Crispr-cas vector system, composition, kit, method for producing prokaryote with modified target DNA, and prokaryote

Abstract

Provided is a technique or the like that enables genome editing in prokaryotes. A CRISPR-Cas vector system for use in prokaryotes according to the present disclosure includes (a) a polynucleotide for encoding guide RNA and a terminator sequence that include a base sequence capable of hybridizing to target DNA in a prokaryote, and (b) a polynucleotide for encoding a Cas protein. The aforementioned (a) and (b) are located on the same or different vectors. The guide RNA and the Cas protein can form a complex. In the aforementioned (a), the terminator sequence is located on the 3'-end side of the guide RNA.

Description

CRISPR-Cas vector system, composition, kit, method for producing a prokaryotic organism having modified target DNA, and prokaryotic organism

[0001] The present disclosure relates to CRISPR-Cas vector systems, compositions, kits, methods for producing prokaryotes with modified target DNA, and prokaryotes.

[0002] Lactic acid bacteria have been reported to promote human health by improving the intestinal environment and boosting immunity, and are expected to be used in a wide range of fields, including food, supplements, and pharmaceuticals. However, while the effects of lactic acid bacteria have been reported, their mechanism of action has not necessarily been elucidated. While the development of highly effective lactic acid bacteria with a clear mechanism of action is desirable, genetic engineering techniques for lactic acid bacteria are still insufficient, and it is necessary to establish technologies and methods that enable more precise genetic modification.

[0003] Furthermore, when genome editing of lactic acid bacteria is performed using CRISPR-Cas9, which is currently the mainstream method for genome editing, CRISPR-Cas9 causes double-stranded DNA breaks, which makes it highly toxic to lactic acid bacteria, and there have been reported cases where editing is not possible depending on the strain or target gene (Non-Patent Document 1).

[0004] Leenay, Ryan T et al. “Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods.” Biotechnology journal vol. 14,3 (2019): e1700583. doi:10.1002 / biot.201700583

[0005] Therefore, an object of the present disclosure is to provide, for example, a CRISPR-Cas vector system for use in prokaryotes that is capable of genome editing in prokaryotes.

[0006] In order to achieve the above-mentioned object, the CRISPR-Cas vector system for use in prokaryotes of the present disclosure comprises: (a) a polynucleotide encoding a guide RNA and a terminator sequence, the polynucleotide comprising a base sequence capable of hybridizing to target DNA in a prokaryote; and (b) a polynucleotide encoding a Cas protein, wherein (a) and (b) are arranged on the same or different vectors, the guide RNA and the Cas protein are capable of forming a complex, and in (a), the terminator sequence is arranged on the 3' end of the guide RNA.

[0007] The composition of the present disclosure comprises (a) a vector and (b) a Cas protein or a nucleic acid encoding the same, wherein the vector comprises a polynucleotide encoding a guide RNA and a terminator sequence, the guide RNA and the Cas protein comprising a base sequence capable of hybridizing to target DNA in a prokaryote, wherein the guide RNA and the Cas protein are capable of forming a complex, and wherein the terminator sequence in the polynucleotide is located on the 3' end of the guide RNA.

[0008] The kits of the present disclosure include a CRISPR-Cas vector system of the present disclosure and / or a composition of the present disclosure.

[0009] The method for producing a prokaryote having modified target DNA of the present disclosure includes an introduction step of introducing the CRISPR-Cas vector system of the present disclosure and / or the composition of the present disclosure into a prokaryote.

[0010] The prokaryotic organisms of the present disclosure comprise a CRISPR-Cas vector system of the present disclosure and / or a composition of the present disclosure.

[0011] According to the present disclosure, for example, it is possible to provide a CRISPR-Cas vector system for use in prokaryotes that is capable of genome editing in prokaryotes.

[0012] FIG. 1A is a schematic diagram showing the arrangement of pYK1 to pYK5 among 10 types (pYK1 to pYK10) of CRISPR-Cas plasmid vectors with different promoter and other configurations. FIG. 1B is a schematic diagram showing the arrangement of pYK6 to pYK10 among 10 types (pYK1 to pYK10) of CRISPR-Cas plasmid vectors with different promoter and other configurations. FIG. 2 is a schematic diagram showing the vector map of CRISPR-Cas plasmid vector pYK8. FIG. 3 is a schematic diagram showing the guide RNA sequence and PAM sequence, as well as the cytosine-to-thymine conversion in the upp gene. FIG. 4 is a graph showing the results of mutagenesis in L. plantarum WCFS1. Figure 5 is a schematic diagram showing the guide RNA sequence, PAM sequence, and cytosine-to-thymine conversion in the non-coding region of the genome of L. gasseri ATCC 33323. Figure 6 is a schematic diagram showing the configuration of CRISPR-Cas plasmid vectors pYK8 and pYK11. Figure 7 is a graph showing the results of mutagenesis in L. plantarum WCFS1. Figure 8 is a schematic diagram showing the configuration of CRISPR-Cas plasmid vectors pYK8, pYK8-dual, and pYK8-triple. Figure 9 is a schematic diagram showing the guide RNA sequence and PAM sequence in the urdA gene. Figure 10 is a graph showing the results of mutagenesis in L. plantarum WCFS1. Figure 11 is a schematic diagram showing the guide RNA sequence, PAM sequence, and cytosine-to-thymine conversion in the urdA gene. Figure 12 is a photograph showing the results of serial dilution of lactic acid bacteria cultured in erythromycin-free medium for vector removal. Figure 13 shows the results of Sanger sequencing of the target sequence of the urdA gene.

[0013] <Definition> As used herein, "CRISPR-Cas" (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) refers to a complex composed of a Cas protein and a guide RNA. The CRISPR-Cas typically specifically recognizes and binds to a target DNA sequence and performs nucleic acid modification, such as cleavage.

[0014] As used herein, "CRISPR-Cas vector system" refers to a vector system comprising a polynucleotide encoding a guide RNA and a polynucleotide encoding a Cas protein.

[0015] As used herein, "guide RNA" refers to an RNA that can target a Cas protein to a target DNA sequence. The guide RNA includes a crRNA (CRISPR RNA) that binds to a target site and, optionally, a tracrRNA (trans-activating crRNA) that is involved in the activity of the CRISPR-Cas system, depending on the type of Cas protein. The guide RNA may be, for example, a single-stranded RNA in which the crRNA and tracrRNA are directly or indirectly linked, or may be two RNAs, the crRNA and the tracrRNA.

[0016] As used herein, "crRNA" refers to an RNA having a base sequence (hereinafter also referred to as "sequence") complementary to a target DNA sequence. The crRNA generally includes a spacer sequence and a repeat sequence. The spacer sequence can also be referred to as a sequence (guide sequence) capable of forming a double strand with the complementary strand of the target DNA sequence.

[0017] As used herein, "tracrRNA" refers to RNA having a sequence complementary to a portion of the crRNA. The sequence complementary to a portion of the crRNA is also referred to as an anti-repeat region (sequence). The tracrRNA includes an anti-repeat region (sequence) (repeat region) followed by one or more hairpin structures, and has a sequence capable of forming a stem loop. The tracrRNA is known to function as a scaffold for binding the Cas protein and crRNA in some Cas proteins.

[0018] As used herein, "hybridize" refers to annealing with a complementary polynucleotide that occurs due to nucleotide complementarity, specifically, base complementarity in nucleotides. That is, it means that two polynucleotides can form a non-covalent pair via hydrogen bonds. The hybridization may be, for example, the binding of two complementary base sequences, or substantially complementary base sequences with one or more mismatched base pairs.

[0019] As used herein, "complementary" or "complementary" means the ability to form nucleotide pairs, i.e., base pairs, between one polynucleotide and another polynucleotide.

[0020] As used herein, "Cas protein" refers to a protein in the CRISPR / Cas system. The Cas protein is known to activate a DNA cleavage domain, for example, by forming a complex with a guide RNA, and cause double-strand breaks (DSBs) through nuclease activity, or single-strand breaks through nickase activity. The Cas protein can also be referred to as, for example, a "Cas nuclease." Furthermore, when a mutation is introduced into the Cas protein, the Cas protein can also be referred to as a "Cas protein mutant."

[0021] As used herein, "targeting" means binding to or accumulating in a target region.

[0022] As used herein, "targeted site" or "targeted region" refers to a site or region in the DNA of a target prokaryote intended to induce a desired effect, such as a mutation (e.g., deletion, substitution, conversion, and / or insertion) of the nucleic acid sequence.

[0023] As used herein, "target nucleic acid" refers to the target nucleotide sequence in a target prokaryotic nucleic acid to which a guide RNA hybridizes.

[0024] As used herein, "target DNA" refers to the target nucleotide sequence in the DNA of a target prokaryote to which a guide RNA hybridizes.

[0025] As used herein, "genomic DNA" refers to deoxyribonucleotides (DNA) in the genome of a prokaryotic organism within its cytoplasm. Furthermore, as used herein, "plasmid DNA" refers to a DNA molecule that exists independently of the genomic DNA and can be replicated independently of the genomic DNA. Plasmid DNA is typically circular, double-stranded DNA.

[0026] As used herein, "protein" or "peptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0027] As used herein, the term "domain" refers to a structurally or functionally organized region in a protein, polypeptide, and / or peptide.

[0028] As used herein, a "vector" means a nucleic acid that can be delivered to a host cell in vitro or in vivo, or a recombinant plasmid or virus that contains such a nucleic acid.

[0029] As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "polynucleotide" is used in conjunction with a specific protein, the "polynucleotide" refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the polynucleotide include genomic DNA, cDNA, and mRNA. The polynucleotide may be, for example, single-stranded or double-stranded. The polynucleotide can be interchangeably referred to as a "nucleic acid" or an "oligonucleotide."

[0030] As used herein, "prokaryote" refers to an organism that does not have a membrane-enclosed nucleus.

[0031] As used herein, "lactic acid bacteria" refers to bacteria that produce lactic acid from sugars through metabolism.

[0032] As used herein, the term "terminator" refers to a region having the function of releasing transcribed RNA from a transcription complex and terminating transcription.

[0033] As used herein, a "promoter" or a "promoter region" refers to a region that is present upstream of DNA encoding a gene or a polynucleotide, contains a (nucleic acid) sequence to which a transcription factor binds, and regulates the amount of transcription of the gene or the polynucleotide. The "promoter" or "promoter region" can also be referred to as, for example, a "transcriptional regulatory region."

[0034] As used herein, "enzymatically active protein" means a protein that has enzymatic activity.

[0035] As used herein, "nucleic acid modifying enzyme" refers to an enzyme that can modify bases (e.g., A, T, C, G, or U) in nucleic acid sequences such as DNA and RNA. The "nucleic acid modification" refers to the substitution, conversion, deletion, insertion, addition, etc. of bases in a nucleic acid sequence.

[0036] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. Furthermore, the nucleic acid sequence of RNA can also be obtained from the corresponding DNA base sequence using appropriate sequence conversion software, etc.

[0037] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure and each embodiment can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0038] <CRISPR-Cas Vector System> In one aspect, the present disclosure provides a CRISPR-Cas vector system for use in prokaryotes. The CRISPR-Cas vector system for use in prokaryotes of the present disclosure comprises: (a) a polynucleotide encoding a guide RNA and a terminator sequence, the polynucleotide encoding the guide RNA and the Cas protein, the polynucleotide comprising a base sequence capable of hybridizing to target DNA in a prokaryote; and (b) a polynucleotide encoding a Cas protein, wherein (a) and (b) are arranged on the same or different vectors, the guide RNA and the Cas protein are capable of forming a complex, and in (a), the terminator sequence is arranged on the 3' end of the guide RNA.

[0039] The present inventors have discovered that genome editing of prokaryotes using the CRISPR-Cas system does not always occur. The present inventors conceived the idea that, in prokaryotes, when a guide RNA is transcribed from a nucleic acid encoding the guide RNA of a CRISPR-Cas vector system, the 3' end of the guide RNA is transcribed longer than the original 3' end of the crRNA or tracrRNA, resulting in failure to target the complex with the Cas protein and preventing genome editing. As a result of extensive research, the present inventors discovered that genome editing of prokaryotes is possible by placing a terminator sequence on the 3' end of the guide RNA, i.e., the 3' end of the crRNA or tracrRNA, in a CRISPR-Cas vector system, and have established the present disclosure. Therefore, according to the present disclosure, genome editing of prokaryotes such as lactic acid bacteria is possible. The mechanism of action of the terminator sequence is a presumed mechanism of action and does not limit the present disclosure in any way.

[0040] In the present disclosure, the polynucleotide (a) encodes a guide RNA and a terminator sequence containing a base sequence capable of hybridizing to a target DNA in the prokaryote. The polynucleotide (a) may consist of the guide RNA and the terminator sequence, or may include other components.

[0041] Examples of the prokaryote include lactic acid bacteria, Bacillus subtilis var. natto, Escherichia coli, and cyanobacteria, preferably lactic acid bacteria. Examples of the prokaryote include prokaryote distributed in the skin, oral cavity, or digestive tract. Examples of the digestive tract include the esophagus, stomach, small intestine (e.g., duodenum, jejunum, ileum), large intestine (e.g., cecum, colon, rectum), and the like.

[0042] Examples of the prokaryote generally distributed in the intestinal tract include the strains shown in Table 1. Examples of the prokaryote distributed in the intestinal tract include enterobacteria and indigenous intestinal bacteria.

[0043]

[0044] Examples of the prokaryote generally distributed in the oral cavity include the strains shown in Table 2. Examples of the prokaryote distributed in the oral cavity include oral bacteria and oral flora.

[0045]

[0046] Examples of the prokaryotes that are generally distributed on the skin include the bacterial strains shown in Table 3 below. Examples of the prokaryotes that are distributed on the skin include normal skin flora.

[0047]

[0048] Examples of the prokaryotes that are generally distributed in areas other than the intestinal tract, oral cavity, and skin include the strains shown in Table 4 below.

[0049]

[0050] Examples of the lactic acid bacteria include lactic acid bacteria classified into genera such as Lactobacillus, Lactiplantibacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Bifidobacterium, Staphylococcus, Carnobacterium, Oenococcus, Tetragenococcus, and Vagococcus.

[0051] The lactic acid bacteria are, for example, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paragasseri, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Ligilactobacillus salivarius, Levilactobacillus brevis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus mundtii, Enterococcus casseliflavus, Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, Pediococcus acidilactici, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, Pediococcus pentosaceus, Pediococcus stilesii、Leuconostoc citreum、Leuconostoc pseudomesenteroides、Leuconostoc lactis、Streptococcus cristatus、Streptococcus gordonii、Streptococcus mutans、StreptococcusExamples of lactic acid bacteria include those classified into species such as Bifidobacterium salivarius, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus pyogenes, Streptococcus constellatus, Streptococcus sanguinis, Bifidobacterium spp., Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium catenulatum, and Bifidobacterium longum.

[0052] The target DNA in the prokaryote is, for example, any sequence present in the genomic DNA present in the prokaryote, plasmid DNA possessed by the prokaryote, invaded phage DNA, artificially introduced DNA, etc., and examples thereof include a gene locus, a sequence encoding a gene, non-coding DNA, a gene control region (promoter sequence, enhancer sequence, terminator sequence, etc.). When the target DNA encodes a gene, the target DNA can also be referred to as, for example, a target gene. The target DNA may be single-stranded (ssDNA) or double-stranded DNA (dsDNA). In the CRISPR-Cas system of the present disclosure, for example, when a nuclease that cleaves RNA in a guide RNA-dependent manner is used as the Cas protein, RNA may be targeted instead of DNA. In this case, the explanation of the present disclosure can be used by replacing "target DNA" with "target RNA" and "DNA" with "RNA". The CRISPR-Cas system of the present disclosure may target, for example, RNA in addition to DNA as the target. In this case, the explanation of the present disclosure can be used by replacing "target DNA" with "target nucleic acid" and "DNA" with "nucleic acid."

[0053] The target DNA to which the guide RNA can hybridize is not particularly limited and can be set, for example, according to the PAM (proto-spacer adjacent motif) of the Cas protein. The length of the target DNA is not particularly limited and, for example, is a length to which the guide RNA can specifically hybridize, specific examples of which include a length of 14 to 30 bases, or a length of 16 to 25 bases.

[0054] The hybridization can be detected, for example, by various hybridization assays under stringent conditions. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." (eds.) Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used.

[0055] The "stringent conditions" may be, for example, low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the degree of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" may be, for example, those described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)].

[0056] The guide RNA includes, for example, a spacer sequence (guide sequence) and a direct repeat sequence. In the guide RNA, the spacer sequence hybridizes, for example, to the target nucleotide sequence of the target DNA. The spacer sequence may be, for example, completely complementary (identical) to the target nucleotide sequence of the target DNA, or may be substantially complementary (identical). The term "substantially complementary" means, for example, that the sequence is composed of a base sequence having 50% or more identity to the nucleotide sequence of the target DNA and is capable of hybridizing to the nucleotide sequence of the target DNA. The "identity" to the nucleotide sequence of the target DNA may be, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The "identity" can be determined by aligning two base sequences or amino acid sequences (the same applies hereinafter). The alignment can be calculated using, for example, BLAST, FASTA, or the like with default parameters. The substantially complementary sequence is, for example, a sequence in which one or two mismatched base pairs exist between the spacer sequence and the target DNA. In the guide RNA, the length of the spacer sequence is not particularly limited as long as it is long enough to hybridize to the target DNA, and examples thereof include a length of 14 to 30 bases, or a length of 16 to 25 bases.

[0057] Preferably, the spacer sequence further includes a seed sequence. The seed sequence is, for example, a sequence on the 5'-end side of the PAM sequence in the spacer sequence, and is important for the specificity of the guide RNA to the target DNA. In the guide RNA, the seed sequence is, for example, substantially or completely complementary to the nucleotide sequence of the target DNA, preferably the latter. When the seed sequence is substantially complementary to the nucleotide sequence of the target DNA, the substantially complementary sequence is, for example, a sequence in which one or two mismatch base pairs exist between the seed sequence and the nucleotide sequence of the target DNA.

[0058] In the guide RNA, the length of the direct repeat sequence is not particularly limited and may be, for example, 19 to 40 bases long or 21 to 38 bases long. In the case of Staphylococcus aureus-derived Cas9 (SpCas9), the length of the direct repeat sequence is, for example, 36 bases long. In the case of Salmonella typhimurium-derived Cas9, the length of the direct repeat sequence is, for example, 21 bases long.

[0059] The length of the guide RNA sequence is set so as to enable the Cas protein to target the target DNA. The length of the guide RNA sequence is not particularly limited, and examples include 30 to 220 bases, 40 to 220 bases, 50 to 220 bases, 60 to 220 bases, 70 to 220 bases, 80 to 220 bases, 90 to 220 bases, 100 to 220 bases, 110 to 220 bases, 110 to 210 bases, 110 to 200 bases, 110 to 190 bases, 110 to 180 bases, 110 to 170 bases, 110 to 160 bases, and 110 to 150 bases. When the guide RNA contains crRNA and tracrRNA in separate RNAs, the length of the guide RNA sequence is, for example, the total length of both RNAs. When the guide RNA contains crRNA and tracrRNA in the same RNA (in the case of sgRNA described below), the length of the sequence of the guide RNA is, for example, the length of the sequence of the sgRNA.When the guide RNA contains crRNA, the length of the sequence of the guide RNA is, for example, the length of the sequence of the crRNA.

[0060] The guide RNA may be one type or multiple types. When there are multiple types of guide RNA, it is preferable that the guide RNAs are configured to be able to hybridize to different target DNAs, for example. When there are multiple types of guide RNA, for example, by configuring the guide RNA to target the target DNA to the 5' and 3' sides of the target region, the CRISPR / Cas system can induce deletion of the target region between the 5' and 3' sides (large deletion). When there are multiple types of guide RNA, for example, by configuring the guide RNA to target the target DNA to the 5' and 3' sides of the target region and introducing donor DNA having sequences complementary to the 5' side region (5' arm region) and the 3' side region (3' arm region), the CRISPR / Cas system can introduce the donor DNA into the region between the 5' side and the 3' side by recombination.

[0061] The guide RNA may contain only crRNA, or may contain crRNA and tracrRNA. When the guide RNA contains crRNA and tracrRNA, the guide RNA may be an sgRNA (single-stranded guide strand) in which the tracrRNA and the crRNA are fused, or may not be fused, that is, may be composed of two RNAs, crRNA and tracrRNA. When the tracrRNA and the crRNA are not fused, it is preferable that the tracrRNA and the crRNA have substantially or completely complementary sequences, such as the repeat sequence and the anti-repeat region. The structure of the guide RNA (single-stranded or double-stranded) and the presence or absence of tracrRNA can be determined depending on the type of Cas protein described below.

[0062] The tracrRNA includes a repeat sequence (anti-repeat region) and one or more hairpin structures. The number of hairpin structures is not particularly limited and may be, for example, 1 to 10, 1 to 5, 1 to 3, or 2. The tracrRNA is not particularly limited as long as it can bind to the crRNA and form a complex.

[0063] The tracrRNA can be designed based on the sequence of the tracrRNA in the bacterial species from which the Cas protein is derived. The tracrRNA can be, for example, the same sequence as the tracrRNA in the bacterial species from which the Cas protein is derived, i.e., a naturally occurring sequence (natural sequence), or it can be a sequence in which one or more bases have been substituted, added, deleted, and / or inserted relative to the natural sequence (artificial sequence). The artificial sequence can be prepared, for example, by deleting bases at the 5' and / or 3' ends of the natural sequence. The tracrRNA sequence can also have, for example, a nucleotide region with other functionality, and, for example, can be modified to include one or more aptamer sequences (see Reference 1 below). Examples of the aptamer sequence include MCP, PCP, Com, SLBP, FXR1, AP205, BZ13, f1, f2, fd, fr, ID2, JP34 / GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, φCb5, φCb8r, φCb12r, φCb23r, Qβ, R17, SP-β, TW18, TW19, and VK. Reference 1: Konermann, Silvana et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex." Nature vol. 517,7536 (2015): 583-8. doi:10.1038 / nature14136

[0064] The length of the tracrRNA sequence is not particularly limited and can be set depending on, for example, the Cas protein. Examples of the length of the tracrRNA sequence include 10 to 200 bases, 20 to 190 bases, 30 to 180 bases, 40 to 180 bases, 50 to 180 bases, 60 to 180 bases, 70 to 180 bases, 70 to 170 bases, 70 to 160 bases, 70 to 150 bases, 70 to 140 bases, 70 to 130 bases, 70 to 120 bases, 70 to 110 bases, 70 to 100 bases, and 70 to 90 bases. As a specific example, in the case of wild-type SpCas9, the length of the tracrRNA sequence is, for example, about 89 bases in length (e.g., 70 to 110 bases in length, 80 to 100 bases in length) or about 171 bases in length (e.g., 160 to 180 bases in length, 150 to 190 bases in length).

[0065] The crRNA includes, for example, a spacer sequence and a direct repeat sequence. The crRNA is not particularly limited as long as it can bind to the tracrRNA and form a complex. The description of the spacer sequence can be applied to the spacer sequence. The length of the direct repeat sequence is not particularly limited, and may be, for example, 35 to 40 bases long.

[0066] The length of the crRNA sequence is not particularly limited and can be set depending on the Cas protein. Examples of the length of the crRNA sequence include 25 to 80 bases, 30 to 80 bases, 35 to 80 bases, 40 to 80 bases, 49 to 80 bases, 40 to 70 bases, and 40 to 60 bases.

[0067] The crRNA sequence can be configured to target a region adjacent to a PAM (proto-spacer adjacent motif) sequence of the target DNA. The PAM sequence varies depending on the species from which the Cas protein is derived. Examples of PAM sequences are shown in Table 5 below. In Table 5 below, N stands for A, T, C, or G, and W stands for A or T.

[0068]

[0069] The spacer sequence constituting the crRNA may be, for example, completely complementary (identical) to the target nucleotide sequence of the target DNA, or may be substantially complementary (identical). The term "substantially complementary" means, for example, that the sequence is composed of a base sequence having 50% or more identity to the target nucleotide sequence and is capable of hybridizing to the target nucleotide sequence. The "identity" to the target nucleotide sequence may be, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The substantially complementary sequence is, for example, a sequence in which one or two mismatched base pairs exist between the spacer sequence and the target DNA. The length of the spacer sequence is not particularly limited as long as it is long enough to hybridize with the target nucleotide, and may be, for example, 14 to 30 bases long.

[0070] Examples of the terminator sequence include an rrnA terminator, an rrnB terminator, a T4 terminator, a T7 terminator, an fd phage terminator, a tet terminator, a trpA terminator, a lac terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, an IE1 terminator, a lambda t0 terminator, an rrnG terminator, a soxR terminator, a T3Te terminator, a T7Te terminator, and a tonB terminator.

[0071] The terminator sequence may be derived from, for example, a prokaryote, such as Escherichia coli, T4 phage, T7 phage, λ phage, Brevibacterium lactofermentum, or the lactic acid bacteria. The origin of the terminator sequence can be determined, for example, from the description of the origin of the Cas protein, which will be described later. The terminator sequence may be derived from, for example, the same species as the prokaryote, or from a different species.

[0072] The terminator sequence may be located, for example, at the 3'-end of the guide RNA, and is preferably functionally linked to the 3'-end of the guide RNA, i.e., linked so as to be able to terminate transcription. The terminator sequence is located, for example, within 1 to 30 bases, 1 to 20 bases, or 1 to 10 bases from the 3'-end of the crRNA and / or tracrRNA, with the base at the 3'-end as the reference base (base 0), and is preferably located within 1 to 10 bases.

[0073] The terminator sequence may be located, for example, at the 3' end of the crRNA and / or the tracrRNA. The terminator sequence is located, for example, within 1 to 30 bases, 1 to 20 bases, or 1 to 10 bases, preferably within 1 to 10 bases, from the 3' end of the crRNA and / or the tracrRNA, with the base at the 3' end of the crRNA and / or the tracrRNA as the reference base (base 0). When the guide RNA includes a crRNA and a tracrRNA, the terminator sequence is located at the 3' end of at least one of the crRNA and the tracrRNA. The terminator is preferably located at the 3' end of the RNA located at the 3' end of the crRNA or the tracrRNA. The terminator sequence may be located, for example, at the 3' end of both the crRNA and the tracrRNA.

[0074] The polynucleotide (b) encodes a Cas protein. The type of the Cas protein is not particularly limited and may be, for example, a Cas protein constituting a CRISPR-Cas system classified into either class 1 or class 2 (see References 2-3 below). The Cas protein may be composed of, for example, one Cas protein or multiple Cas proteins depending on the CRISPR-Cas system. In the class 1 CRISPR-Cas system, a Cas-RNA complex containing multiple Cas proteins is known to be responsible for the degradation of exogenous nucleic acids. In the class 2 CRISPR-Cas system, a complex containing a single Cas protein and a guide RNA is known to cleave exogenous nucleic acids. Reference 2: Makarova KS et.al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?”, CRISPR J. 2018 Oct;1(5):325-336. Reference 3: Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJM, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas C, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020 Feb;18(2):67-83. doi: 10.1038 / s41579-019-0299-x. Epub 2019 Dec 19. PMID: 31857715; PMCID: PMC8905525.

[0075] Known examples of the class 1 CRISPR-Cas system include type I, type III, and type IV CRISPR-Cas systems. The type I CRISPR-Cas system is known to involve a complex of Cas3, a cascade, and crRNA. The type III CRISPR-Cas system is known to include, for example, Cas10 as the Cas protein. The type IV CRISPR-Cas system is known to include, for example, Csf1 as the Cas protein. Type I is further classified into six types, type IA, type IB, type IC, type ID, and type IE, as well as type IG, a subtype of type IB, depending on the type of cascade protein. In type IA, the cascade includes, for example, Cas5, Cas6, Cas7, Cas8a1, and Cas11 (Csa5). In the IB type, the cascade includes, for example, Cas5, Cas6, Cas7, and Cas8b1. In the IC type, the cascade includes, for example, Cas5, Cas7, and Cas8c. In the ID type, the cascade includes, for example, Cas5 (Csc1), Cas6, Cas7 (Csc2), and Cas10d. In the IE type, the cascade includes, for example, Cas5, Cas6, Cas7, Cas8e (Cse1), and Cas11 (Cse2). In the IF type, the cascade includes, for example, Cas5f1 (Csy2), Cas6f, Cas7f1 (Csy3), and Cas8f (Csy1). In the IG type, the cascade includes, for example, Cas5, Cas6, Cas7 (Cst2), and Cas8a1 (Cst1).

[0076] Examples of the class 2 CRISPR-Cas system include type II, type V, and type VI. The type II can be classified into subtypes of type II-A, type II-B, and type II-C, for example, based on molecular phylogeny. An example of the type II is Cas9. An example of the type V is Cas12a (Cpf1). An example of the type VI is Cas13.

[0077] Specific examples of the Cas protein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas12b, Cas12c, Cas12d (CasY), Cas12e (CasX), and other Cas12 proteins; Cas13a, Cas13b, Cas13c, Cas13d, and other Cas13 proteins; Cas14a (Cas12f), CasΦ, and Csy. 1, Csy2, Csy3, Cse1, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or variants thereof.

[0078] The origin of the Cas protein is not particularly limited, and examples thereof include bacteria of the genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, and Azospirillum. spirillum), Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospira ceae), Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helicococcus Examples of the prokaryotic bacteria include bacteria of the genus Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Acidaminococcus, etc. The origin of the Cas protein may be, for example, the same species as the prokaryote, or a different species.

[0079] When the Cas protein is Cas9, for example, Cas9 derived from Streptococcus pyogenes (Streptococcus pyogenes) (SpCas9), Cas9 derived from Streptococcus thermophilus (StCas9), Cas9 derived from Streptococcus pneumoniae (SpCas9), Cas9 derived from Neisseria meningitidis (NmCas9), Cas9 derived from Staphylococcus aureus (Staphylococcus aureus) (SaCas9), Cas9 derived from Campylobacter jejuni (CjCas9), Cas9 derived from Francisella novicida (Francisella When the Cas protein is Cpf1, examples thereof include Cpf1 (FnCpf1) derived from Francisella novicida, Cpf1 (AsCpf1) derived from Acidaminococcus sp., and Cpf1 (LbCpf1) derived from Lachnospiraceae bacterium.

[0080] The Cas protein may or may not cleave the double strands constituting the target DNA. When the Cas protein cleaves the double strands constituting the target DNA, the Cas protein may cleave at least one of the double strands constituting the target DNA, or may cleave only one of the strands. That is, the Cas protein may be a Cas protein having nuclease activity that cleaves both double strands constituting the DNA, a Cas protein having nickase activity that cleaves only one of the double strands constituting the DNA, or a Cas protein in which the ability to cleave both double strands constituting the DNA has been inactivated. Examples of Cas proteins having nuclease activity that cleave both double strands constituting the DNA include the above-mentioned Cas proteins. A Cas protein having nickase activity that cleaves only one of the double strands constituting the DNA can be prepared, for example, by introducing a mutation into the nuclease domain of the Cas protein. When the Cas protein is Cas9, examples of the nuclease domain include RuvC (e.g., RuvC1) or an HNH-like nuclease domain. Examples of the Cas9 (nCas9) with nickase activity include Cas9 mutants in which mutations have been introduced into the RuvC1 or HNH-like nuclease domain, specifically mutants with D10A or H840A mutations. Examples of the Cas9 (dCas9) that does not have double-strand cleavage activity include Cas9 mutants in which mutations have been introduced into the RuvC1 and HNH-like nuclease domains, specifically mutants with both D10A and H840A mutations. When the Cas protein is Cas12a (Cpf1), examples of the nuclease domain include a RuvC-like nuclease domain. Examples of the Cas12 (nCas12) having nickase activity include Cas12 mutants in which a mutation has been introduced into the RuvC nuclease domain, and specific examples include AsCpf1 mutants in which an R1226A mutation has been introduced.Cas12a in which the cleavage ability of both double strands constituting the DNA has been inactivated includes, for example, FnCpf1 mutants, mutants in which D917A or E1006A mutations have been introduced into the RuvC-like domain, AsCpf1 mutants, mutants in which D908A, E993A, and D1263A mutations have been introduced into the RuvC-like domain, LbCpf1 mutants, mutants in which D832A, E925A, D947A, or D1180A mutations have been introduced into the RuvC-like domain, etc. In addition, Cas13 in which the nuclease activity has been inactivated includes, for example, mutants of Cas13 in which a mutation has been introduced into HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) having nuclease activity (dCas13) and the like. When the Cas protein is Cas14, the nuclease domain can be, for example, a RuvC nuclease domain. The Cas protein having nickase activity or the Cas protein not having double-strand cleavage activity can be prepared, for example, by identifying the nuclease domain between Cas proteins by homology search and introducing a mutation into the identified nuclease domain.

[0081] The terminator sequence may be located, for example, at the 3'-end of the polynucleotide encoding the Cas protein, and is preferably operably linked to the 3'-end of the polynucleotide encoding the Cas protein. The terminator sequence is located, for example, within 1 to 30 bases, 1 to 20 bases, or 1 to 10 bases from the 3'-end of the polynucleotide encoding the Cas protein, with the base at the 3'-end (base 0) as the reference base, and is preferably located within 1 to 10 bases.

[0082] The polynucleotide (a) and the polynucleotide (b) are configured to be transcribed and / or translated within the prokaryote and capable of forming a complex. The Cas protein can form a Cas protein / guide RNA complex, for example, by binding to crRNA and / or tracrRNA. The complex can target the Cas protein to the target DNA, for example, depending on the sequence of the guide RNA, and modify the target DNA depending on the activity of the Cas protein or the enzymatic protein described below. The modification can be, for example, double-stranded breaks or single-stranded breaks in the target DNA, or the conversion, substitution, deletion, or insertion of one or more nucleotides. The modification can be, for example, modification of the base, sugar, or phosphate moiety of the target DNA. The complex of the guide RNA and the Cas protein can, for example, convert, replace, delete, or insert one or more nucleotides at the targeted site. The one or more can be, for example, 1 to 10 or 1 to 5.

[0083] The vector system of the present disclosure includes (a) and (b), and (a) and (b) may be arranged on the same vector or on different vectors. When (a) and (b) are arranged on the same vector, the arrangement of (a) and (b) is not particularly limited, and (b) may be arranged on the 3'-end side of (a), or (b) may be arranged on the 5'-end side of (a). When (b) is arranged on the 3'-end side of (a), it is preferable that the terminator sequence is arranged between the polynucleotide encoding the guide RNA and the polynucleotide encoding the Cas protein.

[0084] In the vector system of the present disclosure, the guide RNA may include the crRNA, or may include the crRNA and the tracrRNA. When the guide RNA includes the crRNA, the crRNA may be located on the same vector as (b) or on a different vector. When the crRNA and (b) are located on the same vector, the location of the crRNA and (b) is not particularly limited, and (b) may be located on the 3' side of the crRNA, or (b) may be located on the 5' side of the crRNA. When (b) is located on the 3' side of the crRNA, it is preferable that the terminator sequence be located between the polynucleotide encoding the crRNA and the polynucleotide encoding the Cas protein. When the guide RNA comprises the crRNA and the tracrRNA, and the crRNA and / or the tracrRNA is arranged on the same vector as (b), the arrangement of the crRNA, the tracrRNA, and the (b) is not particularly limited, and examples thereof include the crRNA, the tracrRNA, and the (b); the crRNA, the (b), and the tracrRNA; the (b), the crRNA, and the tracrRNA; the tracrRNA, the crRNA, and the (b); the tracrRNA, the (b), and the crRNA; or the (b), the tracrRNA, and the crRNA. The crRNA and the tracrRNA may each have the terminator sequence arranged at the 3' end. When the crRNA and the tracrRNA are arranged consecutively, the terminator sequence may be arranged at the 3' end of each RNA, or at the 3' end of either RNA. When (b) is arranged at the 3' end of the crRNA or the tracrRNA, it is preferable that the terminator sequence be arranged between the polynucleotide encoding the crRNA or the tracrRNA and the polynucleotide encoding the Cas protein.

[0085] The vector system of the present disclosure may include, for example, a promoter sequence on the vector. The promoter can be appropriately set depending on, for example, the type of prokaryote in which the polynucleotide encoding the Cas protein and / or the guide RNA is expressed. Examples of the promoter include a T3 promoter, a T7 promoter, an sp6 promoter, an EF1α promoter, an SRα promoter, an SV40 (Simian virus) promoter, an LTR promoter, a CMV (Cytomegalovirus) promoter, an RSV (Respiratory syncytial virus) promoter, an HSV-tk promoter, a cauliflower mosaic virus (CaMV) 35S promoter, an actin promoter, a heat shock promoter, a REF (Rubber Elongation Receptor) promoter, and the like. Factor) promoter, polyhedrin promoter, p10 promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter, GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, SPO1 promoter, SPO2 promoter, penP promoter, gyrA-ldh promoter, pgm promoter, rrn4 promoter, P23 promoter, araBAD promoter, cat promoter, cspA promoter, EM7 promoter, J23119 promoter, T5 promoter, tac promoter, etc.

[0086] The promoter sequence may be the above-mentioned unidirectional promoter or a bidirectional promoter. Examples of the bidirectional promoter include the gyrA-ldh bidirectional promoter. When multiple components of the polynucleotide encoding the guide RNA and the Cas protein are placed on the same expression vector, the bidirectional promoter can, for example, efficiently induce the expression of each component.

[0087] The origin of the promoter sequence can be determined, for example, from the same species as the prokaryote or from a different species.

[0088] The promoter sequence is located, for example, near the 5' end of the polynucleotide encoding the guide RNA (e.g., the crRNA and / or the tracrRNA) and / or the Cas protein. The promoter sequence is preferably operably linked to each of the guide RNA and the polynucleotide encoding the Cas protein so as to be capable of inducing transcription thereof. When the promoter sequence is located near the 5' end of the guide RNA (e.g., the crRNA and / or the tracrRNA) and / or the polynucleotide encoding the Cas protein, the promoter is not particularly limited as long as it is capable of promoting transcription at the 3' end of the promoter sequence; for example, a pgm promoter can be used. The promoter sequence is preferably located near the 5' end of the guide RNA (e.g., the crRNA and / or the tracrRNA) or the polynucleotide encoding the Cas protein. As a specific example, the promoter sequence is positioned within 1 to 50 bases, 1 to 40 bases, 1 to 30 bases, 1 to 20 bases, or 1 to 10 bases from the 5'-end of the polynucleotide encoding the guide RNA (e.g., the crRNA and / or the tracrRNA) or the Cas protein, using the base at the 5'-end as the reference (base 0), and preferably within 1 to 10 bases.

[0089] The vector system of the present disclosure may further include, for example, (c) a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof. Examples of the enzymatic activity of the enzymatically active protein include nucleic acid modification activities such as methylase activity, demethylase activity, base modification activity, histone modification activity, RNA cleavage activity, and DNA cleavage activity; transcription activation activity, transcription repression activity, transcription deactivation factor activity, DNA integration activity, and nucleic acid binding activity. The enzymatically active protein may be, for example, a peptide fragment thereof, as long as it has catalytic activity. The enzymatically active protein may be derived from the same species as the Cas protein or from a different species.

[0090] The enzymatically active protein is preferably a nucleic acid modifying enzyme having nucleic acid modifying activity. Examples of the nucleic acid modifying enzyme include enzymes that modify nucleic acids, thereby directly or indirectly modifying DNA, such as nucleases, nucleobase conversion enzymes, and DNA glycosylases. Examples of DNA modification include a DNA strand cleavage reaction catalyzed by the nuclease to cleave a DNA strand, a nucleobase conversion reaction catalyzed by the nucleobase conversion enzyme to convert a substituent on a purine or pyrimidine ring of a nucleobase to another group or the like without cleaving the DNA strand, and an abasic reaction catalyzed by the DNA glucosidase to hydrolyze an N-glycosidic bond in DNA.

[0091] Examples of the nuclease include nucleases such as Cas proteins and endonucleases such as restriction enzymes; recombinases; DNA gyrases; DNA topoisomerases; and transposases.

[0092] Examples of the nucleic acid base conversion enzyme include deaminases. Examples of the deaminases include cytidine deaminase, adenosine deaminase, guanosine deaminase, etc. The nucleic acid base conversion enzyme is not particularly limited, and examples thereof include lamprey-derived PmCDA1 (Petromyzon marinus cytosine deaminase 1), vertebrate-derived AID (Activation-induced cytidine deaminase: AICDA), APOBEC family, ADAR family, TadA, etc.

[0093] Examples of the DNA glycosylase include thymine DNA glycosylase, oxoguanine glycosylase, and alkyladenine DNA glycosylase.

[0094] The vector system may further include a base excision repair inhibitory protein or a polynucleotide encoding the same as the polynucleotide (c), for example, to improve the modification efficiency of the nucleic acid modifying enzyme. The base excision repair inhibitory protein is not particularly limited as long as it inhibits the base excision repair pathway, and a DNA glycosylase inhibitor located upstream of the base excision repair pathway is preferred. Examples of the DNA glycosylase inhibitor include a thymine DNA glycosylase inhibitor, a uracil DNA glycosylase inhibitor, an oxoguanine DNA glycosylase inhibitor, and an alkylguanine DNA glycosylase inhibitor. For example, when cytidine deaminase is used as the nucleic acid modifying enzyme, it is preferable to use a uracil DNA glycosylase inhibitor to inhibit the repair of U:G or G:U mismatches in DNA caused by mutations. Examples of the uracil DNA glycosylase inhibitor include a uracil DNA glycosylase inhibitor (UGI) derived from PBS1, a Bacillus subtilis bacteriophage, and a uracil DNA glycosylase inhibitor (UGI) derived from PBS2, a Bacillus subtilis bacteriophage. The PBS2-derived UGI is known to have the effect of making it difficult for mutations, cleavage, and recombination from cytosine to other than thymine in DNA to occur, and therefore it is preferable to use a PBS2-derived UGI.

[0095] For example, (c) is directly or indirectly linked to (b). The direct link is, for example, a covalent link, and for example, the Cas protein and the enzymatically active protein or its enzymatically active domain constitute a fusion protein. The indirect link is, for example, a link via a binding tag-binding partner. The binding tag-binding partner is, for example, a combination of substances that specifically bind to each other. Examples of the binding tag-binding partner include a combination of biotin and avidin or streptavidin, a combination of an SH3 domain (SH3) and an SH ligand, a combination of nickel and a His tag, and a combination of an epitope tag such as a flag™ tag, HA tag, T7 tag, V5 peptide tag, and / or Myc tag with an antibody against the tag. In this case, the Cas protein and the enzymatically active protein or its enzymatically active domain are linked (bound) via the binding tag-binding partner, for example, after transcription and translation from the polynucleotide.

[0096] The polynucleotide (a), the polynucleotide (b), and the polynucleotide (c) are configured, for example, to be transcribed and / or translated within the prokaryote and capable of forming a complex. The Cas protein can form a Cas protein / guide RNA / enzymatic protein or enzymatic domain complex by, for example, binding to crRNA or tracrRNA and the enzymatic protein or its enzymatic domain. The complex can target the Cas protein to the target DNA, for example, depending on the sequence of the guide RNA, and modify the target DNA depending on the activity of the Cas protein or the enzymatic protein. The modification can, for example, cause double-stranded or single-stranded breaks in the target DNA, or the conversion, deletion, or insertion of one or more nucleotides. The modification can also, for example, modify the base, sugar, or phosphate moiety of the target DNA. The complex of the guide RNA and the Cas protein can, for example, convert, delete, or insert one or more nucleotides at the targeted site. The one or more may be, for example, 1 to 10 or 1 to 5.

[0097] In the present disclosure, when the vector system includes (c), the (a), (b), and (c) may be arranged, for example, on the same vector or on different vectors. For example, in the description of the arrangement of the polynucleotides (a) and (b), the polynucleotide (c) is preferably arranged contiguously with the polynucleotide (b).

[0098] The vector system of the present disclosure may include, for example, a multicloning site, an enhancer, a splicing signal, a polyA addition signal, a drug resistance gene, an auxotrophy complementing gene, a replication origin, and the like on the vector.

[0099] In the vector system of the present disclosure, for example, (a) and (b), or (a), (b), and / or (c) are inserted into an expression vector. The vector can also be said to have, for example, (a) and (b), or (a), (b), and / or (c) operably linked thereto.

[0100] The vector can be prepared, for example, by inserting the polynucleotides (a) and (b), or (a), (b), and / or (c), into a backbone vector (hereinafter also referred to as a "basic vector"). The type of expression vector is not particularly limited and can be determined appropriately depending on, for example, the type of host. Specifically, when the vector is synthesized by genetic engineering techniques, the vector is first synthesized by, for example, designing and synthesizing the polynucleotides (a) and (b), or (a), (b), and / or (c). The design and synthesis can be performed, for example, by PCR using a vector or the like containing polynucleotides encoding the polynucleotides (a) and (b), or (a), (b), and / or (c) as a template and primers designed to synthesize the desired nucleic acid region. The resulting polynucleotide is then ligated to an appropriate vector to obtain a recombinant vector for protein expression (expression vector), and this recombinant vector is introduced into a host so that the target gene can be expressed to obtain a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012))).

[0101] The vector can be appropriately designed depending on, for example, the prokaryote to be introduced and the method of introduction. Examples of the vector include a phage vector, a plasmid vector, a virus vector, a retrovirus vector, a chromosomal vector, an episomal vector, and a virus-derived vector.

[0102] When transforming a host using the heat shock method as the introduction method, the vector may be, for example, a binary vector. Examples of the expression vector include pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as Escherichia coli, the expression vector may be, for example, pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When transforming yeast, the expression vector may be, for example, pYepSec1, pMFa, and pYES2. When transforming lactic acid bacteria, the expression vector may be, for example, pTRKH2 and pTRK1203.

[0103] The vector may include, for example, regulatory sequences such as an enhancer sequence, a polyadenylation signal sequence, and an origin of replication sequence (ori) in addition to the promoter sequence and terminator sequence described above. The location of the regulatory sequence in the vector is not particularly limited. For example, the regulatory sequence may be a sequence already present in the original vector, or the regulatory sequence may be further inserted into the vector, or the regulatory sequence present in the base vector may be replaced with another regulatory sequence. The enhancer sequence is, for example, a sequence that enhances transcription from DNA. Examples of the enhancer sequence include the SV40 enhancer. The poly A addition sequence is, for example, a sequence that stabilizes transcribed RNA. Examples of the poly A addition sequence include the SV40 virus-derived poly A addition sequence.

[0104] According to the vector system of the present disclosure, for example, genome editing can be suitably performed on the prokaryote.

[0105] <Composition> In another aspect, the present disclosure provides a composition capable of modifying target DNA in a prokaryote. The composition of the present disclosure includes (a) a vector and (b) a Cas protein or a nucleic acid encoding the same, wherein the vector includes a guide RNA including a base sequence capable of hybridizing to target DNA in a prokaryote and a polynucleotide encoding a terminator sequence, wherein the guide RNA and the Cas protein are capable of forming a complex, and the terminator sequence in the polynucleotide is located on the 3' end of the guide RNA. The composition of the present disclosure can be applied to the composition of the present disclosure as described above for the CRISPR-Cas vector system.

[0106] In the present disclosure, the composition may further comprise, for example, (c) a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof.

[0107] In the present disclosure, when the composition includes (c), the guide RNA including a base sequence hybridizable to the target DNA in the prokaryote and the polynucleotide encoding the terminator sequence, and the polynucleotide encoding the enzymatic activity domain thereof may be placed, for example, on the same vector or on different vectors.

[0108] <Kit> In another aspect, the present disclosure provides a kit capable of modifying target DNA in a prokaryote. The kit of the present disclosure includes a CRISPR-Cas vector system of the present disclosure and / or a composition of the present disclosure. The kit of the present disclosure can incorporate the descriptions of the CRISPR-Cas vector system and composition of the present disclosure.

[0109] The kit of the present disclosure may include, for example, a prokaryote. The prokaryote may be one type or multiple types. The kit of the present disclosure may also include, for example, instructions or manuals.

[0110] The kit of the present disclosure may include, for example, a culture medium. Examples of the culture medium include YM medium, YPD medium, PD medium, DOB medium, SD medium, LB medium, NB medium, SCD medium, MRS medium, and BHI medium. The culture medium may include additives such as carbon sources such as glucose, dextrin, soluble starch, and sucrose; nitrogen sources such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract; inorganic substances such as calcium chloride, sodium dihydrogen phosphate, and magnesium chloride; vitamins; and growth-promoting factors. When the kit of the present disclosure includes the additive, the additive may be added to the culture medium before culture or may be added during culture. The addition may be continuous or intermittent.

[0111] The kit of the present disclosure may further include, for example, a container for storing the CRISPR-Cas vector system of the present disclosure and / or the composition of the present disclosure, and a culture medium.

[0112] In the kit of the present disclosure, for example, each component may be contained separately, or some or all of the components may be contained in a mixed or unmixed state. In the kit of the present disclosure, when all of the components are contained in a mixed or unmixed state in a single container, the kit of the present disclosure can also be referred to as, for example, a culture medium.

[0113] The kits of the present disclosure can also be suitably used as test kits or research kits, for example, for use in genome editing of prokaryotes.

[0114] <Method for producing a prokaryote having modified target DNA> In another aspect, the present disclosure provides a method for producing a prokaryote having modified target DNA. The method for producing a prokaryote having modified target DNA of the present disclosure includes an introduction step of introducing a CRISPR-Cas vector system and / or a composition of the present disclosure into a prokaryote. The explanations of the CRISPR-Cas vector system and composition of the present disclosure can be used for the production method of the present disclosure.

[0115] In the present disclosure, in the introduction step, for example, a complex of the Cas protein and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the targeting site.

[0116] In the present disclosure, the introduction method can be carried out by a known method capable of introducing (transfecting) an exogenous protein or exogenous DNA into host cells such as prokaryotes, and specific examples include, for example, introduction using a gene gun such as a particle gun, microinjection, calcium phosphate method, polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid introduction, DEAE-dextran method, direct injection using microglass tubes or the like, hydrodynamic method, cationic liposome method, lysozyme method, competent method, PEG method, method using an introduction adjuvant, Agrobacterium-mediated method, protoplast method, etc. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction adjuvant include atelocollagen, nanoparticles, polymers, etc.

[0117] In the present disclosure, the introduction step may further include, for example, introducing a polynucleotide encoding the (c) enzymatically active protein or an enzymatically active domain thereof. When the polynucleotide encoding the enzymatically active domain is introduced, the polynucleotide encoding the guide RNA and terminator sequence, which include a base sequence hybridizable to the target DNA in the prokaryote, and the polynucleotide encoding the enzymatically active domain may be placed on the same vector or on different vectors, for example.

[0118] The introduction step may include a step of culturing the prokaryote. The culturing can be performed by a known method or a modified method under conditions depending on the type of the prokaryote. The medium used in the culturing can be the same as that described in the kit.

[0119] In the culturing step, the culture pH is not particularly limited as long as it is the optimal pH for the growth of the prokaryote. When the prokaryote is a lactic acid bacterium, the pH is, for example, pH 5 to pH 8, or pH 6 to pH 7.

[0120] In the culturing step, the culture temperature is not particularly limited as long as it is the optimal temperature for the growth of the prokaryote. When the prokaryote is a lactic acid bacterium, the temperature is, for example, 25°C to 40°C, or 30°C to 37°C.

[0121] In the culturing step, the number of days for which the culture is carried out is not particularly limited as long as it is long enough for the complex to be expressed, and may be, for example, 16 hours to 10 days.

[0122] The culture may be performed, for example, by static culture or by shaking culture.

[0123] The present disclosure may further include a step of removing the CRISPR-Cas vector system of the present disclosure from the prokaryote after the introduction step. The removal can be carried out, for example, by a known method.

[0124] <Prokaryote> In another aspect, the present disclosure provides a prokaryote. The prokaryote of the present disclosure comprises a CRISPR-Cas vector system of the present disclosure and / or a composition of the present disclosure. The prokaryote of the present disclosure can use the same descriptions of the CRISPR-Cas vector system and composition of the present disclosure.

[0125] Examples of prokaryotes of the present disclosure include lactic acid bacteria, Bacillus subtilis var. natto, Escherichia coli, and cyanobacteria.

[0126] The prokaryote of the present disclosure may have a modified target DNA.

[0127] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0128] Example 1 Using the CRISPR-Cas vector system of the present disclosure, prokaryotes in which target DNA was modified could be produced.

[0129] (1) Construction of a CRISPR-Cas Vector System First, a CRISPR-Cas vector system was constructed to obtain lactic acid bacteria with modified target DNA.

[0130] (1-1) Design of CRISPR-Cas Plasmid Vectors CRISPR-Cas plasmid vectors were designed. Specifically, the CRISPR-Cas plasmid vectors were designed using genetic engineering experiment support software Snapgene (manufactured by GSL Biotech). Ten types of CRISPR-Cas plasmid vectors were prepared with different promoter and other configurations to determine the vector structure optimally expressed in lactic acid bacteria. The configurations of the 10 types (pYK1 to pYK10) are shown in Figures 1A to 1B, and the vector map of one of them (pYK8) is shown in Figure 2. Note that the vector maps of pYK1 to pYK7, pYK9, and pYK10 are the same as the vector map of pYK8, with tracrRNA replaced with crRNA and tracrRNA replaced with crRNA in each of the vectors shown in Figures 1A to 1B. As shown in Figures 1A to 1B, the terminators of pYK7 and pYK10 were the rrnA terminator derived from L. sakei and were positioned at the 3' end of the tracrRNA. The terminators of pYK8 to pYK10 were the rrnB terminator derived from E. coli and were positioned at the 3' end of the crRNA. The amino acid sequences or nucleotide sequences of each domain shown in Figures 1A to 1B are shown in SEQ ID NOS: 1 to 13. In pYK8 to pYK10, the distance from the crRNA or tracrRNA to the terminator sequence (distance from the 3' end) was designed to be 1 to 30 bases.

[0131] crRNA base sequence (SEQ ID NO: 1): 5'-NGTTTAAGAGCTATGCTGTTTTGAATGGTCCCAAAAC-3' (N = 14 to 30 bases, N = A, T, C, or G)

[0132] TracrRNA base sequence (SEQ ID NO: 2): 5'-GGAACCATTCAAAACAGCATAGCAAGTTtAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3'

[0133]

[0134] Amino acid sequence of PmCDA-SH Ligand (SEQ ID NO: 4): MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV-GGGGSPPPALPPKRRR

[0135] Amino acid sequence of UGI (SEQ ID NO: 5) MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0136] Nucleotide sequence of gyrA-ldh promoter (SEQ ID NO: 6): 5'-CTTTTATATTATATCACAAATAAGGCTCTTTTTCAGCTATTCTACTATAGTTTTCCGCTGAGAAAGGTAAATATTAGTGACTTTCTTAACAAAAAGTGTTAGAATGAAAATGTATAGAATATATACTTAATAAATTATAA-3'

[0137] Nucleotide sequence of p23 promoter (SEQ ID NO: 7): 5'-CTTGTTCCTAAAAAGGAATAAGCGttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtctt-3'

[0138] Base sequence of ldh promoter (SEQ ID NO: 8) 5'-atggacggattttgccttttccgtccagagcagtataccatactgacgccttgcgtctgtgcgagttgaccgtgggaactgttgaatttcccacattcgtgaaaacagatgctggaaacgcttacgttattggaagcttgcttgaaacagga ttcacaagtcttgctgtagtaaggctcgacgccattttttgacaatggcaaaatcatgaaaaagtctatcaaatttgtttcagggaattgataatgtgttatactcaacgtgaaatgcagtttgcatgcacataagaaaggatgatatcacc-3'

[0139] Nucleotide sequence of pgm promoter (SEQ ID NO: 9): 5'-GTTAGCACAAAAAAGCAGAAAATAAAAAGTAGAAATAAAAAAAGATGTTTTTTTGCCCATATCTCTATGAAAAAAACTGTGAAATGTGTAAAATATGGATGAAACATTGAATTTAAAAGGAGA-3'

[0140] Nucleotide sequence of rrn4b promoter (SEQ ID NO: 10): 5'-attggattaattgttgacagtttcataactggctggtatattagtaaacgttgct-3'

[0141] Nucleotide sequence of rrn3a promoter (SEQ ID NO: 11) 5'-ttataaaaagatgttgacagcttgttctgatgatgataaactttaatagttgcg-3'

[0142] Nucleotide sequence of rrnA terminator (SEQ ID NO: 12) 5'-aaaaaggttgaaccttagggttcagccttttt-3'

[0143] Nucleotide sequence of rrnB terminator (SEQ ID NO: 13) 5'-AACATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT-3'

[0144] (1-2) Preparation of DNA Fragments A CRISPR-Cas plasmid vector was constructed. Specifically, DNA fragments were prepared for each region constituting the CRISPR-Cas plasmid vector. First, a treatment solution was prepared under the conditions listed in Table 6 below, and the target DNA was treated with a restriction enzyme. The restriction enzyme treatment was carried out at 37°C for 2 hours. In addition, 1 μl of BAP was added to the restriction enzyme treatment as needed, and a dephosphorylation treatment was carried out. The restriction enzymes and buffers listed in Table 6 below were products of NEB.

[0145]

[0146] The DNA after the restriction enzyme treatment was stained using Midori green direct (manufactured by Nippon Genetics Co., Ltd.). After staining, electrophoresis was performed, and a fluorescent band having the desired size was excised. After the excision, purification was performed using a Gel / PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.). After the purification, the target DNA was amplified by PCR using the primers shown in Table 7 below. The PCR conditions are shown in Tables 8 and 9 below. PrimeSTAR (registered trademark) Max DNA Polymerase (manufactured by Takara Bio Inc.) was used for the PCR. The reaction solution after the PCR was stained using Midori green direct (manufactured by Nippon Genetics Co., Ltd.). After staining, electrophoresis was performed, and a fluorescent band having the desired size was excised. After the excision, purification was performed using a Gel / PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.).

[0147]

[0148]

[0149]

[0150] After the purification, the oligos were phosphorylated. A phosphorylation reaction solution was prepared under the conditions shown in Table 10 below, and phosphorylation was carried out at 37°C for 30 minutes. When multiple oligos were treated simultaneously in the phosphorylation treatment, the front strand and back strand were treated separately. Then, an annealing reaction solution was prepared under the conditions shown in Table 11 below, and annealing was carried out for 1 hour by slowly cooling from 97°C to 37°C.

[0151]

[0152]

[0153] (1-3) Ligation or Gibson Assembly After the annealing in Example 1 (1-2), a product was obtained by joining the DNA fragments by ligation or Gibson assembly. The ligation was carried out using Ligation High Ver. 2 (manufactured by Toyobo Co., Ltd.) at 16°C for 2 hours. The Gibson assembly was carried out using Gibson Assembly (registered trademark) Master Mix (manufactured by New England Biolabs) at 50°C for 1 hour. In the ligation and Gibson assembly methods, the molar ratio of the DNA fragment on the vector side to the DNA fragment to be inserted was adjusted to 1:3.

[0154] (1-4) Transformation into Escherichia coli Next, the product obtained in Example 1 (1-3) was transformed into Escherichia coli. The Escherichia coli used was the E. coli HST08 strain (manufactured by Takara Bio Inc.). Specifically, competent cells stored at -80°C were thawed on ice. After thawing, 50 μl of the competent cells were dispensed into a 1.5 ml tube, and 5 μl of the product was added. After the addition, the tube was cooled on ice for 10 minutes. After the ice cooling, the tube was heated at 42°C for 40 to 45 seconds using a heat block. After the heating, the tube was cooled on ice for 2 minutes. After the ice cooling, 500 μl of SOC medium was added, and the tube was subjected to shaking culture using a maximizer at 1500 rpm, 37°C, and 1 hour. After the shaking culture, the tube was centrifuged, and the culture supernatant was removed so that the total volume was approximately 100 μl. After the removal, the pellet was resuspended, and the entire amount was smeared on LB agar medium supplemented with antibiotics and cultured at 37°C overnight. After the culture, colony PCR was performed on the formed colonies. For the colony PCR, EmeraldAmp® PCR Master Mix (manufactured by Takara Bio Inc.) was used. The conditions for the colony PCR are shown in Tables 12 and 13 below. Primer A and Primer B shown in Table 12 below were appropriately selected and used, as they were capable of distinguishing between DNA fragments before and after ligation.

[0155]

[0156]

[0157] (1-5) Plasmid Extraction and Purification: Individuals that successfully performed PCR using the colony PCR in Example 1 (1-4) were inoculated into LB liquid medium supplemented with antibiotics and cultured for 18 hours at 37°C, 180 rpm, using a bioshaker. After the culture, the cells were centrifuged, the supernatant was removed, and an E. coli precipitate was obtained. Plasmids were extracted from the precipitate. The plasmids were extracted using a Plasmid Mini Kit (manufactured by Nippon Genetics Co., Ltd.). Specifically, a cell lysis solution containing Tris (pH 8.0), EDTA, and glucose was added to dissolve the precipitate. After the dissolution, a lysis buffer containing NaOH and SDS was added and the mixture was slowly mixed by inversion. After the inversion, an acidic salt solution (neutralization buffer) such as ammonium acetate was added and mixed by inversion. After the inversion, a 1:1 mixture of phenol and chloroform was added. After the addition, the mixture was centrifuged, and the aqueous layer was collected to obtain purified plasmids.

[0158] To confirm that the plasmid was the desired plasmid, restriction enzyme treatment and electrophoresis were performed. Appropriate restriction enzymes were selected for the restriction enzyme treatment using Snapgene. The restriction enzyme treatment was performed under the recommended conditions for the restriction enzyme, using a treatment solution prepared under the conditions listed in Table 14 below. After the restriction enzyme treatment, electrophoresis was performed to confirm the band sizes.

[0159]

[0160] The obtained plasmid was analyzed and confirmed using a DNA sequencer. Specifically, the analysis of the plasmid was outsourced to Genewiz, and the obtained signals were imported into Snapgene for alignment. This confirmed that it was the desired plasmid.

[0161] (2) Genome editing of lactic acid bacteria Using the plasmid obtained in Example 1 (1-5), lactic acid bacteria with modified target DNA were constructed.

[0162] (2-1) Manipulation of Lactic Acid Bacteria Lactic acid bacteria were cultured in MRS medium under static conditions at 30°C. Selection was performed using antibiotic concentrations of erythromycin (10 μg / ml) and chloramphenicol (10 μg / ml), and 5-fluorouracil was added to the medium to a concentration of 100 μg / ml.

[0163] (2-2) Selection of Lactic Acid Bacteria Strains In this example, two types of lactic acid bacteria strains were used: Lactiplantibacillus plantarum WCFS1 (L. plantarum WCFS1) and Lactobacillus gasseri ATCC 33323 (L. gasseri ATCC 33323).

[0164] (2-3) Transformation into Lactic Acid Bacteria 2 ml of MRS liquid medium was dispensed into a 15 ml Corning tube, and a single colony was collected and cultured overnight at 37°C under static conditions. After the culture, 1 ml of the preculture solution and 25 ml of 0.41 mol / l glycine-containing MRS medium were added to a 50 ml Corning tube, and the culture was continued at 37°C under static conditions. The culture was continued until the OD600 reached 0.85 to 1.00. After the culture, the mixture was centrifuged at 4°C, 5000 rpm, and 10 minutes, and the supernatant was removed. After the removal, 5 ml of MgCl ２ The pellet was resuspended in 5 ml of SacGly solution (10% glycerol, 0.5 mol / L sucrose) and centrifuged at 4°C, 5,000 rpm, and 10 minutes, and the supernatant was removed. This process was repeated. The pellet was then resuspended in 5 ml of SacGly solution (10% glycerol, 0.5 mol / L sucrose) and centrifuged at 4°C, 5,000 rpm, and 10 minutes, and the supernatant was removed. After the removal, the pellet was resuspended in 1 ml of SacGly solution, added to a 1.5 ml Eppendorf tube, and centrifuged at 4°C, 15,000 rpm, and 1 minute, and the supernatant was removed. After the removal, the pellet was resuspended in 0.5 ml of SacGly solution and dispensed into 60 μl aliquots to obtain competent cells.

[0165] Next, electroporation was performed. Specifically, 10 μl of DNA solution (2 to 5 μg) and 60 μl of the competent cells were added to a cuvette (manufactured by Nepa Gene Co., Ltd.) with an electrode distance of 0.1 cm. After the addition, the cuvette was set in an electroporator (manufactured by Nepa Gene Co., Ltd.), and electroporation was performed under the following conditions. After the electroporation, 1 ml of MRS medium was added, and the cells were cultured at 30°C overnight. After the culture, 250 μl of the culture medium was smeared on an erythromycin-containing MRS agar medium and cultured until colonies were formed (L. plantarum WCFS1: 2 to 4 days, L. gasseri ATCC 33323: 2 to 9 days). <Electroporation conditions> Poling pulse: voltage 1250V, pulse width 2.5msec, pulse interval 50ms, pulse count 1, polarity + Transfer pulse: voltage 150V, pulse width 50msec, pulse interval 50ms, pulse count 5, polarity + / -

[0166] (2-4) Study of Mutation Introduction Efficiency in L. plantarum WCFS1 After electroporation, the efficiency of mutation introduction in L. plantarum WCFS1 was examined. Specifically, four transformants were selected from the electroporated transformants in Example 1 (2-3) above and cultured overnight in 500 μl of erythromycin-containing MRS medium. After the culture, the transformants were isolated from the culture onto erythromycin-containing MRS agar medium. Two colonies of each of the isolated cells (a total of eight colonies) were selected, and colony PCR was performed using primers that amplify the target DNA sequence. 1 μl of ExoSAP-IT™ Express (Thermo Fisher Scientific) and 5 μl of PCR treatment solution were mixed and incubated at 37°C for 8 minutes. After the incubation, the enzyme was inactivated at 80°C for 1 minute. Genewiz was commissioned to sequence 1 μl of the treatment solution, and mutations in the target sequence of the eight colonies were confirmed. Using the online tool EditR, the cytosine (C) to thymine (T) conversion rate was calculated from the data obtained by Sanger sequencing. The target sequence was the upp gene. The guide RNA (gRNA) sequence, PAM sequence, and cytosine to thymine conversion are shown in Figure 3. As shown in Figure 3, the proximal base from the PAM sequence was numbered as 1. The cytosine to thymine conversion rate was calculated as the conversion rate from the target cytosine at position 18 to thymine, as shown in Figure 3. After the calculation, the editing efficiency was calculated as the average value of the eight colonies. These results are shown in Figure 4.

[0167] Figure 4 is a graph showing the results of mutagenesis in L. plantarum WCFS1. In Figure 4, the vertical axis indicates editing efficiency (%), and the horizontal axis indicates the type of CRISPR-Cas plasmid vector. In Figure 4, the error bars indicate the standard error of the mean (mean ± SEM). As shown in Figure 4, pYK7 to pYK10 showed higher editing efficiency than pYK1 to pYK6. Furthermore, the editing efficiency of pYK8 to pYK10 was close to 100%. These results demonstrate that by positioning a terminator sequence downstream of the rRNA sequence, L. plantarum WCFS1 exhibits an editing efficiency close to 100%.

[0168] (2-5) Study of Mutation Introduction Efficiency in L. gasseri ATCC 33323 We investigated whether the CRISPR-Cas vector system of the present disclosure would also exhibit high mutation introduction efficiency in L. gasseri ATCC 33323, a strain different from L. plantarum WCFS1. The cytosine (C) to thymine (T) conversion rate was calculated when pYK9, which showed high editing efficiency in L. plantarum WCFS1, was transformed. The target sequence was selected from a non-coding region in the genome of L. gasseri ATCC 33323. The guide RNA sequence, PAM sequence, and cytosine to thymine conversion are shown in Figure 5. As shown in Figure 5, the base proximal to the PAM sequence was numbered as 1. The cytosine to thymine conversion rate was calculated as the conversion rate from the target cytosine at position 18 to thymine, as shown in Figure 5. After this calculation, the editing efficiency was calculated as the average value for the eight colonies. As a result, it was found that high editing efficiency was also observed in L. gasseri ATCC 33323.

[0169] (2-6) Examination of Mutation Introduction Efficiency When dCAS Is Used The editing efficiency in lactic acid bacteria was examined when the nCas of the CRISPR-Cas vector system of the present disclosure was changed to an inactive Cas9 (dCas) in which a mutation was introduced into the nuclease domain of Cas9. Specifically, the nCas9 in nCas9-SH3 of pYK8 was changed to dCas9 (SpCas9 D10A, H840A), and a new plasmid vector pYK11 was constructed. Figure 6 shows the configuration of pYK8 and pYK11. pYK8 or pYK11 was transformed into L. plantarum WCFS1 using the same method as in Example 1 (2-3), and the mutation introduction efficiency was examined using the same method as in Example 1 (2-4). These results are shown in Figure 7.

[0170] Figure 7 is a graph showing the results of mutagenesis in L. plantarum WCFS1. In Figure 7, the vertical axis indicates editing efficiency (%), and the horizontal axis indicates the type of CRISPR-Cas plasmid vector. As shown in Figure 7, pYK11 was found to exhibit high editing efficiency, similar to pYK8. These results indicate that high editing efficiency was observed in L. plantarum WCFS1 even when nCas9 in nCas9-SH3 was replaced with dCas9.

[0171] (2-7) Study of Mutation Introduction Efficiency in Multiple Editing The editing efficiency in lactic acid bacteria was examined when the number of guide RNAs in the CRISPR-Cas vector system of the present disclosure was changed to multiple. Specifically, the guide RNAs in pYK8 were changed to two or three, and new plasmid vectors pYK8-dual (number of guide RNAs: two) and pYK8-triple (number of guide RNAs: three) were constructed. Figure 8 shows the configurations of pYK8, pYK8-dual, and pYK8-triple. Figure 9 also shows the guide RNA sequence and PAM sequence. L. plantarum WCFS1 was transformed with pYK8, pYK8-dual, or pYK8-triple using the same method as in Example 1 (2-3), and the mutation introduction efficiency was examined using the same method as in Example 1 (2-4). These results are shown in Figure 10.

[0172] Figure 10 is a graph showing the results of mutagenesis in L. plantarum WCFS1. In Figure 10, the vertical axis shows editing efficiency (%), and the horizontal axis shows numbers where the proximal base from the PAM sequence is numbered as 1. In Figure 10, (A) shows the editing efficiency in the first guide RNA, (B) shows the editing efficiency in the second guide RNA, and (C) shows the editing efficiency in the third guide RNA. As shown in Figure 10, pYK8, pYK8-dual, and pYK8-triple showed high editing efficiency from the target cytosine at position 18 to thymine in the first guide RNA. pYK8, pYK8-dual, and pYK8-triple showed high editing efficiency from the target cytosine at positions 17, 18, and 20 to thymine in the second guide RNA, and also showed high editing efficiency from the target cytosine at position 18 to thymine. pYK8, pYK8-dual, and pYK8-triple showed high editing efficiency of the third guide RNA, converting the target cytosine at position 19 to thymine. These results demonstrate that high editing efficiency is achieved in L. plantarum WCFS1 even when multiple guide RNAs are used for multiple editing.

[0173] (2-8) Construction of urdA Gene-Deficient L. plantarum WCFS1 Imidazolepropionic acid (ImP) is a metabolic substance produced by intestinal bacteria from histidine and is known to disrupt glucose metabolism and insulin signaling. We investigated the construction of L. plantarum with reduced ImpP production by deleting the urdA gene, which encodes the ImpP synthase urdA. Specifically, a CRISPR-Cas plasmid vector was constructed based on pYK8 to target a stop codon into the first half of the urdA gene. The guide RNA sequence, PAM sequence, and cytosine-to-thymine conversion are shown in Figure 11. As shown in Figure 11, the base proximal to the PAM sequence was numbered as 1. After construction, the plasmid vector was transformed into L. plantarum using the same method as in Example 1 (2-3). After transformation, the resulting single colonies were examined for mutation introduction using the same method as in Example 1 (2-4). As a result, it was found that a mutation had been introduced, introducing a stop codon early into the target urdA gene. Next, the CRISPR-Cas plasmid vector remaining in the lactic acid bacteria was removed. The removal was carried out by culturing in a medium without erythromycin. Figure 12 is a photograph showing the results of serial dilution of lactic acid bacteria cultured in a medium without erythromycin for vector removal. Next, for the lactic acid bacteria for which the removal was confirmed, the state of mutation introduction was confirmed using the same method as in Example 1 (2-4). These results are shown in Figure 13.

[0174] Figure 13 shows the results of Sanger sequencing of the target sequence of the urdA gene. As shown in Figure 13, it was found that the introduced target mutation was maintained in the lactic acid bacteria after removal of the CRISPR-Cas plasmid vector.

[0175] (2-9) Investigation of Imp Production in UrdA Gene-Deficient L. plantarum WCFS1 We investigated whether the amount of Imp produced was reduced in the urdA gene-defective L. plantarum WCFS1. Specifically, three clones each of the urdA gene-defective L. plantarum WCFS1 and wild-type L. plantarum WCFS1 obtained in Example 1 (2-7) were cultured. After the culture, quantitative measurement of Imp in the culture supernatant was performed using LC / MS. The results are shown in Table 15 below. As shown in Table 15 below, the amount of Imp produced in the urdA gene-defective L. plantarum WCFS1 was more than 10-fold lower than that in the wild-type L. plantarum WCFS1. These results indicated that a lactic acid bacterial strain with low Imp production ability had been obtained.

[0176]

[0177] It was also confirmed that the urdA gene-deficient L. plantarum WCFS1 was capable of milk fermentation and yogurt production, just like the parent strain, by adding the additives required for the parent strain L. plantarum WCFS1 (Reference 4). Reference 4: Ma C, Cheng G et al., "Determination of the essential nutrients required for milk fermentation by Lactobacillus plantarum. LWT 65," LWT, 2016, Volume 65, Pages 884-889, doi.org / 10.1016 / j.lwt.2015.09.003.

[0178] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0179] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0180] This application claims priority based on Japanese Patent Application No. 2024-092650, filed on June 6, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0181] <Supplementary Notes> Some or all of the above embodiments and examples can be described as in the following supplementary notes, but are not limited to the following. <CRISPR-Cas Vector System> (Supplementary Note 1) A CRISPR-Cas vector system for use in prokaryotes, comprising: (a) a polynucleotide encoding a guide RNA and a terminator sequence, the polynucleotide including a base sequence capable of hybridizing to target DNA in a prokaryote; and (b) a polynucleotide encoding a Cas protein, wherein (a) and (b) are arranged on the same or different vectors, the guide RNA and the Cas protein are capable of forming a complex, and in (a), the terminator sequence is arranged on the 3' end of the guide RNA. (Supplementary Note 2) A CRISPR-Cas vector system according to Supplementary Note 1, wherein the complex converts, deletes, or inserts one or more nucleotides at a targeted site. (Supplementary Note 3) The CRISPR-Cas vector system according to Supplementary Note 1 or 2, wherein the guide RNA comprises a crRNA, and the terminator sequence is located on the 3'-end of the crRNA. (Supplementary Note 4) The CRISPR-Cas vector system according to Supplementary Note 3, wherein the terminator sequence is located within 30 bases from the 3'-end of the crRNA, based on the base at the 3'-end of the crRNA. (Supplementary Note 5) The CRISPR-Cas vector system according to any of Supplementary Notes 1 to 4, wherein the guide RNA comprises a crRNA and a tracrRNA, and the terminator sequence is located on the 3'-end of the crRNA and / or the tracrRNA, and the crRNA and the tracrRNA are located on the same or different vectors. (Supplementary Note 6) The CRISPR-Cas vector system according to Supplementary Note 5, wherein the terminator sequence is located within 30 bases from the 3' end of the crRNA and / or the tracrRNA, based on the base at the 3' end. (Supplementary Note 7) The CRISPR-Cas vector system according to Supplementary Note 5 or 6, wherein a promoter sequence is located at the 5' end of the crRNA and / or the tracrRNA.(Appendix 8) The CRISPR-Cas vector system according to any one of Appendices 1 to 7, comprising a polynucleotide encoding (c) an enzymatically active protein or an enzymatically active domain thereof, wherein (a), (b), and (c) are arranged on the same or different vectors, and wherein the Cas protein, the guide RNA, and the enzymatically active protein or an enzymatically active domain thereof are capable of forming a complex. (Appendix 9) The CRISPR-Cas vector system according to any one of Appendices 1 to 8, wherein the polynucleotide encoding the (b) Cas protein and the polynucleotide encoding the (c) enzymatically active protein or an enzymatically active domain thereof are linked. (Appendix 10) The CRISPR-Cas vector system according to Appendices 8 or 9, wherein the enzymatically active protein is a nucleic acid-modifying enzyme. (Appendix 11) The CRISPR-Cas vector system according to Appendices 10, wherein the nucleic acid-modifying enzyme comprises a nucleobase-converting enzyme. (Appendix 12) The CRISPR-Cas vector system according to Appendices 11, wherein the nucleic acid base conversion enzyme includes a deaminase. (Appendix 13) The CRISPR-Cas vector system according to any one of Appendices 1 to 12, wherein the Cas protein cleaves at least one of the two strands making up the target DNA. (Appendix 14) The CRISPR-Cas vector system according to Appendices 13, wherein the Cas protein cleaves one of the two strands making up the target DNA. (Appendix 15) The CRISPR-Cas vector system according to any one of Appendices 1 to 12, wherein the Cas protein does not cleave the two strands making up the target DNA. (Appendix 16) The CRISPR-Cas vector system according to any one of Appendices 1 to 15, wherein the Cas protein is a Cas9 protein or a Cas12 protein. (Appendix 17) The CRISPR-Cas vector system according to any one of Appendices 1 to 16, wherein the prokaryote is a lactic acid bacterium. (Appendix 18) The CRISPR-Cas vector system according to any one of Appendices 1 to 17, wherein the prokaryote is of a heterologous species to which the Cas protein is derived.<Composition> (Appendix 19) A composition comprising (a) a vector and (b) a Cas protein or a nucleic acid encoding the same, wherein the vector comprises a polynucleotide encoding a guide RNA and a terminator sequence, the polynucleotide encoding the guide RNA and the Cas protein comprising a base sequence hybridizable to target DNA in a prokaryote, and the terminator sequence is located on the 3' end of the guide RNA. (Appendix 20) The composition according to Appendix 19, wherein the complex converts, deletes, or inserts one or more nucleotides at a targeted site. (Appendix 21) The composition according to Appendix 19 or 20, wherein the guide RNA comprises a crRNA, and the terminator sequence is located on the 3' end of the crRNA. (Appendix 22) The composition according to Appendix 21, wherein the terminator sequence is located within 30 bases from the 3' end of the crRNA and / or tracrRNA. (Appendix 23) The composition according to any one of Appendices 19 to 22, wherein the guide RNA comprises a crRNA and a tracrRNA, the terminator sequence is located on the 3' end of the crRNA and / or the tracrRNA, and the crRNA and the tracrRNA are located on the same or different vectors. (Appendix 24) The composition according to Appendices 23, wherein the terminator sequence is located within 30 bases from the 3' end of the crRNA and / or the tracrRNA, based on the base at the 3' end. (Appendix 25) The composition according to Appendices 23 or 24, wherein a promoter sequence is located on the 5' end of the crRNA and / or tracrRNA. (Supplementary Note 26) (c) The composition according to any one of Supplementary Notes 19 to 25, comprising a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof, wherein the polynucleotide encoding a guide RNA and a terminator sequence comprising a base sequence hybridizable to a target DNA in a prokaryote, and the polynucleotide encoding the enzymatically active domain are located on the same or different vectors. (Supplementary Note 27) The composition according to Supplementary Note 26, wherein the enzymatically active protein is a nucleic acid-modifying enzyme.(Appendix 28) The composition according to Appendix 27, wherein the nucleic acid modifying enzyme comprises a nucleobase conversion enzyme. (Appendix 29) The composition according to Appendix 28, wherein the nucleobase conversion enzyme comprises a deaminase. (Appendix 30) The composition according to any one of Appendixes 19 to 29, wherein the Cas protein cleaves at least one of the two strands constituting the target DNA. (Appendix 31) The composition according to Appendix 30, wherein the Cas protein cleaves one of the two strands constituting the target DNA. (Appendix 32) The composition according to any one of Appendixes 19 to 29, wherein the Cas protein does not cleave the two strands constituting the target DNA. (Appendix 33) The composition according to any one of Appendixes 19 to 32, wherein the Cas protein is a Cas9 protein or a Cas12 protein. (Appendix 34) The composition according to any one of Appendixes 19 to 33, wherein the prokaryote is a lactic acid bacterium. (Appendix 35) The composition according to any one of Appendices 19 to 34, wherein the prokaryote is of a species heterologous to the origin of the Cas protein. <Kit> (Appendix 36) A kit comprising the CRISPR-Cas vector system according to any one of Appendices 1 to 18 and / or the composition according to any one of Appendices 19 to 35. <Method for producing a prokaryote whose target DNA has been modified> (Appendix 37) A method for producing a prokaryote whose target DNA has been modified, comprising an introduction step of introducing into a prokaryote the CRISPR-Cas vector system according to any one of Appendices 1 to 18 and / or the composition according to any one of Appendices 19 to 35. (Appendix 38) The production method according to Appendix 37, wherein in the introduction step, a complex of the Cas protein and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the targeting site. (Appendix 39) The production method according to Appendix 37 or 38, wherein the introduction step further comprises introducing a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof, and the polynucleotide encoding a guide RNA and a terminator sequence comprising a base sequence hybridizable to the target DNA in the prokaryote and the polynucleotide encoding the enzymatically active domain are placed on the same or different vectors.(Appendix 40) The production method according to any one of Appendices 37 to 39, wherein the modification is a double-strand break, a single-strand break, a conversion, deletion, and / or insertion of one or more nucleotides in the target DNA. <Prokaryote> (Appendix 41) A prokaryote comprising a CRISPR-Cas vector system according to any one of Appendices 1 to 18 and / or a composition according to any one of Appendices 19 to 35. (Appendix 42) The prokaryote according to Appendice 41, wherein the prokaryote is a lactic acid bacterium. (Appendix 43) The prokaryote according to Appendices 41 or 42, wherein the target DNA is modified. (Appendix 44) The prokaryote according to any one of Appendices 41 to 43, wherein the modification is a double-strand break, a single-strand break, a conversion, deletion, and / or insertion of one or more nucleotides in the target DNA.

[0182] As described above, the present disclosure can provide a technology that enables genome editing in prokaryotes, etc. Therefore, the present disclosure can be said to be extremely useful, for example, in the fields of food and medicine.

Claims

1. A CRISPR-Cas vector system for use in prokaryotes, comprising: (a) a polynucleotide encoding a guide RNA and a terminator sequence, the polynucleotide comprising a base sequence capable of hybridizing to target DNA in prokaryotes; and (b) a polynucleotide encoding a Cas protein, wherein (a) and (b) are arranged on the same or different vectors, the guide RNA and the Cas protein are capable of forming a complex, and in (a), the terminator sequence is arranged on the 3' end of the guide RNA.

2. The CRISPR-Cas vector system of claim 1, wherein the complex converts, deletes, or inserts one or more nucleotides at the targeted site.

3. The CRISPR-Cas vector system according to claim 1 or 2, wherein the guide RNA comprises a crRNA, and the terminator sequence is located on the 3' end of the crRNA.

4. The CRISPR-Cas vector system of claim 3, wherein the terminator sequence is positioned within 30 bases from the 3' end of the crRNA.

5. The CRISPR-Cas vector system of any one of claims 1 to 4, wherein the guide RNA comprises a crRNA and a tracrRNA, the terminator sequence is located on the 3' end of the crRNA and / or the tracrRNA, and the crRNA and the tracrRNA are located on the same or different vectors.

6. The CRISPR-Cas vector system of claim 5, wherein the terminator sequence is positioned within 30 bases from the 3' end of the crRNA and / or the tracrRNA.

7. The CRISPR-Cas vector system of any one of claims 1 to 6, further comprising (c) a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof, wherein (a), (b), and (c) are arranged on the same or different vectors, and the Cas protein, the guide RNA, and the enzymatically active protein or an enzymatically active domain thereof are capable of forming a complex.

8. A CRISPR-Cas vector system according to any one of claims 1 to 7, wherein the (b) polynucleotide encoding the Cas protein and the (c) polynucleotide encoding the enzymatically active protein or an enzymatically active domain thereof are linked.

9. The CRISPR-Cas vector system of claim 7 or 8, wherein the enzymatically active protein is a nucleic acid-modifying enzyme.

10. The CRISPR-Cas vector system of claim 9, wherein the nucleic acid modifying enzyme comprises a nucleic acid base conversion enzyme.

11. The CRISPR-Cas vector system of claim 10, wherein the nucleic acid base conversion enzyme comprises a deaminase.

12. A CRISPR-Cas vector system described in any one of claims 1 to 11, wherein the Cas protein cleaves at least one of the two strands that make up the target DNA.

13. The CRISPR-Cas vector system of claim 12, wherein the Cas protein cleaves one of the two strands that make up the target DNA.

14. A CRISPR-Cas vector system according to any one of claims 1 to 11, wherein the Cas protein does not cleave the double strand that constitutes the target DNA.

15. A CRISPR-Cas vector system described in any one of claims 1 to 14, wherein the Cas protein is a Cas9 protein or a Cas12 protein.

16. The CRISPR-Cas vector system of any one of claims 1 to 15, wherein the prokaryote is a lactic acid bacterium.

17. The CRISPR-Cas vector system of any one of claims 1 to 16, wherein the prokaryote is heterologous to the origin of the Cas protein.

18. A composition comprising: (a) a vector; and (b) a Cas protein or a nucleic acid encoding the same; wherein the vector comprises a polynucleotide encoding a guide RNA and a terminator sequence, the guide RNA and the Cas protein comprising a base sequence capable of hybridizing to target DNA in a prokaryote; wherein the guide RNA and the Cas protein are capable of forming a complex; and wherein the terminator sequence in the polynucleotide is located on the 3' end of the guide RNA.

19. A kit comprising the CRISPR-Cas vector system of any one of claims 1 to 17 and / or the composition of claim 18.

20. A method for producing a prokaryote in which target DNA has been modified, comprising an introduction step of introducing into a prokaryote a CRISPR-Cas vector system according to any one of claims 1 to 17 and / or a composition according to claim 18.

21. The manufacturing method described in claim 20, wherein in the introduction step, a complex of the Cas protein and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the targeting site.

22. The manufacturing method described in claim 20 or 21, wherein the introduction step further comprises introducing a polynucleotide encoding an enzymatically active protein or an enzymatically active domain thereof, and the polynucleotide encoding a guide RNA and a terminator sequence containing a base sequence capable of hybridizing to the target DNA in the prokaryote and the polynucleotide encoding the enzymatically active domain are placed on the same or different vectors.

23. A method of production according to any one of claims 20 to 22, wherein the modification is a double-strand break, a single-strand break, a conversion, deletion and / or insertion of one or more nucleotides in the target DNA.

24. A prokaryote comprising a CRISPR-Cas vector system according to any one of claims 1 to 17 and / or a composition according to claim 18.

25. The prokaryote of claim 24, wherein the prokaryote is a lactic acid bacterium.

26. The prokaryote of claim 24 or 25, wherein the target DNA of the prokaryote is modified.

27. A prokaryote according to any one of claims 24 to 26, wherein the modification is a double-strand break, a single-strand break, a conversion, deletion and / or insertion of one or more nucleotides in the target DNA.

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