiple target genomic regions have a sequence identity of 95% or more with each other, DNA fragments can be inserted into the multiple target genomic regions simultaneously. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of the sequence identity is not particularly limited, but may be 100%.

[0042] In this specification, the sequence identity of a subject base sequence to a reference base sequence can be determined, for example, as follows. First, the reference base sequence and the subject base sequence are aligned. Here, gaps may be included in each base sequence to maximize sequence identity. Next, the number of matching bases in the reference base sequence and the subject base sequence is calculated, and the sequence identity can be determined according to the following formula (1): Sequence identity (%) = number of matching bases / total number of bases in the subject base sequence × 100 (1) The value of sequence identity of a base sequence can be obtained by calculation based on the alignment obtained by known homology search software such as BLASTN.

[0043] The target genome region is preferably present within a transposon-like sequence. The genomes of many organisms contain numerous transposon-like sequences. Generally, transposon-like sequences are not essential for the survival of target cells, and even if a DNA fragment is inserted into a transposon-like sequence, the target cell's viability is not affected, making it easy to obtain target cells into which the DNA fragment has been inserted.

[0044] The genome of Aspergillus oryzae contains transposon-like sequences set forth in SEQ ID NOs: 1 to 13. There is one transposon on chromosome 1, three on chromosome 2, two on chromosome 3, one on each of chromosomes 4 to 6, and two on each of chromosomes 7 and 8, for a total of 13 transposons. The sequence set forth in SEQ ID NO: 1 is a transposon-like sequence present on chromosome 1. The sequences set forth in SEQ ID NOs: 2 to 4 are transposon-like sequences present on chromosome 2. The sequences set forth in SEQ ID NOs: 5 to 6 are transposon-like sequences present on chromosome 3. The sequence set forth in SEQ ID NO: 7 is a transposon-like sequence present on chromosome 4. The sequence set forth in SEQ ID NO: 8 is a transposon-like sequence present on chromosome 5. The sequence set forth in SEQ ID NO: 9 is a transposon-like sequence present on chromosome 6. The sequences set forth in SEQ ID NOs: 10 to 11 are transposon-like sequences present on chromosome 7. The sequences set forth in SEQ ID NOs: 12 to 13 are transposon-like sequences present on chromosome 8.

[0045] When the biological species of the target cell is Aspergillus oryzae and the target genomic region is present within a transposon-like sequence, the transposon-like sequence preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 13. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0046] The genome of Emericella nidulans contains the transposon-like sequences set forth in SEQ ID NOs: 17 to 32. There are a total of 16 transposon-like sequences: one on chromosome 1, two on chromosome 2, four on chromosome 3, one each on chromosomes 4 and 5, two on chromosome 6, one on chromosome 7, and three on chromosome 8.

[0047] The sequences set forth in SEQ ID NOs: 17 to 18 are transposon-like sequences present on chromosome 1. The sequences set forth in SEQ ID NOs: 19 to 20 are transposon-like sequences present on chromosome 2. The sequences set forth in SEQ ID NOs: 21 to 24 are transposon-like sequences present on chromosome 3. The sequence set forth in SEQ ID NO: 25 is a transposon-like sequence present on chromosome 4. The sequence set forth in SEQ ID NO: 26 is a transposon-like sequence present on chromosome 5. The sequences set forth in SEQ ID NOs: 27 to 28 are transposon-like sequences present on chromosome 6. The sequence set forth in SEQ ID NO: 29 is a transposon-like sequence present on chromosome 7. The sequences set forth in SEQ ID NOs: 30 to 32 are transposon-like sequences present on chromosome 8.

[0048] When the biological species of the target cell is Emericella nidulans and the target genomic region is present within a transposon-like sequence, the transposon-like sequence preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by any of SEQ ID NOs: 17 to 32. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0049] The vector is not particularly limited as long as it exhibits the effects of the present invention, and may be, for example, a plasmid, a phagemid, a cosmid, an artificial chromosome, a bacteriophage, a viral vector, or the like.

[0050] The vector preferably has an autonomous replication sequence. When the vector has an autonomous replication sequence, the programmable endonuclease expression cassette and the donor DNA cassette are replicated in the target cell into which the vector is introduced. In general genome editing, the programmable endonuclease expression cassette has an autonomous replication sequence, but the donor DNA cassette does not have an autonomous replication sequence. When the vector according to this embodiment has an autonomous replication sequence, both the programmable endonuclease expression cassette and the donor DNA cassette are replicated in the target cell, thereby improving the efficiency of genome editing.

[0051] The vector preferably contains a selection marker gene expression cassette, which makes it easier to efficiently select target cells into which the vector has been introduced.

[0052] <Programmable endonuclease expression cassette> The structure of the programmable endonuclease expression cassette is not particularly limited as long as the effects of the present invention are achieved, but it may include a promoter, a DNA region encoding the programmable endonuclease, and a terminator.

[0053] The programmable endonuclease has endonuclease activity or nickase activity. The programmable endonuclease includes wild-type programmable endonucleases and mutants thereof, so long as they exhibit endonuclease activity or nickase activity.

[0054] The programmable endonuclease is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cas proteins, preferably Cas proteins. Examples of Cas proteins include Cas9, Cas12, Cas13, and Cas14.

[0055] When the programmable endonuclease is a ZFN, the zinc finger binding domain of the ZFN can be appropriately designed so that the ZFN can recognize a target sequence in a target genomic region and cleave the target genomic region.

[0056] When the programmable endonuclease is a TALEN, by appropriately designing the TALE domain, the TALEN can recognize a specific target sequence in a target genomic region and cleave the target genomic region.

[0057] As described below, when the programmable endonuclease is a Cas protein, by appropriately designing the spacer sequence of the guide RNA, the Cas protein-guide RNA complex can recognize the target sequence in the target genomic region and cleave the target genomic region.

[0058] The promoter that the programmable endonuclease expression cassette may have is not particularly limited as long as the effects of the present invention are achieved, but an inducible promoter is preferred, which enables time-specific enhancement of programmable endonuclease expression.

[0059] <Donor DNA Cassette> The donor DNA cassette contains a DNA fragment to be inserted into a target genomic region of a subject cell. The sequence of the DNA fragment contained in the donor DNA cassette is not particularly limited as long as the effects of the present invention are achieved.

[0060] The donor DNA cassette preferably comprises a 5' homology arm and a 3' homology arm. Here, the 5' homology arm comprises a sequence capable of homologous recombination with a base sequence near the 5' upstream of one DNA strand of the target genomic region. The 3' homology arm comprises a sequence capable of homologous recombination with a base sequence near the 3' downstream of one DNA strand of the target genomic region. In the target genomic region, the target sequence is located between the base sequence near the 5' upstream of one DNA strand of the target genomic region and the base sequence near the 3' downstream of one DNA strand of the target genomic region.

[0061] The 5' homology arm and 3' homology arm that the donor DNA cassette may have undergo homologous recombination with the base sequence near the 5' upstream and the base sequence near the 3' downstream, respectively, and the DNA fragment contained in the donor DNA cassette is inserted into the target genomic region of the target cell.

[0062] The 5' homology arm preferably contains a sequence having 95% or more sequence identity with the base sequence near the 5' upstream of one DNA strand of the target genomic region, and the sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of the sequence identity is not particularly limited, but may be 100%.

[0063] The 3' homology arm preferably contains a sequence having 95% or more sequence identity with the base sequence near the 3' downstream of one DNA strand of the target genomic region, although the sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, and may be 100%.

[0064] When the biological species of the target cell is Aspergillus oryzae, the 5' homology arm preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 14. The nucleotide sequence represented by SEQ ID NO: 14 is a part of a transposon-like sequence of Aspergillus oryzae. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0065] When the biological species of the target cell is Aspergillus oryzae, the 3' homology arm preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 15. The nucleotide sequence represented by SEQ ID NO: 15 is a part of a transposon-like sequence of Aspergillus oryzae. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0066] When the target cell species is Emericella nidulans, the 5' homology arm preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 33. The nucleotide sequence represented by SEQ ID NO: 33 is a part of a transposon-like sequence of Emericella nidulans. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0067] When the target cell species is Emericella nidulans, the 3' homology arm preferably contains a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 34. The nucleotide sequence represented by SEQ ID NO: 34 is a part of a transposon-like sequence of Emericella nidulans. The sequence identity may be 97% or more, 98% or more, or 99% or more. The upper limit of sequence identity is not particularly limited, but may be 100%.

[0068] The donor DNA cassette may include a promoter, a DNA fragment encoding a protein of interest, and a terminator, in which case the donor DNA cassette is inserted into a target genomic region, allowing the protein of interest to be expressed in a target cell.

[0069] <<Guide RNA Expression Cassette>> When the programmable endonuclease is a Cas protein, the vector preferably further comprises a guide RNA expression cassette. The guide RNA expressed from the guide RNA expression cassette forms base pairs with the complementary strand of the target sequence in the target genomic region.

[0070] The guide RNA expression cassette may include a promoter, a DNA region encoding the guide RNA, and a terminator. The promoter of the guide RNA expression cassette is not particularly limited as long as the effects of the present invention are achieved, but an RNA polymerase III-dependent promoter is preferred. The terminator of the guide RNA expression cassette is not particularly limited as long as the effects of the present invention are achieved, but the terminator of the U6 snRNA gene is preferred.

[0071] The guide RNA (gRNA) preferably comprises a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), where the crRNA is involved in binding to a target sequence in a target genomic region, and the tracrRNA is involved in binding to a Cas protein.

[0072] The crRNA preferably comprises a spacer sequence and a repeat sequence. The spacer sequence is identical to the target sequence of the target genomic region. The spacer sequence is located on the 5' side of the crRNA.

[0073] The tracrRNA preferably comprises an anti-repeat sequence and a 3' tail sequence, wherein the anti-repeat sequence has a sequence complementary to the repeat sequence of the crRNA and forms base pairs with the repeat sequence, and the 3' tail sequence forms a stem-loop.

[0074] The guide RNA may be a single-stranded guide RNA (sgRNA) in which the 5' end of the tracrRNA is linked to the 3' end of the crRNA. Alternatively, the guide RNA may be one in which the crRNA and tracrRNA are separate RNA molecules and base-paired at the repeat and anti-repeat sequences.

[0075] When the programmable endonuclease is a Cas protein, the target sequence in the target genome region to be cleaved by the Cas protein must be a sequence adjacent to the 5' side of a protospacer adjacent motif (PAM). Those skilled in the art can appropriately design the target sequence depending on the type of Cas protein.

[0076] PAM refers to a sequence recognized by the Cas protein during DNA cleavage by the Cas protein. The sequence and position of the PAM vary depending on the type of Cas protein. For example, in the case of the Cas9 protein, the PAM must be immediately adjacent to the 3' side of the target sequence. The PAM sequence corresponding to the Cas9 protein varies depending on the bacterial species from which the Cas9 protein is derived. For example, the PAM corresponding to the Cas9 protein of S. pyogenes is "NGG", the PAM corresponding to the Cas9 protein of S. thermophilus is "NNAGAA", the PAM corresponding to the Cas9 protein of S. aureus is "NNGRRT" or "NNGRR(N)", the PAM corresponding to the Cas9 protein of N. meningitidis is "NNNNGATT", and the PAM corresponding to the Cas9 protein of T. The sequence is "NAAAC" (where "R" is A or G; "N" is A, T, G, or C), which corresponds to the Cas9 protein of B. denticola.

[0077] The repeat sequence of the crRNA and the sequence of the tracrRNA can be selected appropriately depending on the type of Cas protein, and those derived from the same bacterial species as the Cas protein can be used.

[0078] When the target cell is Aspergillus oryzae and the target genomic region is present within a transposon-like sequence, the spacer sequence of the guide RNA may be the base sequence represented by SEQ ID NO:16.

[0079] When the target cell is Emericella nidulans and the target genomic region is present within a transposon-like sequence, the spacer sequence of the guide RNA may be the base sequence represented by SEQ ID NO:35.

[0080] <Amount Used> The vector according to this embodiment may be dissolved in a liquid and then introduced into target cells. Examples of the liquid include water, a buffer solution, and a known transfection reagent.

[0081] The concentration of the vector in the liquid to be brought into contact with the target cells is not particularly limited as long as the effects of the present invention are achieved, but may be, for example, 1 pg / μL to 10 μg / μL.

[0082] (Second Aspect: Production Method) The production method according to the second aspect is a method for producing a recombinant in which a DNA fragment is inserted into a target genomic region of a target cell, comprising the step of introducing the vector according to the first aspect into the target cell.

[0083] According to the production method of the second aspect, by using the vector of the first aspect, a recombinant in which a DNA fragment has been inserted into a target genome region can be obtained with high efficiency.

[0084] The biological species of the target cells and the target genomic region of the target cells are the same as those described above in the first embodiment.

[0085] In the production method according to the second aspect, the vector according to the first aspect is introduced into target cells by contacting the target cells with the vector. The method for introducing the vector into target cells is not particularly limited as long as the effects of the present invention are achieved, and a person skilled in the art can adopt an appropriate method depending on the type of target cells. The concentration of the vector contacted with the target cells may be the same as the concentration described above in the first aspect.

[0086] (Third Aspect: Production Method) The production method according to the third aspect is a method for producing a recombinant in which DNA fragments are inserted into multiple target genomic regions of a target cell. The production method includes the step of introducing an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragments. The programmable endonuclease recognizes the target genomic regions.

[0087] According to the production method of the third aspect, DNA fragments can be simultaneously inserted into multiple target genomic regions of a target cell, and recombinants thereof can be obtained.

[0088] The biological species of the target cell and the multiple target genomic regions of the target cell are the same as those described above in the first aspect.

[0089] The programmable endonuclease expression cassette and the donor DNA cassette containing the DNA fragment are the same as those described above in the first embodiment, and these two cassettes may be carried on a single vector or on different vectors.

[0090] The vector is not particularly limited as long as it exhibits the effects of the present invention, and may be, for example, a plasmid, a phagemid, a cosmid, an artificial chromosome, a bacteriophage, a viral vector, or the like.

[0091] In the production method according to the third aspect, the method for introducing the vector into the target cells and the concentration of the vector brought into contact with the target cells may be the same as those described above in the first and second aspects.

[0092] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0093] (Materials and Methods) <Construction of A. oryzae Strains> The ligD gene of A. oryzae is a homolog of human DNA ligase IV. The ΔligD strain is unable to repair non-homologous end DNA ligation and is suitable for genome editing.

[0094] The formation of mycelial masses in A. oryzae involves α-1,3-glucan (AG) and the extracellular polysaccharide galactosaminogalactan (GAG). The agsA, agsB, and agsC genes are required for AG synthesis. The sphZ and ugeZ genes are required for GAG synthesis. The ΔagsAΔagsBΔagsCΔsphZΔugeZ strain, which lacks AG and AGA (referred to as the ΔAG-GAG strain), is suitable for culture in liquid media because its mycelia disperse in the liquid medium and no mycelial masses are formed.

[0095] The ΔligDΔagsAΔagsBΔagsCΔsphZΔugeZ strain (referred to as the ΔligDΔAG-GAG strain) is unable to repair non-homologous end-DNA ligation, and the hyphae disperse in the medium. The ΔligDΔAG-GAG strain is suitable for genome editing, and in liquid culture it grows to high density and exhibits excellent enzyme productivity.

[0096] The ΔligDΔAG-GAG strain was produced by the following method: A. oryzae ΔadeA strain was prepared. The genotype of this strain was ΔligD::sCΔadeA::ptrA niaD － For details of this strain, see Zhang, S., Ban, A., Ebara, N., Mizutani, O., Tanaka, M., Shintani, T., Gomi, K., Self-excising Cre / mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae, J. Biosci. Bioeng., 2017, 123(4):403-411.

[0097] The ΔAG-GAGΔadeA strain was created using the A. oryzae ΔadeA strain by a marker recycling system. The genotype of the ΔAG-GAGΔadeA strain was ΔligD::sCΔadeA::ptrAΔagsA::loxPΔagsB::loxPΔagsC::loxPΔsphZugeZ::loxP adeA － niaD － It is. For the production method, see Miyazawa K, Yoshimi A, Sano M, Tabata F, Sugahara A, Kasahara S, Koizumi A, Yano S, Nakajima T, Abe K., Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillus oryzae hyphae in liquid culture. Front Microbiol. 2019, 10:2090 can be referred to.

[0098] The minimal medium (MM) for culturing A. oryzae contained 0.5% (NH ４ ) ２ SO ４, 0.05% KCl, 0.2% KH ２ P.O. ４ , 0.05% MgSO ４ , trace amounts of FeSO ４ , ZnSO ４ , CuSO ４ , MnSO ４ , Na ２ B ４ O ７ and (NH ４ ) ６ Mo ７ O ２４ Czapek-Dox (CD) medium containing 0.5% yeast extract and 1% carbon source was used. For the culture of the ΔAG-GAGΔadeA strain, 0.01% adenine was added to this medium. To test enzyme production in liquid medium, YP complete medium containing 0.5% yeast extract and 1% Bacto peptone (Thermo Fisher Science) and supplemented with 3% carbon source was used. A. oryzae was cultured at 30°C.

[0099] <Creation of E. nidulans Strain> The Emericella nidulans ΔagsB strain was prepared. The genotype of this strain was biA1 pyrG89 wA3 argB2 pyroA4 ligD::ptrA ΔagsB::argB. Regarding this strain, Yoshimi, A., Sano, M., Inaba, A., Kokubun, Y., Fujioka, T., Mizutani, O., Hagiwara, D., Fujikawa, T., Nishimura, M., Yano, S., Kasahara, S., Shimizu, K., Yamaguchi, M., Kawakami, K and Abe, K., Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: AgsB is the major α-1,3-glucan synthase in this fungus. PLoS One, 2013, 8(1):e54893).

[0100] The minimal medium (MM) for culturing E. nidulans contained 0.6% NaNO . ３ , 0.05% KCl, 0.2% KH ２ P.O. ４ , 0.05% MgSO ４ , trace amounts of FeSO ４ , ZnSO ４ , CuSO ４ , MnSO ４ , Na ２ B ４ O ７ and (NH ４ ) ６ Mo ７ O ２ Czapek-Dox (CD) medium containing 1% carbon source was used. Biotin (0.02 mg / L), pyridoxine (0.5 g / L), uridine (1.22 g / L), and uracil (1.12 g / L) were added depending on nutritional requirements. E. nidulans was cultured at 37°C.

[0101] <PCR> The target DNA was amplified using KOD One® PCR Master Mix (TOYOBO) or KOD FX Neo (TOYOBO) as the DNA polymerase, and a T100 thermal cycler (BIO-RAD) was used as the amplification device.

[0102] <In-Fusion Cloning> Plasmids were constructed using In-Fusion (registered trademark) HD ​​Cloning Kit (TaKaRa) according to the attached protocol.

[0103] As described below, a plasmid for introducing multiple copies of a xylanase gene expression cassette into A. oryzae and a plasmid for introducing multiple copies of a xylanase gene expression cassette into E. nidulans were prepared as vectors carrying an expression cassette for a programmable endonuclease and a donor DNA cassette containing a DNA fragment. The primers used in the vector construction are shown in Tables 1 and 2.

[0104]

[0105]

[0106] <1. A. Preparation of a Plasmid for Introducing a Multicopy Xylanase Gene Expression Cassette in oryzae> <1-1. Preparation of Plasmid pXPcas9aA> In order to prepare a multicopy gene introduction plasmid for oryzae, the plasmid pXPcas9aA was first prepared by the following method. The Cas9 expression plasmid ppAsAcas9 and the plasmid ppAsAcas9gpG containing a gRNA expression cassette for pyrG gene editing were provided by Dr. Junichi Maruyama (University of Tokyo). For details about these plasmids, please refer to Katayama, T., Nakamura, H., Zhang, Y., Pascal, A., Fujii, W., Maruyama, JI, Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion / Integration in the Industrial Filamentous Fungus Aspergillus oryzae., Applied and Environmental Microbiology, 2019, 85, e01896-18.

[0107] The Cas9 expression plasmid ppAsAcas9 uses the pyrithiamine resistance gene ptrA as a selection marker. This selection marker was replaced with the adeA marker, which can be selected by hyphal color. The adeA marker was amplified by PCR using primers P1 and P2 with the E. nidulans genome as a template. The sequence AMA1, required for autonomous plasmid replication in Aspergillus oryzae, was amplified by PCR with primers P3 and P4 with ppAsAcas9 as a template. En. adeA and AMA1 were ligated by fusion PCR using primers P1 and P4, and inserted into the ppAsAcas9 vector digested with SbfI and MfeI using in-fusion cloning to obtain the plasmid pCas9aA.

[0108] The promoter used for Cas9 expression was the promoter (PxylP) of the endoxylanase gene (xylP) from Penicillium chrysogenum. PxylP promoter activity is induced in a medium containing xylose as the sole carbon source and significantly repressed by glucose. PxylP was amplified by PCR using the plasmid pAAAXP-Cre as a template and primers P5 and P6. For details on PxylP, see Zhang, S., Ban, A., Ebara, N., Mizutani, O., Tanaka, M., Shintani, T., Gomi, K., Self-excising Cre / mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae., J. Biosci. Bioeng., 2017, 123(4):403-411.

[0109] Using the above-mentioned plasmid pCas9aA as a template, vector DNA amplified by PCR using primers P7 and P8 was ligated to the PxylP fragment by in-fusion cloning to obtain plasmid pXPcas9aA.

[0110] <1-2. Preparation of Plasmid pXPcas9aAgTp> Plasmid pXPcas9aAgTp was prepared by inserting a gRNA expression cassette into the plasmid pXPcas9aA using the following method. The promoter and terminator for gRNA expression were the U6 RNA polymerase III promoter (PU6) and the terminator (TU6), respectively, and a spacer sequence was designed to cleave near the center of the transposon-like sequence. The spacer sequence is the base sequence of SEQ ID NO: 16.

[0111] Using ppAsAcas9gpG as a template and primers P9 and P10, a PCR product containing PU6 and a spacer sequence was obtained. Using ppAsAcas9gpG as a template and primers P11 and P12, a PCR product containing a spacer sequence, a scaffold sequence, and TU6 was obtained. The scaffold sequence is a sequence other than the spacer sequence contained in the gRNA sequence. Using primers P9 and P12, the two PCR products were linked by fusion PCR, and then inserted into the plasmid pXPCas9aA by the in-fusion cloning method to obtain the plasmid pXPCas9aAgTp.

[0112] <1-3. Preparation of Plasmid pXPCas9aAgTpDon> The 5' homology arm and 3' homology arm were inserted into the plasmid pXPcas9aAgTp by the following method to prepare the plasmid pXPCas9aAgTpDon.

[0113] The 300 bp (excluding the PAM sequence) 5' upstream and 3' downstream of the genome breakpoint in the A. oryzae transposon-like sequence were designated the 5' homology arm (Tpup) and the 3' homology arm (Tpdown). The sequence of one DNA strand of Tpup consists of the base sequence represented by SEQ ID NO:14, and the sequence of one DNA strand of Tpdown consists of the base sequence represented by SEQ ID NO:15.

[0114] Using the A. oryzae genome as a template, the Tpup fragment was amplified by PCR using primers P13 and P14, and the Tpdown fragment was amplified by PCR using primers P15 and P16. Next, the Tpup fragment and the Tpdown fragment were ligated by fusion PCR using primers P13 and P16. Next, the ligated Tpup fragment and Tpdown fragment were inserted into the above-mentioned plasmid pXPCas9aAgTp by in-fusion cloning to create the plasmid pXPCas9aAgTpDon.

[0115] <1-4. Preparation of Plasmid pXPcas9aADoXF> Plasmid pXPcas9aADoXF was prepared by inserting a xylanase gene expression cassette into plasmid pXPCas9aAgTpDon as follows. The xylanase gene expression cassette used the xylanase biosynthetic gene xynF1 and the high-expression promoter PglaA142. The promoter activity of PglaA142 is induced by glucose and maltose, with maltose inducing promoter activity more strongly than glucose.

[0116] The xylanase gene expression cassette was amplified by PCR using the plasmid pNGA142-xynF1 (described below) as a template and primers P17 and P18. This expression cassette fragment was inserted into the pXPCas9aAgTpDon vector treated with the restriction enzyme SfoI by in-fusion cloning to produce the plasmid pXPcas9aADoXF.

[0117] <2. Preparation of a Plasmid for Introducing Multicopies of a Xylanase Gene Expression Cassette into E. nidulans> <2-1. Preparation of Plasmid pXPcas9pG-EN> A multicopy gene introduction plasmid for E. nidulans was prepared by the following method. The uracil-requiring marker pyrG was amplified using the A. oryzae genome as a template and primers P21 and P22. The sequence AMA1 required for autonomous replication of the plasmid was amplified by PCR using primers P3 and P4 and ppAsAcas9 as a template. The pyrG fragment and AMA1 fragment were ligated by fusion PCR and then inserted into the pCas9aA vector digested with SbfI and SmaI to prepare the plasmid pXPcas9pG-EN.

[0118] <2-2. Preparation of plasmid pXPcas9pGgTp-EN> A gRNA expression cassette was inserted into the plasmid pXPcas9pG-EN by the following method to prepare the plasmid pXPcas9pGgTp-EN.

[0119] The promoter and terminator for gRNA expression were the U6 RNA polymerase III promoter (PU6) and the terminator (TU6), and the spacer sequence was designed to cleave near the center of the transposon-like sequence of E. nidulans. The spacer sequence is the base sequence of SEQ ID NO: 35.

[0120] Using pXPcas9aAgTp as a template and primers P9 and P23, a PCR product containing PU6 and a spacer sequence was obtained. Using pXPcas9aAgTp as a template and primers P24 and P25, a PCR product containing a spacer sequence, a scaffold sequence, and TU6 was obtained. The two PCR products were ligated by fusion PCR using primers P9 and P25, and then inserted into the pXPCas9pG-EN vector by in-fusion cloning to create the plasmid pXPCas9pGgTp-EN.

[0121] 2-3. Preparation of Plasmid pXPCas9pGgTpDon-EN Plasmid pXPCas9pGgTpDon-EN was prepared by inserting the 5′ homology arm and 3′ homology arm into the plasmid pXPcas9pGgTp-EN according to the following method.

[0122] In the transposon-like sequence of E. nidulans, the upstream and downstream 300 bp (excluding the PAM sequence) from the genome breakpoint were designated as the 5' homology arm (EnTpup) and the 3' homology arm (EnTpdown). The sequence of one DNA strand of EnTpup consists of the base sequence represented by SEQ ID NO: 33, and the sequence of one DNA strand of EnTpdown consists of the base sequence represented by SEQ ID NO: 34.

[0123] Using the E. nidulans genome as a template, the EnTpup fragment was amplified by PCR using primers P26 and P27, and the AnTpdown fragment was amplified by PCR using primers P28 and P29. The EnTpup fragment and the EnTpdown fragment were then ligated by fusion PCR using primers P26 and P29. The EnTpup fragment and the EnTpdown fragment were then inserted into the pXPCas9pGgTp-EN vector by in-fusion cloning to create the plasmid pXPCas9pGgTpDon-EN.

[0124] 2-4. Preparation of Plasmid pXPcas9pGgTpDoXF-EN Plasmid pXPcas9pGgTpDoXF-EN was prepared by inserting a xylanase gene expression cassette into the plasmid pXPCas9pGgTpDon-EN as follows: The xylanase gene expression cassette used was the xylanase biosynthetic gene xynF1 and the high-expression promoter PglaA142.

[0125] The xylanase gene expression cassette was amplified by PCR using the plasmid pNGA142-xynF1 (described below) as a template and primers P30 and P31. This expression cassette fragment was inserted into the plasmid pXPCas9pGgTpDonEN, which had been treated with the restriction enzyme SmaI, by in-fusion cloning to prepare pXPcas9pGgTpDoXF-EN.

[0126] <Preparation of A. oryzae strain into which one copy of xylanase gene expression cassette has been introduced> The plasmid into which one copy was introduced was the Aspergillus oryzae high expression vector pNGA142. For details of pNGA142, see Minetoki T, Tsuboi H, Koda A, Ozeki K (2003) "Development of high expression system with the improved promoter using the cis-acting element in Aspergillus species." J. Biol. Macromol., 3(3) 89-96.

[0127] The xylanase gene xynF1 was amplified using primers P19 and P20 from the A. oryzae genome as a template. This fragment was digested with restriction enzymes HindIII and SpeI and inserted by ligation into the pNGA142 vector (pNGA142-xynF1) that had been treated with HindIII and SpeI. The constructed plasmid pNGA142-xynF1 was treated with restriction enzyme HpaI and one copy was introduced into the niaD locus on the A. oryzae chromosome.

[0128] <Transformation> Transformation of A. oryzae and E. nidulans was carried out according to the method of Gomi et al. (Gomi, K., Iimura, Y., Hara, S., 1987. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51, 2549-2555). Transformation and purification of candidate transformant strains were carried out by culturing them on agar medium containing glucose as a carbon source.

[0129] <Conidial PCR> Conidia were removed from the plates on which the strains were cultured using a toothpick and thoroughly suspended in Buffer A (1.2% Tris, 7.45% KCl, pH 9.5). The mixture was then vortexed for 3 minutes, warmed in a microwave oven (500W, 20 seconds), and placed on ice for 5 minutes. The resulting solution, which was vortexed again for 3 minutes, was used as a template for PCR. The primers used for the conidial PCR of Aspergillus oryzae are shown in Table 3, and the primers used for the conidial PCR of Emericella nidulans are shown in Table 4. In Tables 3 and 4, the forward primer (Fw) was a sequence located outside each transposon-like sequence. The reverse primer (Rv) was a single sequence located inside each transposon-like sequence.

[0130]

[0131]

[0132] <Xylanase enzyme production experiment> The strain was cultured for 48 or 96 hours in a liquid medium containing 0.5% yeast extract, 1% Bacto peptone, and 3% carbon source, and the culture supernatant and bacterial cells were collected. The culture supernatant was subjected to SDS-PAGE. Glucose or maltose was used as the carbon source. SDS-PAGE was performed as described (Tanaka, M., Tokuoka, M., Shintani, T., Gomi, K., 2012. Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae. Appl. Microbiol. Biotechnol. 96, 1275-1282.). Xylanase production was calculated by quantifying the bands on SDS-PAGE using ImageJ, using bovine serum albumin as a standard protein.

[0133] <Analysis of Xylanase Gene Transcription Levels> RNA was extracted using NucleoSpin® RNA Plus Kit (Takara). cDNA was synthesized using PrimeScript® II 1st Strand cDNA Synthesis Kit (Takara). Reactions and analysis were performed using Fast SYBR® Green Master Mix (Life Technologies) and StepOnePlus™ Real-Time PCR system (Life Technologies). The histone H4 gene was used as a reference, and the relative expression level was calculated using the ΔCT method. The sequences of the primers used in RT-PCR analysis are shown in Table 5.

[0134]

[0135] <Creation of an A. oryzae strain incorporating one copy of a kojic acid synthase gene expression cassette> The plasmid used to introduce one copy was the Aspergillus oryzae high expression vector pNGA142. The kojA gene (NCBI Gene ID: AO090113000136) was amplified using primers P32 and P33, using the A. oryzae genome as a template. The sequences of primers P32 and P33 are shown in Table 6. The amplified DNA fragment was digested with restriction enzymes HindIII and NdeI and inserted by ligation into pNGA142 vector (pNGA142-kojA) that had been treated with HindIII and NdeI. The constructed plasmid pNGA142-kojA was treated with restriction enzyme MfeI and one copy was introduced into the niaD locus on the chromosome of A. oryzae.

[0136] 3. Preparation of a Plasmid for Introducing Multiple Copies of the Kojic Acid Synthase Gene into A. oryzae Using the plasmid pNGA142-kojA as a template, an expression cassette fragment was amplified using primers P34 and P35. The sequences of primers P34 and P35 are shown in Table 6. Using the in-fusion cloning method, this expression cassette fragment was inserted into the pXPCas9aAgTpDon vector treated with the restriction enzyme SfoI to obtain the plasmid pXPcas9aAgTpkojA.

[0137]

[0138] <Kojic acid production experiment> Kojic acid production medium (10% carbon source, 0.25% yeast extract, 0.1% K ２ HPO ４ ,0.05%MgSO ４ The koji mold was cultured in a medium containing 100% ethanol (pH 6.0) at 30°C, and the culture supernatant was collected. Glucose or maltose was used as the carbon source. 1 μl of 1 M FeCl was added to 50 μl of the culture supernatant. ３ The amount of kojic acid produced was calculated using commercially available kojic acid (Fujifilm Wako Pure Chemical Industries, Ltd., 114-00493) as a standard.

[0139] (Experimental Example 1) As described below, the A. oryzae ΔAG-GAGΔadeA strain was transformed using the vector pXPcas9aADoXF for introducing multiple copies of a xylanase gene expression cassette into A. oryzae. Next, multiple copies of the xylanase gene were introduced by genome editing.

[0140] As shown in Figure 1A, the A. oryzae genome contains 13 DNA transposon-like sequences. One transposon-like sequence is present on chromosome 1, three on chromosome 2, two on chromosome 3, one on each of chromosomes 4 to 6, and two on each of chromosomes 7 and 8. In Figure 1A, "100%" and "99%" refer to the sequence identity of each transposon-like sequence based on the sequence set forth in SEQ ID NO: 5.

[0141] Figure 1B is a diagram showing the process of introducing a xylanase gene expression cassette into a transposon-like sequence by genome editing using a plasmid for introducing multiple copies of the xylanase gene expression cassette. In Figure 1B, "Tp" stands for transposon-like sequence, and "HR" stands for homologous recombination.

[0142] The A. oryzae ΔAG-GAGΔadeA strain was transformed with the pXPcas9aADoXF plasmid for introducing multiple copies of the xylanase gene into A. oryzae. The strains were cultured on an agar medium containing glucose as a carbon source, and 20 candidate transformant strains were obtained in which the plasmid had been introduced into the koji mold cells based on the adeA marker.

[0143] To confirm that the target DNA fragment (i.e., the xylanase gene expression cassette) contained in the donor DNA cassette was inserted into each DNA transposon-like sequence by genome editing, forward and reverse primers for PCR were designed as shown in Figure 1C. The forward primers were 13 sequences located outside each transposon-like sequence. The reverse primer was one sequence located inside each transposon-like sequence. These primers are shown in Table 3.

[0144] For example, by using the Ao. TpChr1 Fw primer and the Ao. Tp-Co Rv primer, it is possible to determine whether a target DNA fragment has been inserted into a transposon-like sequence on chromosome 1. Ao. TpChr2-1 Fw, Ao. TpChr2-2 Fw, and Ao. TpChr2-3 Fw are primers for determining whether a target DNA fragment has been introduced into three DNA transposon-like sequences on chromosome 2. The same applies to DNA transposon-like sequences on other chromosomes. If a target DNA fragment has not been introduced, the PCR product is 1.5 to 2 kb. If a target DNA fragment has been introduced, the PCR product is 5 to 6 kb.

[0145] First, conidia were collected from A. oryzae before the introduction of the target DNA fragment, and PCR was performed. Next, the PCR products were electrophoresed using agarose gel. The results are shown in Figure 1D. In Figure 1D, Chr1 shows the results of amplifying a transposon-like sequence on chromosome 1, while Chr2-1, Chr2-2, and Chr2-3 show the results of amplifying three transposon-like sequences on chromosome 2. The same applies to transposon-like sequences on other chromosomes.

[0146] Twenty candidate transformant strains were purified by culturing them on agar medium containing glucose as a carbon source for three days, and 12 strains grew. Conidia were collected from one of these 12 strains, PCR was performed, and the PCR product was electrophoresed. The results are shown in Figure 1E.

[0147] The results shown in Figure 1E show that when cultured in a glucose-containing medium, Cas9 expression was not induced, indicating that the target DNA was inserted only in three or four DNA transposon-like sequences. For example, it was confirmed that the target DNA fragment was inserted into Chr7-2 (chromosome 7) and Chr8-2 (chromosome 8).

[0148] Twelve strains grown on glucose agar medium were transferred to agar medium containing xylose as the sole carbon source and cultured for three days to induce Cas9 expression. Eight strains grew (these are called xylose-grown strains 1 to 8). Conidia from xylose-grown strain 8 were collected, PCR was performed, and the PCR products were electrophoresed. The results are shown in Figure 1F.

[0149] The results shown in Figure 1F indicate that when cultured in a medium containing xylose, Cas9 expression was induced, and thus the target DNA fragment was introduced into the 12 transposon-like sequences.

[0150] Furthermore, for each of the xylose-grown strains 1 to 7, the introduction of the target DNA fragment into 13 transposon-like sequences was analyzed. As a result, it was confirmed that the target DNA fragment was introduced into 12 transposon-like sequences in all of the xylose-grown strains 1 to 6. It was confirmed that the target DNA fragment was introduced into 13 transposon-like sequences in the xylose-grown strain 7. In other words, this shows that genome editing can be performed with high efficiency by using a vector containing both a Cas9 expression cassette and a donor DNA cassette.

[0151] A. oryzae was transformed with a DNA solution containing a mixture of two types of plasmids: a plasmid carrying a Cas9 expression cassette and a plasmid carrying a donor DNA cassette, and the efficiency of genome editing was examined.

[0152] Here, the Cas9 expression plasmid has the same Cas9 expression cassette as the Cas9 expression cassette contained in the plasmid pXPcas9aADoXF. The donor DNA plasmid has the same donor DNA cassette as the donor DNA cassette contained in the plasmid pXPcas9aADoXF. When these two types of plasmids were used, it was not possible to obtain a koji mold strain in which the target DNA fragment was inserted into the transposon-like sequence by genome editing.

[0153] (Experimental Example 2) As described below, the expression level of xylanase was quantified using the strain (13-copy introduced strain) produced in Experimental Example 1 in which the xylanase gene expression cassette was inserted into all 13 transposon-like sequences, and the A. oryzae strain in which one copy of the xylanase gene expression cassette was introduced.

[0154] The xylanase gene expression cassette of the 13-copy-introduced strain is the same as that of the 1-copy-introduced strain. The promoter of the xylanase gene expression cassette is PglaA142. The promoter activity of PglaA142 is induced by glucose and maltose, with maltose inducing the promoter activity more strongly than glucose.

[0155] The 13-copy-introduced strain and the 1-copy-introduced strain were cultured in YPD (3% glucose) medium or YPM (3% maltose) medium for 48 or 96 hours. 2.5 μL of the supernatant after culture was subjected to SDS-PAGE, and the expression level of the xylanase protein was quantified using ImageJ. The results of SDS-PAGE are shown in Figures 2A and 2B. Figure 2A shows the results when YPD medium was used, and Figure 2B shows the results when YPM medium was used. The quantification results using ImageJ are shown in Table 7.

[0156]

[0157] The results of Figures 2A and 2B and Table 7 show that the expression level of the xylanase protein was significantly increased in the 13-copy-introduced strain compared to the 1-copy-introduced strain, regardless of whether YPD medium or YPM medium was used.

[0158] The 13-copy and 1-copy strains were cultured in YPD (3% glucose) or YPM (3% maltose) medium for 48 hours. The expression levels of the xylanase gene in each culture supernatant were analyzed by real-time PCR. The results are shown in Figures 2C and 2D.

[0159] When cultured in YPD medium (3% glucose), the expression level of the 13-copy-introduced strain was 5-fold or more compared to that of the 1-copy-introduced strain.When cultured in YPM medium (3% maltose), the expression level of the 13-copy-introduced strain was 20-fold or more compared to that of the 1-copy-introduced strain.

[0160] (Experimental Example 3) As described below, E. nidulans biA1 pyrG89 wA3 argB2 pyroA4 ligD::ptrA ΔagsB::argB was transformed using the plasmid pXPcas9pGgTpDoXF-EN for introducing multiple copies of a xylanase gene expression cassette into E. nidulans as a vector. Next, multiple copies of the xylanase gene expression cassette were introduced by genome editing.

[0161] There are 16 DNA transposons in the E. nidulans genome: one on chromosomes 4, 5, and 7, two on chromosomes 1, 2, and 6, three on chromosome 8, and four on chromosome 3.

[0162] Similar to the case where multiple copies of the xylanase gene expression cassette were introduced into A. oryzae in Experimental Example 1, E. nidulans was transformed with a plasmid for introducing multiple copies of the xylanase gene expression cassette into E. nidulans, and forward and reverse primers were designed for PCR to confirm that the target DNA (i.e., the xylanase gene expression cassette) had been introduced into each DNA transposon-like sequence by genome editing. The forward primers were 16 different sequences located outside each transposon-like sequence. The reverse primer was one sequence located inside each transposon-like sequence. The primers are shown in Table 4. When the target DNA was not introduced, the PCR product was 1.5 to 2 kb. When the target DNA was introduced, the PCR product was 5 to 6 kb.

[0163] First, conidia were collected from E. nidulans before the introduction of the target DNA, and PCR was performed. The PCR products were then electrophoresed using agarose gel. The results are shown in Figure 3A. In Figure 3A, Chr1-1 and Chr1-2 show the results of amplifying two transposon-like sequences on chromosome 1. The same applies to transposon-like sequences on other chromosomes.

[0164] E. nidulans was transformed using the above-described plasmid for introducing multiple copies of the xylanase gene expression cassette into E. nidulans. The transformation candidate strain was transferred to a plate medium containing xylose as the sole carbon source and cultured for 7 days to induce Cas9 expression. Conidia were collected, PCR was performed, and the PCR products were electrophoresed. The results are shown in Figure 3B.

[0165] The results in FIG. 3B show that the xylanase gene expression cassette was inserted into 6 of the 16 transposon-like sequences.

[0166] (Experimental Example 4) As described below, the expression level of kojic acid synthase was quantified using a strain in which a kojic acid synthase expression cassette was inserted into all 13 transposon-like sequences by genome editing (a strain with 13 copies introduced), and an A. oryzae strain in which one copy of the kojic acid synthase expression cassette was introduced.

[0167] The A. oryzae strain ΔAG-GAGΔadeA was transformed with the plasmid pXPcas9aAgTpkojA. To purify the candidate transformant strain, it was cultured for 3 days on agar medium containing glucose as a carbon source. It was then transferred to agar medium containing xylose as the sole carbon source and cultured for 3 days to induce Cas9 gene expression.

[0168] Conidia were collected from the grown strains, and PCR was performed using the primers shown in Table 3 to determine whether or not a kojic acid synthase gene expression cassette had been inserted into each DNA transposon-like sequence. The results are shown in Figure 4A. Although the amplified DNA fragment of Chr8-1 was weak, a signal was observed. The results shown in Figure 4A indicate that a kojic acid synthase gene expression cassette had been inserted into 13 DNA transposon-like sequences.

[0169] The amount of kojic acid produced was compared between a strain into which one copy of the kojic acid synthase gene expression cassette had been introduced and a strain into which 13 copies of the above-mentioned kojic acid synthase gene expression cassette had been introduced in a kojic acid production medium. The results are shown in Figure 4B.

[0170] When cultured for 96 hours using glucose as a carbon source, the 13-copy-introduced strain produced approximately 18 times more kojic acid than the 1-copy-introduced strain. When maltose was used as a carbon source, the 13-copy-introduced strain produced approximately three times more kojic acid than when glucose was used as a carbon source.

[0171] The vectors and production methods of the present invention are suitable for inserting donor DNA into multiple target genomic regions.

Claims

1. A vector for inserting a DNA fragment into a target genomic region of a target cell, comprising an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragment, wherein the programmable endonuclease recognizes the target genomic region.

2. The vector of claim 1, wherein the programmable endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease, or a Cas protein.

3. The vector of claim 2, wherein the programmable endonuclease is a Cas protein, and the vector further comprises an expression cassette for a guide RNA.

4. The vector of claim 1, wherein the target cell has a plurality of target genomic regions, and the plurality of target genomic regions are dispersed throughout the genome of the target cell.

5. The vector of claim 4, wherein the plurality of target genomic regions have 95% or more sequence identity with each other.

6. The vector of claim 1, wherein the target genomic region is present within a transposon-like sequence.

7. The vector of claim 1, wherein the vector is a plasmid, phagemid, cosmid, artificial chromosome, bacteriophage, or viral vector.

8. The vector of claim 1, wherein the target cell is a cell of a filamentous fungus.

9. The vector of claim 8, wherein the filamentous fungus belongs to the genus Aspergillus, Emericella, Penicillium, Trichoderma, Cephalosporium, or Acremonium.

10. The vector according to claim 9, wherein the filamentous fungus belongs to the genus Aspergillus or Emericella.

11. The vector of claim 10, wherein the filamentous fungus is Aspergillus oryzae, Aspergillus nidulans, or Emericella nidulans.

12. A method for producing a recombinant in which a DNA fragment has been inserted into a target genomic region of a target cell, the method comprising the step of introducing the vector according to any one of claims 1 to 11 into the target cell.

13. A method for producing a recombinant in which DNA fragments are inserted into multiple target genomic regions of a target cell, the method comprising the step of introducing into the target cell an expression cassette for a programmable endonuclease and a donor DNA cassette containing the DNA fragments, wherein the programmable endonuclease recognizes the target genomic regions.

14. The method of claim 13, wherein the plurality of target genomic regions have 95% or more sequence identity with each other.

15. The method of claim 13, wherein the target genomic region is present within a transposon-like sequence.

16. The method of claim 13, wherein the target cell is a cell of a filamentous fungus.

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