Method for producing genome-edited prokaryotic cell

Abstract

A method for producing a genome-edited prokaryotic cell, the method comprising a step for introducing, into a prokaryotic cell, gRNA in which at least one nucleotide is added to the 5'end of a spacer sequence or a DNA construct for expressing the gRNA and a Cas protein or a DNA construct for expressing the protein.

Description

Method for producing a genome-edited prokaryotic cell

[0001] The present invention relates to a method for producing a genome-edited prokaryotic cell. The present invention also relates to a DNA construct used in the production method.

[0002] Genome editing technology is a technology that uses sequence-specific nucleases and the like to cleave a specific sequence (sequence of a target region) on a genome and modify a gene (target gene) containing the sequence. More specifically, it includes cleaving a target gene using a CRISPR / Cas system or the like and introducing mutations to disrupt the gene, or site-specifically inserting or replacing a DNA fragment. Furthermore, according to genome editing, by ligating fragments homologous to the sequences on both sides of the cleavage sequence to both sides of the DNA fragment to be introduced into that site, efficient site-specific insertion or replacement of a DNA fragment becomes possible.

[0003] In higher organisms, various gene modifications have already been carried out using genome editing technology, and its practical application in the improvement of agricultural, forestry, livestock, and fishery products and gene therapy is underway. On the other hand, in prokaryotes as well, attempts have been made to use genome editing technology for the production of useful substances. However, since prokaryotes have almost no DNA repair pathway by non-homologous end joining, cleavage of genomic DNA in genome editing results in lethality to prokaryotes unless there is a homologous sequence for repair. Even when a homologous sequence is added, due to the weak repair activity of prokaryotes, genome editing is difficult in many prokaryotes.

[0004] As a countermeasure to such problems, attempts have been made to use Cas12a, which is easily repaired after cleavage, or to use a host with enhanced repair activity (see the item "3.5. RecE / T-Assisted CRISPR / Cas9 System" in Non-Patent Document 1). However, even when such improved technologies are used, the efficiency of genome editing in prokaryotes has not yet been sufficient.

[0005] International Publication No. 2014 / 065596 International Publication No. 2020 / 122195

[0006] Yulin Mu et al., Int J Mol Sci., October 25, 2022; 23(21): 12852.

[0007] This invention has been made in view of the problems of the prior art described above, and aims to provide a method for genome editing in prokaryotic cells, that is, a method for producing genome-edited prokaryotic cells.

[0008] As a result of diligent research to achieve the above objective, the present inventors have found that in genome editing using CRISPR-Cas9 in prokaryotic cells (Lactiplantybacillus plantarum), when using guide RNA (guide RNA (gRNA) without a nucleotide attached to the 5' end of the spacer sequence) in the conventional method, DNA cleavage by Cas9 exhibits lethal toxicity to prokaryotic cells, making it impossible to obtain transformed cells or detect the introduction of mutations by genome editing.

[0009] On the other hand, it was found that genome-edited prokaryotic cells can be obtained by using gRNA in which at least one nucleotide has been added to the 5' end of the spacer sequence. Furthermore, it was found that the genome editing efficiency in prokaryotic cells is significantly improved by adding 3 to 40 consecutive identical nucleotides to such gRNA compared to adding just one nucleotide.

[0010] Furthermore, even when using gRNA in which a portion of the additional sequence consisting only of identical nucleotides was substituted with other nucleotides, genome editing was observed in prokaryotic cells, just as before the substitution. In addition, for gRNA containing an additional sequence consisting only of identical nucleotides, the genome editing efficiency in prokaryotic cells was highest when all the nucleotides in the additional sequence were adenine.

[0011] Furthermore, regarding the genome-edited prokaryotic cells obtained in this way, it was revealed that these colonies may contain a mixture of mutants with genome-edited mutations and wild-type cells without such mutations (mix mutants). In order to isolate colonies composed solely of mutants (pure mutants) from these mixed mutants, further intensive research was conducted. As a result, it was discovered that by culturing the obtained mix mutant colonies in a liquid medium containing a selection marker under conditions corresponding to the selection marker gene in the plasmid vector for expressing the gRNA, and then performing solid-state culture under those conditions, colonies consisting of pure mutants can be obtained, thus completing the present invention.

[0012] In other words, the present invention provides the following embodiments.

[0013] [1] A method for producing genome-edited prokaryotic cells, comprising the steps of introducing into prokaryotic cells a gRNA having at least one nucleotide attached to the 5' end of a spacer sequence, or a DNA construct for expressing said gRNA, and a Cas protein, or a DNA construct for expressing said protein.

[0014] [2] The method for producing the product according to [1], wherein at least one nucleotide added to the 5' end of the spacer sequence in the gRNA is removed, and two consecutive guanines are removed from that nucleotide.

[0015] [3] The manufacturing method according to [1], wherein an oligonucleotide consisting of at least three nucleotides is attached to the 5' end of the spacer sequence.

[0016] [4] The manufacturing method according to [1], wherein an oligonucleotide consisting of 3 to 40 nucleotides is attached to the 5' end of the spacer sequence.

[0017] [5] The manufacturing method according to [1], wherein an oligonucleotide consisting of at least 10 or more identical nucleotides, or an oligonucleotide in which 50% or less of the oligonucleotides are replaced with other nucleotides, is attached to the 5' end of the spacer sequence.

[0018] [6] The method for producing the same nucleotide as described in [5], wherein the same nucleotide is adenine.

[0019] [7] The manufacturing method according to any one of [1] to [6], wherein the Cas protein is a Cas9 protein.

[0020] [8] The manufacturing method according to any one of [1] to [7], wherein the DNA construct for expressing the gRNA includes a selection marker gene, and further comprises the steps of: liquid-culturing prokaryotic cells into which the DNA construct and the Cas protein or a DNA construct for expressing the protein have been introduced under conditions corresponding to the selection marker gene; and solid-culture the liquid-cultivated prokaryotic cells under conditions corresponding to the selection marker gene to select genome-edited prokaryotic cells.

[0021] [9] A DNA construct for encoding a gRNA having at least one nucleotide attached to the 5' end of a spacer sequence, and for expressing the said gRNA in a prokaryotic cell.

[0022]

[10] The DNA construct according to [9], wherein at least one nucleotide added to the 5' end of the spacer sequence in the gRNA has two consecutive guanines removed.

[0023]

[11] The DNA construct according to [9], wherein an oligonucleotide consisting of at least three nucleotides is attached to the 5' end of a spacer sequence.

[0024]

[12] The DNA construct described in [9], wherein an oligonucleotide consisting of 3 to 40 nucleotides is attached to the 5' end of a spacer sequence.

[0025]

[13] The DNA construct according to [9], wherein an oligonucleotide consisting of at least 10 identical nucleotides, or an oligonucleotide in which 50% or less of the oligonucleotides are replaced by other nucleotides, is attached to the 5' end of the spacer sequence.

[0026]

[14] The DNA construct according to

[13] , wherein the same nucleotide is adenine.

[0027]

[15] A DNA construct according to any one of [9] to

[14] , which further encodes a Cas protein and is used to express the said protein.

[0028]

[16] The DNA construct according to

[15] , wherein the Cas protein is the Cas9 protein.

[0029]

[17] A DNA construct according to any one of [9] to

[16] , further comprising a selection marker gene.

[0030] Furthermore, techniques using gRNA with at least one nucleotide added to the 5' end of the spacer sequence are also disclosed in Patent Documents 1 and 2. However, Patent Document 1 is limited to genome editing in eukaryotic cells, and the added nucleotides are further limited to two guanines. In addition, the invention described in Patent Document 2 is limited to genome editing of only one allele, and furthermore, no examples of prokaryotic cells are shown in its examples. Therefore, this invention is also substantially limited to eukaryotic cells, and the fact that genome editing efficiency in prokaryotic cells is improved by adding a nucleotide to the 5' end of the spacer sequence, as in the present invention, is not disclosed or suggested in any way in any of the Patent Documents.

[0031] According to the present invention, it is possible to produce genome-edited prokaryotic cells.

[0032] This figure shows an overview of the plasmid vectors used in the examples described below. This graph shows the genome editing efficiency when gRNA with a sequence of 1 to 40 consecutive adenines is applied to the Lactiplantybacillus plantarum WCFS1 strain. This graph shows the genome editing efficiency when gRNA with a sequence of 10 or 30 consecutive adenines, thymine, guanine, or cytosine is applied to the Lactiplantybacillus plantarum WCFS1 strain. This graph shows the genome editing efficiency when gRNA with a sequence of 10 consecutive adenines, in which one of the positions 5, 6, or 7 in that sequence is replaced with thymine, guanine, or cytosine, is applied to Lactiplantybacillus plantarum WCFS1. This graph shows the genome editing efficiency when a gRNA with a sequence of 10 consecutive adenines is treated with a gRNA in which one of the positions 1, 3, 9, or 10 in the sequence is replaced with thymine, or with a gRNA in which an odd or even position in the sequence is replaced with thymine, was applied to Lactipruntivebacillus plantarum WCFS1. This graph shows the genome editing efficiency when a gRNA with a sequence of 10 or 30 consecutive adenines, thymine, guanine, or cytosine is treated with Lactipruntivebacillus plantarum Rec strain. This figure shows an overview of the plasmid vectors used in the examples described later. In the figure, "repeatable sequence" represents two direct repeats derived from pCas9, "spacer" represents a site for inserting a spacer sequence, "sequence downstream of repeatable sequence" represents a sequence derived from pCas9 downstream of the direct repeat, "scaffold downstream sequence" represents a scaffold downstream sequence derived from pHSB06X, and "tracr downstream sequence" represents a tracrRNA downstream sequence derived from pCas9. This graph shows the genome editing efficiency of bimolecule gRNA with added nucleotides. In the figure, the added base "-" indicates the genome editing efficiency of bimolecule gRNA without added nucleotides, and "10A" indicates the genome editing efficiency of bimolecule gRNA with a sequence of 10 consecutive adenines added.

[0033] As shown in the examples described below, the inventors have demonstrated that using a guide RNA (gRNA) in which at least one nucleotide is attached to the 5' end of the spacer sequence improves the genome editing efficiency in prokaryotic cells.

[0034] Therefore, the present invention relates to a method for producing genome-edited prokaryotic cells, comprising the steps of introducing a gRNA having at least one nucleotide attached to the 5' end of a spacer sequence, or a DNA construct for expressing said gRNA, and a Cas protein, or a DNA construct for expressing said protein, into prokaryotic cells.

[0035] In the present invention, the CRISPR-Cas system used for genome editing in prokaryotic cells uses, as components, at least a Cas protein, which is a nuclease (RGN; RNA-guided nuclease), and its gRNA. By introducing this system into cells, the gRNA binds to the sequence of the target region, and the Cas protein induced at the binding sequence can cleave the DNA.

[0036] (Guide RNA) In this invention, "guide RNA (gRNA)" is an RNA that contains a nucleotide sequence that interacts with the Cas protein and a nucleotide sequence (spacer sequence) that is complementary to the target region sequence. The morphology of the gRNA varies depending on the type of CRISPR-Cas system, and includes a morphology that contains both crRNA containing a spacer sequence and tracrRNA that interacts with the Cas protein, and a morphology that consists only of crRNA. The crRNA is selected to target the PAM (proto-spacer adjust motif) sequence and the region adjacent to it. In the morphology that contains both crRNA and tracrRNA, the crRNA contains a sequence on the 3' end that can interact (hybridize) with tracrRNA, while the tracrRNA contains a sequence on the 5' end that can interact (hybridize) with a part of the crRNA sequence. The double-stranded RNA formed by the interaction of these sequences interacts with the Cas protein. The gRNA according to the present invention may be in the form of a single gRNA (sgRNA) containing both crRNA and tracrRNA, or in the form of a bimolecule gRNA consisting of a combination of their respective fragments.

[0037] Furthermore, genome editing using the Cas9 protein, described later, requires a combination of crRNA and tracrRNA. On the other hand, the Cas12a protein, described later, does not require tracrRNA and can perform genome editing through interaction with crRNA alone.

[0038] In the present invention, as shown in the examples described later, by adding at least one nucleotide to the 5' end of the spacer sequence, the DNA cleavage activity by the CRISPR-Cas system is suppressed, thereby reducing lethal toxicity and enabling the production of genome-edited prokaryotic cells.

[0039] The length of the sequence added to the 5' end of the spacer sequence (hereinafter also simply referred to as the "additional sequence") may be at least one nucleotide (for example, two or more), preferably three or more (for example, four or more, five or more, six or more, seven or more, eight or more, nine or more), and more preferably ten or more (for example, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty or thirty-five or more). Furthermore, there is no particular upper limit to the length of the additional sequence, as long as it is possible to produce genome-edited prokaryotic cells, but it is preferably 40.

[0040] The nucleotides constituting the additional sequence may all be the same nucleotide (adenine only, uracil only, guanine only, or cytosine only). In such cases, from the viewpoint of genome editing efficiency in prokaryotic cells, the same nucleotide is preferably adenine, guanine, or uracil, and more preferably adenine. On the other hand, in the additional sequence according to the present invention, it is desirable to exclude sequences consisting of two consecutive guanines.

[0041] Furthermore, the additional sequence according to the present invention may not be limited to sequences consisting only of the same nucleotide, but may also be an oligonucleotide consisting of multiple types of nucleotides (i.e., an oligonucleotide consisting of at least one nucleotide selected from adenine, uracil, guanine, and cytosine). For example, an oligonucleotide in which a part of the same nucleotide is substituted with another nucleotide may be included. In such an oligonucleotide, the "other nucleotide" may be any nucleotide different from the same nucleotide, and may be one type, two types, or three types. The number of nucleotides that are substituted by other nucleotides must be at least one, and the ratio of these substitutions (substitution rate: the ratio of the total number of other nucleotides to the total number of nucleotides constituting the added sequence) can be, for example, 75% or less, 70% or less, 67% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 34% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Note that the substitution rate here is shown as a value rounded up to the nearest whole number. Furthermore, "identical nucleotide" here refers to the nucleotide with the highest content in the added sequence after the substitution. Furthermore, if there are multiple nucleotides with the highest content, any of these multiple nucleotides may be considered the same nucleotide (for example, if there are three other nucleotides (i.e., four nucleotides that make up the added sequence after substitution), and their content ratios are 30%, 30%, 30%, and 10%, any of the three nucleotides with a 30% content ratio may be designated as the same nucleotide). Furthermore, there are no particular restrictions on the substitution sites of other nucleotides in the added sequence, as long as it is possible to produce genome-edited prokaryotic cells.

[0042] The length of the spacer sequence in the gRNA of the present invention is not particularly limited as long as genome editing occurs in prokaryotic cells. For example, when using the Cas9 protein described below, it is usually 12 to 50 nucleotides, preferably 17 to 30 nucleotides, and more preferably 17 to 25 nucleotides. Also, when using the Cas12a protein described below, the length of the spacer sequence is usually 21 to 24 nucleotides.

[0043] The sequence of the target region complementary to the spacer sequence may be an endogenous sequence to prokaryotic cells or an exogenous sequence (for example, the sequence of a plasmid vector introduced into prokaryotic cells). The sequence of such a target region is arbitrarily selected and not particularly limited.

[0044] The gRNA may be introduced into prokaryotic cells in the form of RNA, or may be introduced into prokaryotic cells in the form of a DNA construct described below and expressed intracellularly.

[0045] Examples of the method for preparing the gRNA in the form of RNA include a method of synthesizing it by an in vitro transcription reaction by preparing a construct in which a promoter such as T7 is added upstream of the DNA encoding the gRNA, and a method of chemical synthesis. When chemically synthesizing the gRNA, only natural nucleotides may be used, or non-natural nucleotides may also be used. Examples of the "non-natural nucleotides" include analogs of natural nucleotides, nucleotides in which at least one of the base part, sugar part, and phosphate part is modified (for example, phosphorothioate backbone). That is, the gRNA of the present invention is not limited to a polymer consisting only of natural nucleotides, but may be a polymer consisting of natural nucleotides and non-natural nucleotides, or may be a polymer consisting only of non-natural nucleotides.

[0046] (Cas Protein) As the "Cas protein" in the present invention, for example, Cas proteins of the Class 2 system (more specifically, type II Cas proteins such as Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), and Cas14 of type V Cas proteins, Cas13a (C2c2), Cas13b, Cas13c, etc. of type VI Cas proteins) can be mentioned. Preferably, they are Cas9 protein and Cas12a protein, and more preferably, Cas9 protein.

[0047] Regarding the origin of the Cas protein, there is no particular limitation. However, in the case of the Cas9 protein, for example, Cas9 protein derived from Streptococcus pyogenes (SpCas9), Cas9 protein derived from Francisella novicida (FnCas9), Cas9 protein derived from Staphylococcus aureus (SaCas9), Cas9 protein derived from Campylobacter jejuni (CjCas9), and Cas9 protein derived from Neisseria meningitidis (NmCas9) can be mentioned. Also, as the origin of the Cas12a protein, for example, Acidaminococcus, Ruminococcus, Chlamydomonas, and Francisella - novicida can be mentioned. Thus, in the present invention, Cas proteins from various origins can be used.

[0048] Incidentally, typical amino acid sequences and base sequences of Cas proteins are registered in publicly available databases, for example, GenBank (http: / / www.ncbi.nlm.nih.gov), and these can be utilized in the present invention. For example, the typical amino acid sequence and base sequence of the Cas9 protein derived from Streptococcus pyogenes are the respective sequences described in NCBI Reference Sequence: NP_269215 and NC_002737. Also, for example, the typical amino acid sequence of the Cas12a protein derived from Acidaminococcus is the sequence described in NCBI Reference Sequence: WP_021736722.

[0049] In the present invention, the "Cas protein" may be an artificial variant of a natural Cas protein, its homolog, ortholog, or partial peptide, as long as it can form a complex with a guide RNA and edit a specific sequence in a target prokaryotic cell. Examples of "homologists" and "orthologs" include proteins consisting of amino acid sequences that have 85% or more, preferably 90% or more, and more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence of the target Cas protein (e.g., the amino acid sequence described in NP_269215). Sequence identity can be evaluated by a numerical value calculated using BLAST or the like (e.g., default i.e., initial parameters).

[0050] A "mutant" consists of an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence of the referenced natural Cas protein (for example, the amino acid sequence described in NP_269215). Here, "multiple" means, for example, 2 to 150 amino acids, preferably 2 to 100, and more preferably 2 to 50 amino acids (for example, 2 to 30, 2 to 10, 2 to 5, 2 to 3, or 2).

[0051] Examples of mutations that can be introduced include those that cause partial or complete loss of nuclease activity in the Cas protein. A mutant of the Cas protein that has partially lost nuclease activity is called an nCas protein, and a mutant that has completely lost nuclease activity is called a dCas protein.

[0052] When using nCas proteins or dCas proteins, they can be fused with other effector proteins, allowing the DNA sequence within prokaryotic cells to be edited by the activity of the effector protein. The "Cas protein" of the present invention includes such fusion proteins. The activities of the fused effector proteins include, but are not limited to, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, photorecovery enzyme activity, and glycosylase activity. The method for applying deaminase to the CRISPR-Cas system is publicly known (Nishida K. et al., Targeted nucleotide editing using hybrid prokalyotic and vertebrate adaptive immunosystems, Science, DOI: 10.1126 / science.aaf8729, (2016)), and this method can be applied to the present invention.

[0053] Furthermore, mutations introduced into the Cas protein include those that modify PAM recognition (Benjamin, P. et al., Nature 523, 481-485 (2015); Hirano, S. et al., Molecular Cell 61, 886-894 (2016)). By introducing such mutations, the number of base pairs in the PAM sequence recognized by the Cas protein can be reduced, thereby expanding the genomic region targeted by the CRISPR-Cas system.

[0054] The Cas protein may be introduced into prokaryotic cells in the form of a protein, or it may be introduced into prokaryotic cells in the form of a DNA construct as described later and expressed within the cell.

[0055] Those skilled in the art can express the Cas protein according to the present invention as a recombinant protein by loading the DNA encoding the Cas protein onto a suitable vector and introducing it into host cells such as E. coli, animal cells, insect cells, or plant cells, or into a cell-free protein synthesis system (e.g., reticulocyte extract, wheat germ extract). Furthermore, the recombinant protein expressed in host cells, etc., can be purified by known peptide purification methods. In addition, the Cas protein according to the present invention can also be prepared by chemical synthesis using a commercially available polypeptide synthesizer based on its amino acid sequence.

[0056] (DNA constructs) As shown in the examples described below and as described above, in the manufacturing method of the present invention, a DNA construct for expressing the gRNA of the present invention and a DNA construct for expressing the Cas protein are used.

[0057] The DNA construct of the present invention is not particularly limited as long as it can express the gRNA, etc. of the present invention in prokaryotic cells, but it can take the form of an expression vector such as a plasmid vector or a phage vector. Examples of plasmid vectors include pKD46, pHCE1LB, pHY300PLK, pWVO1, pAMβ1, and pSECE1. An example of a phage vector is φFSV. Such an expression vector has a prokaryotic cell replication origin and further has a promoter sequence for expressing the gRNA, etc. of the present invention. Such a "promoter sequence" may be a constitutive promoter that enables constitutive expression, or an inducible promoter that enables inducible expression. Examples include sequences containing a TATA box or a TATA box-like region, more specifically, the promoter sequence of a S-layer protein (surface protein) gene (e.g., a sequence containing PslpA), the promoter sequence of a dehydrogenase gene, and the promoter sequence of a ClpC ATPase chaperone. Furthermore, in the present invention, the expression vector may also include regulatory sequences such as operators, terminators, and enhancers to regulate the expression. The promoter sequence and the regulatory sequences are operatively bound to the target DNA to be expressed (gRNA and / or DNA encoding the Cas protein). Here, "operatively bound" means that the target DNA is expressibly bound to these sequences. The expression vector may also include a selection marker gene, as described later.

[0058] In the present invention, when introducing gRNA and / or Cas protein into prokaryotic cells using such DNA constructs, the DNA construct encoding gRNA and the DNA construct encoding Cas protein may be separate and independent, or they may be a single DNA construct (for example, they may take the form of an all-in-one vector).

[0059] In the present invention, the DNA construct may also include two homologous sequences (homologus arms) adjacent to the left and right of the target site, in order to insert foreign DNA into the target site cleaved by the genome editing system. This results in homologous recombination with the target gene, and the foreign DNA is inserted into the target site cleaved in the target gene. There are no particular restrictions on the length of such homologous sequences as long as homologous recombination can occur, and those skilled in the art can adjust them as appropriate. Furthermore, the "foreign DNA" placed between the two homologous sequences may be any gene (DNA encoding any protein, etc.) or non-coding DNA. In addition, it may also include expression control sequences (promoter sequences, regulatory sequences) for expressing such any gene.

[0060] Such DNA constructs can be prepared by methods known to those skilled in the art. For example, they can be prepared by cleaving purified target DNA with an appropriate restriction enzyme and ligating it to a multi-cloning site or the like of a suitable vector that forms the backbone. Alternatively, the target DNA may be inserted into an intermediate vector by double cross-over recombination, and the expression vector according to the present invention can also be prepared using methods such as LR, in-fusion cloning, or TA cloning. Furthermore, the nucleotide sequences arranged in the expression vector according to the present invention may be optimized to codons suited to the prokaryotic cells into which they are introduced, in order to efficiently express their translation products in those cells.

[0061] (Prokaryotic Cells) The "prokaryotic cells" targeted by the method of the present invention refer to single-celled organisms that do not have a nucleus or other membrane-bound organelles, such as bacteria (eubacteria) and archaea. The bacteria may be Gram-positive or Gram-negative. Examples of "Gram-negative bacteria" include Escherichia coli, Pseudomonas, Salmonella, Campylobacter, and Helicobacter. Examples of "Gram-positive bacteria" include lactic acid bacteria, Bacillus (such as Bacillus subtilis), Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, and Clostridium.Furthermore, examples of "lactic acid bacteria" include bacteria belonging to the genera Lactiplantibacillus, Lacticaseibacillus, Leviractobacillus, Lydilactobacillus or Rimosilactobacillus (all formerly known as Lactobacillus), Lactococcus, Enterococcus, Pediococcus, Leuconostoc, Streptococcus, or Bifidobacterium. More specifically, Lactiplantibacillus plantarum (formerly known as Lactobacillus plantarum) and Lacticaseibacillus paracasei Lactobacillus paracasei (formerly known as Lactobacillus paracasei), Lactobacillus casei (formerly known as Lactobacillus casei), Lactobacillus rhamnosus (formerly known as Lactobacillus rhamnosus), Leviractobacillus brevis (formerly known as Lactobacillus brevis), Ligilactobacillus salivarius Lactobacillus salivarius, Limosilactobacillus reuteri, Limosilactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus gasseri, Lactococcus lactis Examples include *Lactis*.

[0062] Furthermore, as shown in the examples described later, from the viewpoint of further improving genome editing efficiency, prokaryotic cells with enhanced DNA repair activity are preferred as the target of the method of the present invention. Enhancement of DNA repair activity can be performed, for example, by activating a system for promoting homologous recombination in prokaryotic cells (RecE / T, Redα / β, etc.), and more specifically, it can be performed, for example, by introducing the Lp_0640-42 gene, introducing the sapK-sappR gene, culturing in the presence of a synthetic peptide (IP-673), or by appropriately combining these.

[0063] The present invention may also take the form of a kit for producing genome-edited prokaryotic cells. The kit of the present invention may include at least the gRNA of the present invention or a DNA construct for expressing said gRNA, but may also include other substances (e.g., the Cas protein or a DNA construct for expressing said protein, or the prokaryotic cells described above). Furthermore, the kit of the present invention may also include instructions for use.

[0064] (Method for producing genome-edited prokaryotic cells) In the method of the present invention, the above-mentioned gRNA or a DNA construct for expressing the gRNA and the above-mentioned Cas protein or a DNA construct for expressing the protein are introduced into such prokaryotic cells.

[0065] Those skilled in the art can select appropriate known methods, taking into consideration the type of cell, the type of substance, etc., to introduce these substances into prokaryotic cells. Examples of such known methods include electroporation, competent cell method, protoplast method, calcium phosphate method, DEAE-dextran method, and cationic liposome method. The DNA constructs introduced by such methods may be retained extrachromosomally within the host prokaryotic cell or incorporated into the chromosome.

[0066] In prokaryotic cells into which gRNA and Cas protein have been introduced in this manner, the detection or selection of genome-edited cells can be performed using methods known to those skilled in the art. Examples of such known methods include DNA sequencing (next-generation sequencing, etc.), PCR (RT-PCR), CAPS, microarray analysis, Southern blotting, and Northern blotting. Using such nucleotide detection methods, it is possible to determine whether or not a mutation has been introduced by genome editing by comparing the sequence or length of the target region or its transcript before and after mutation introduction, and based on this determination, it is possible to detect prokaryotic cells in which the target region has been genome-edited. Furthermore, selection can also be performed by comparing the presence or size of proteins expressed from the target region before and after mutation introduction, not limited to such nucleotide detection methods. Examples of such protein detection methods include immunohistochemistry, ELISA, RIA, dot immunohistochemistry assay, and Western blotting.

[0067] Furthermore, with respect to genome-edited prokaryotic cells obtained by the manufacturing method of the present invention, as shown in the examples described below, these colonies may be in a state where mutant cells with genome-edited mutations and wild-type cells without such mutations coexist (mix mutant). In the manufacturing method of the present invention, by adopting the following embodiments, it becomes possible to isolate colonies composed solely of mutant cells (pure mutant) from the mix mutant.

[0068] (Method for Isolating Pure Mutant 1) A method for producing genome-edited prokaryotic cells, comprising: Step 1: A step of liquid-culturing prokaryotic cells into which a DNA construct containing a selection marker gene and a Cas protein or a DNA construct for expressing said protein have been introduced, under conditions corresponding to the selection marker gene; and Step 2: A step of solid-state-culturing the prokaryotic cells liquid-culturing in Step 1 under conditions corresponding to the selection marker gene, and selecting genome-edited prokaryotic cells.

[0069] The DNA construct for expressing the gRNA of the present invention, which is introduced into prokaryotic cells, has a select marker gene. Examples of the "select marker gene" include drug resistance genes and nutrient requirement genes. Examples of the "drug resistance gene" include erythromycin resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, ampicillin resistance gene, eneomycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blastocydin S resistance gene, bialafos resistance gene, zeosin resistance gene, paromomycin resistance gene, gentamicin resistance gene, and hygromycin resistance gene. Examples of "nutrient requirement genes" include the N-(5'-phosphoribosyl)anthranilate isomerase (TRP1) gene, tryptophan synthase (TRP5) gene, malate β-isopropyl dehydrogenase (LEU2) gene, imidazole glycerol phosphate dehydrogenase (HIS3) gene, histidinol dehydrogenase (HIS4) gene, dihydroorotate dehydrogenase (URA1) gene, and orotidine-5-phosphate decarboxylase (URA3) gene.

[0070] In this context, the selection marker gene only needs to have a DNA construct for expressing guide RNA. However, as mentioned above, in the present invention, the DNA construct for expressing Cas protein may also have such a selection marker gene. In that case, the selection marker genes of the DNA construct for expressing guide RNA and the DNA construct for expressing Cas protein may be the same or different.

[0071] In the present invention, prokaryotic cells that have been subjected to a DNA construct or the like containing a selection marker gene may be used directly in step 1. However, as shown in the examples described later, after the introduction treatment, the presence or absence of genome editing may be detected using the method described above, and prokaryotic cells in which genome editing is detected may be used in step 1. Alternatively, after the introduction treatment, prokaryotic cells selected by solid culture under conditions corresponding to the selection marker gene may be used in step 1.

[0072] The "conditions corresponding to the selection marker gene" refer to, for example, the presence of the drug if the selection marker gene used is a drug resistance gene, or the presence of nutrients biosynthesized by the pathways involved in the gene if it is a nutrient requirement gene. These conditions are usually prepared by adding the drugs and nutrients described later to the culture medium. If the selection marker genes of the DNA construct for expressing gRNA and the DNA construct for expressing Cas protein are different, cultivation is required in the presence of drugs and / or nutrients corresponding to both selection marker genes.

[0073] The "culturing" in steps 1 and 2 can be carried out by a person skilled in the art using methods known to the extent appropriate to the type of prokaryotic cell being cultured. For example, the cells can be cultured using various culture media containing carbon sources, nitrogen sources, organic and inorganic salts, etc., that are commonly used for culturing prokaryotic cells. Examples of carbon sources include glucose, dextrin, soluble starch, sucrose, glycerol, and organic acids. Examples of nitrogen sources include inorganic substances such as ammonium salts and nitrates, and organic substances such as cornstarch liquor, peptone, casein, yeast extract, meat extract, soybean meal and potato extract, and amino acids. Examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, magnesium sulfate, and sodium chloride. Furthermore, yeast extract, vitamins, growth factors, malt extract, tissue extracts, reducing agents, inorganic metal salts such as Fe, Mn, and Mo, and organic compounds containing metals may be added to the culture medium.

[0074] Then, in step 2, culture (liquid culture) is performed using a liquid medium prepared by dissolving these culture medium components in water. The culture time for such liquid culture can be appropriately adjusted by those skilled in the art depending on the type of prokaryotic cells to be cultured, the composition of the culture medium used, the type of drugs or nutrients added, etc., but it is usually 12 to 72 hours, preferably 18 to 28 hours.

[0075] In step 3, a solid medium or semi-solid medium is prepared by adding a gelling agent (agar (agarose), gellan gum, gellite, sodium alginate, carrageenan, etc.) to the liquid medium and solidifying or semi-solidifying it, and then culture (solid culture) is performed using this medium. The solid culture in step 3 should be carried out until prokaryotic cells that can be cultured under conditions corresponding to the selected marker gene (for example, colonies on the solid medium or semi-solid medium formed by these cells) can be detected.

[0076] By following the above cultivation process, it becomes possible to obtain only pure mutant, as shown in the examples described later.

[0077] Furthermore, the present invention is not limited to the culture method described above, and it is also possible to obtain pure mutant by the following method.

[0078] (Method for Isolating Pure Mutant 2) A method for producing genome-edited prokaryotic cells, comprising: Step 1: A step of liquid-culturing prokaryotic cells into which a DNA construct containing a selection marker gene and a Cas protein or a DNA construct for expressing said protein have been introduced, under conditions not corresponding to the selection marker gene; Step 2: A step of solid-culture the prokaryotic cells liquid-culturing in Step 1 under conditions not corresponding to the selection marker gene; and Step 3: A step of solid-culturing the prokaryotic cells solid-culturing in Step 2 under conditions corresponding to and not corresponding to the selection marker gene, and selecting prokaryotic cells that have proliferated only under the conditions not corresponding to the selection marker gene.

[0079] The selection marker gene, the conditions corresponding to the selection marker gene, and the culture (liquid culture, solid culture) are as described above (Method for Isolating Pure Mutant 1), etc. However, "conditions not corresponding to the selection marker gene" means, for example, the absence of the drug if the selection marker gene used is a drug resistance gene, or the absence of nutrients biosynthesized by the pathways involved in the gene if it is a nutrient requirement gene. These are usually prepared by not adding the drugs and nutrients mentioned above to the culture medium. Furthermore, "growth only under these non-corresponding conditions" means, for example, that prokaryotic cells are streak cultured on the above culture medium, and colonies are not detected under the conditions corresponding to the selection marker gene, while colonies can be detected under the conditions not corresponding to the selection marker gene. The "colonies" detected here are preferably those that can be confirmed by visual inspection (for example, colonies that are 1 mm or larger in size (if approximately circular, the longest diameter is 1 mm or larger)).

[0080] Furthermore, by using this isolation method and culturing the DNA in liquid under conditions that do not correspond to the selection marker gene (i.e., there is no selection pressure), the DNA construct containing the selection marker gene is removed, making it possible to obtain a pure mutant with fixed mutations.

[0081] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0082] (Example 1) Construction of a plasmid for genome editing of Lactiplantybacillus plantarum To perform genome editing targeting the L-lactate dehydrogenase gene (ldh) of Lactiplantybacillus plantarum, pHSB04X was modified and used as follows. Note that pHSB04X is a plasmid vector constructed for genome editing of Leviractobacillus brevis ATCC367, and encodes an sgRNA and homologous arms that target the alcohol dehydrogenase gene, and the Cas9 protein (Figure 1a shows an overview of pHSB04X and the plasmid vector modified therefrom that was used in this example). In this example, first, the spacer sequence of the sgRNA that targets the alcohol dehydrogenase gene was replaced with a spacer sequence that targets ldh or a restriction enzyme site (two BspQI recognition sequences) for inserting the spacer sequence to which various oligonucleotides have been added to the 5' end.

[0083] Specifically, using pHSB04X (Addgene #117258) as a template, a DNA fragment of approximately 14kb, with the spacer sequence removed, was amplified by PCR using primers <SEQ ID NO: 1> and <SEQ ID NO: 2>. The primers <SEQ ID NO: 1> and <SEQ ID NO: 2> are designed with the recognition sequence for the restriction enzyme BamHI between the recognition sequences for the restriction enzyme BspQI. pHSB04X-fw (SEQ ID NO: 1) TAACCGAAGTTTAGCGCTTTTGTTAAGAGAAATTTTTTC pHSB04X-rv (SEQ ID NO: 2) CGCTAACTTCCGGTTTATACTATTTGTGAAGAGCGGATCCGCTTCCGTTTTTAGAGCTAG.

[0084] The PCR reaction conditions were as follows: 1.0 ng of template DNA solution, 0.2 U of KOD FX Neo (Toyobo), 5.0 μL of 2× PCR Buffer for KOD FX Neo (Toyobo), 2.0 μL of 2 mM each of dNTP Mix, 0.4 μL each of 10 μM forward and reverse primers, and 0.4 μL of sterile water were mixed. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, followed by thermal denaturation at 98°C for 10 seconds, annealing at an appropriate temperature for 30 seconds, and extension at 68°C for 30 seconds per kb plus a 30-second buffer time. This process was repeated for 25-35 cycles, after which the mixture was stored at 4°C.

[0085] These DNA fragments were ligated using the In-Fusion® reaction to produce pHSB05X. The infusion reaction solution consisted of 0.4 μL of 5×In-Fusion® mix (TaKaRa) and 1.6 μL of DNA solution. The mixture was incubated at 50°C for 15 minutes using a thermal cycler or a constant temperature bath.

[0086] pHSB05X was introduced into E. coli XL10-Gold using the Hanahan method to obtain the transformed strain E. coli XL10-Gold (pHSB05X), and the plasmid was recovered. For plasmid recovery, first, 200 μL of Resuscitation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) 500 μL was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate containing the plasmid recovered in this manner was stored.

[0087] pHSB05X was digested with ApaI to prepare fragments of approximately 12 kb. Restriction enzyme digestion was performed by mixing DNA solution (400 ng in the case of plasmids), 15 U of restriction enzyme stock solution, 1.0 μL of 10× buffer, 1.0 μL of 10× BSA if necessary, and sterile water to a total volume of 10 μL, and incubating in a 37°C incubator for 1 hour to overnight.

[0088] These fragments were linked by a ligation reaction to produce pHSB06X. The ligation reaction was performed by mixing 1.0 μL of the vector DNA solution with 1.0 μL of 2×Ligation mix (manufactured by NIPPON GENE) and incubating it in a 16°C incubator for 1 hour.

[0089] pHSB06X was introduced into E. coli XL10-Gold using the Hanahan method to obtain the transformed strain E. coli XL10-Gold (pHSB06X), and the plasmid was recovered. For plasmid recovery, first, 200 μL of Resuscitation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. 500 μL of Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied to the column and centrifuged at 13,000 rpm at 4°C for 1 minute, and the filtrate was discarded. 750 μL of Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 13,000 rpm at 4°C for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 13,000 rpm at 4°C for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate containing the plasmid recovered in this manner was stored.

[0090] 0.5 μL each of oligoDNA (100 μM) containing the sense and antisense strands of a spacer sequence targeting LDH of Lactiplantybacillus plantarum WCFS1 strain, 1.0 μL of 10× annealing buffer, and 8.0 μL of sterile water were mixed. Using a thermal cycler, the mixture was incubated at 95°C for 5 minutes, then the temperature was lowered to 25°C over 90 minutes using the slope function to anneal the two oligoDNAs. The annealed oligoDNA combinations were <SEQ ID NO: 3> and <SEQ ID NO: 4>. The 10× annealing buffer was prepared by mixing 2 mL of 1 M Tris acetate (pH 7.8), 1 mL of 1 M magnesium acetate, 2.5 mL of 4 M potassium glutamate, 0.2 mL of 50 mM NAD, 1 mL of 1 M ammonium sulfate, and 3.3 mL of distilled water. ldh_sense (sequence number: 3) TTCTTCCCAGTTCGCGAATGTTCGCTAG ldh_antisense (sequence number: 4) AAACTAGCGGAACATCGCGGAACTGGGAA.

[0091] pHSB06X was digested with BspQI to obtain a fragment of approximately 12 kb. Restriction enzyme digestion was performed by mixing DNA solution (400 ng in the case of plasmid), 15 U of restriction enzyme stock solution, 1.0 μL of 10× buffer, 1.0 μL of 10× BSA if necessary, and sterile water to a total volume of 10 μL, and incubating in a 50°C incubator for 1 hour to overnight. pHSB0 was prepared by ligating the digested pHSB06X with the annealing oligonucleotide. The ligation reaction was performed by mixing 0.4 μL of vector-side DNA solution, 0.6 μL of insert-side DNA solution, and 1.0 μL of 2× Ligation mix (manufactured by NIPPON GENE) and incubating in a 16°C incubator for 1 hour.

[0092] pHSB0 was introduced into E. coli XL10-Gold using the Hanahan method to obtain the transformed strain E. coli XL10-Gold (pHSB0), and the plasmid was recovered. For plasmid recovery, first, 200 μL of Resussion Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) 500 μL was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate containing the plasmid recovered in this manner was stored.

[0093] One strain of Lactiplantilachilus plantarum WCFS was inoculated into MRS liquid medium and incubated at 37°C for 21 hours. The resulting culture was collected by centrifugation, and genomic DNA was extracted from the bacterial cells. The genomic DNA was obtained by suspending 14 g of wet Lactiplantilachilus plantarum WCFS strain 1 in 180 ml of suspension buffer (10 mM Tris-HCl (pH 7.5); 1 mM EDTA; 10 mM NaCl). To this, 9 ml of lysis buffer (20% Sucross; 50 mM EDTA; 50 mM Tris-HCl (pH 7.5)) and 135 mg of lysozyme were added and incubated at 37°C for 10 minutes. Then, 90 ml of suspension buffer, 9 ml of 25% SDS, 18 ml of 5 M NaCl, 180 ml of phenol, and 32 ml of chloroform were added and slowly mixed thoroughly. After that, the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature and the upper layer was collected. Next, an equal volume of chloroform was added to the upper layer and mixed thoroughly, and the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature and the upper layer was collected. An equal volume of ethanol was added to this and mixed thoroughly, and the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature. The obtained precipitate was dried in a vacuum desiccator for 20 minutes and then dissolved in a small amount of TE buffer (10 mM Tris-HCl (pH 8.0); 1 mM EDTA). 20 μL of RNaseA solution was added, and the mixture was incubated at 37°C for 15 hours. Then, 1.2 ml of chloroform was added, and the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature, and the upper layer was collected. 6 ml of chloroform was added, and the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature, and the upper layer was collected. 6 ml of isopropanol was added and thoroughly mixed, then incubated at room temperature for 30 minutes, and the mixture was centrifuged at 3,500 x g for 20 minutes at room temperature. The obtained precipitate was washed with 70% ethanol, dried in a vacuum desiccator, and dissolved in TE buffer to obtain genomic DNA.

[0094] Using the genomic DNA of *Lactiplantybacillus plantarum* WCFS1 strain as a template, approximately 1 kb of DNA fragments were amplified by PCR using primers <SEQ ID NO: 5> and <SEQ ID NO: 6>, and <SEQ ID NO: 7> and <SEQ ID NO: 8> to amplify homologous arm-1 and homologous arm-2. During this process, nucleotides 603 to 608 of the ldh ORF (SEQ ID NO: 9) were modified from TAAGG to GTGAT. ldh-1-fw (Sequence number: 5) TGCTTTTTTTGGGCCATGTTTGCCTAAACCCAACGACATTTTC ldh-1-rv (Sequence number: 6) CAGAAAACGCCCTTGTTTATCAGCGCGACATC ldh-2-fw (Sequence number: 7) AACAAGGCGTTTCTGAACGAAAGATTTAGC ldh-2-rv (Sequence number: 8) CTTTTTCTAAACTAGTGGGCGTTTTTCAGTTTTTGTTCGTTTTG.

[0095] The PCR reaction conditions were as follows: 2.0 μL of template DNA solution, 0.2 μL of KOD FX Neo (Toyobo), 5.0 μL of 2× PCR Buffer for KOD FX Neo (Toyobo), 2.0 μL of 2 mM each of dNTP Mix, 0.4 μL each of 10 μM forward and reverse primers, and 0.4 μL of sterile water were mixed. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, followed by thermal denaturation at 98°C for 10 seconds, annealing at an appropriate temperature for 30 seconds, and extension at 68°C for 30 seconds per kb plus a 30-second buffer time. This process was repeated for 25-35 cycles, after which the mixture was stored at 4°C.

[0096] pHSB06XP and pHSB0P were prepared by inserting PCR reaction products (approximately 1 kb each) into the ApaI cleavage sites of pHSB06X and pHSB0, respectively, via infusion reaction. The infusion reaction solution was based on 0.4 μL of 5×In-Fusion® mix (TaKaRa), 0.8 μL of vector DNA solution, and 0.4 μL of insert DNA solution. The mixture was incubated at 50°C for 15 minutes using a thermal cycler or incubator.

[0097] pHSB06XP or pHSB0P was introduced into E. coli XL10-Gold by the Hanahan method to obtain transformed E. coli XL10-Gold strains (pHSB0XP, pHSB0P), and the plasmids were recovered. In plasmid recovery, first, 200 μL of Resussion Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) 500 μL was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate containing the plasmid recovered in this manner was stored.

[0098] (Example 2) To effectively perform genome editing at the target site for creating a Lactipruntilbacillus plantarum strain with enhanced DNA repair activity, we attempted to construct a Lactipruntilbacillus plantarum strain (Rec strain) with enhanced DNA repair activity by transforming the Lactipruntilbacillus plantarum WCFS1 strain with a plasmid vector (pLH01) encoding the plantarum WCFS1 recombinase. Specifically, the Lactipruntilbacillus plantarum WCFS1 strain was cultured in MRS medium at 37°C for 20 hours, and then cultured in fresh MRS medium containing 0.75 M sorbitol and 2% glycine at 37°C until the turbidity was 0.4 to 0.6. After collecting the cells, they were washed with electroporation buffer (952 mM sucrose, 3.5 mM magnesium chloride) and suspended in 1 / 62.5 volume of the same buffer.

[0099] 0.5 μg of pLH01 (Addgene #117261; an overview of pLH01 is shown in Figure 1b) was mixed with 80 μL of the aforementioned suspension cells, and the mixture was placed in a Bio-Rad Gene Pulsar cuvette (0.2 cm cuvette) and incubated on ice for 15 minutes. Subsequently, electroporation was performed using a Gene Pulsar under conditions of 2.0 kV, 25 μF, and 400 Ω. The cells were diluted in 1 mL of MRS medium containing 0.5 M sucrose and 0.1 M magnesium chloride, incubated at 37°C for 3 hours, and seeded onto MRS agar medium containing 10 μg / mL chloramphenicol. Positive colonies were selected, and a strain of Lactiplantibacillus plantarum WCFS1 transformed with pLH01 was obtained. This strain was designated as the Rec strain.

[0100] (Example 3) Genome editing of Lactiplantybacillus plantarum using gRNA without nucleotide addition We attempted to edit the genome of Lactiplantybacillus plantarum WCFS1 strain using gRNA (without nucleotide addition to the 5' end) in a conventional method. Specifically, Lactiplantybacillus plantarum WCFS1 strain cultured in the same manner as in Example 2 was suspended in electroporation buffer and pHSB06XP or pHSB0P was introduced. At this time, positive colonies were selected on MRS agar medium containing 10 μg / mL erythromycin, and colony-forming units (CFUs) were measured to calculate the transformation efficiency of the plasmid. As a result, pHSB06XP showed 496 cfus, while pHSB0P did not form colonies, suggesting that DNA cleavage by Cas9 exhibits lethal toxicity to bacteria.

[0101] (Example 4) Construction of a plasmid encoding nucleotide-added gRNA The purpose was to evaluate the effect of gRNA with nucleotides added to the 5' end, and in order to construct a plasmid encoding it, a plasmid with a spacer sequence in which adenine was continuously added to the 5' end was constructed based on pHSB06XP. Specifically, a plasmid expressing gRNA with a sequence of 1 to 40 adenine units added to the 5' end of the spacer sequence was constructed. OligoDNA was annealed in the same manner as in Example 1, except that <Sequence ID: 10> and <Sequence ID: 11>, <Sequence ID: 12> and <Sequence ID: 13>, <Sequence ID: 14> and <Sequence ID: 15>, <Sequence ID: 16> and <Sequence ID: 17>, <Sequence ID: 18> and <Sequence ID: 19>, <Sequence ID: 20> and <Sequence ID: 21>, and <Sequence ID: 22> and <Sequence ID: 23> were used.ldh-1A-sense (Sequence number: 10) TTCTATCCCAGTTCGCGAATGTTCGCTG ldh-1A-antisense (Sequence number: 11) AAACAGCGCACATTCGCGAACTGGATA ldh-3A-sense (Sequence number: 12) TTCTAAATCCCAGTTTTCGCGAATGTTCGCTG ldh-3A-antisense (Sequence number: 13) AAACAGCGCACATTCGCGAACTGGAATTTTA ldh-7A-sense (Sequence number: 14) TTCTAAAAAAAAATCCCAGTTTTCGCGAATGTTCGCTG ldh-7A-antisense (Sequence number: 15) AAACAGCGACATCGCGGAACTGGGAATTTTTTTTTTA ldh-10A-sense (Sequence number: 16) TTCTAAAAAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-antisense (Sequence number: 17) AAACAGCGACATCCGCGAACTGGGAATTTTTTTTTTTTA ldh-30A-sense (Sequence number: 18) TTCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCCCAGTTCGCCGGAATGTTCGCTG ldh-30A-antisense (Sequence number: 19) AAACAGCGACATCGCGGAACTGGGAATTT TTCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCCCAGTTCGCGAATGTTCGCTG ldh-40A-atlishens0 (Sequence number: 23) AAACAGCGAACATGCCGGAACTGGGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTA.

[0102] pHSB06XP was cleaved with BspQI and linked with an annealing oligo in the same manner as in Example 1 to produce pHSB1AP, pHSB3AP, pHSB7AP, pHSB10A, pHSB30A, pHSB35AP, and pHSB40AP.

[0103] (Example 5) Genome editing of Lactiplantybacillus plantarum WCFS1 strain using nucleotide-added gRNA We attempted to modify the DNA sequence in the ldh gene of Lactiplantybacillus plantarum WCFS1 strain using the nucleotide-added gRNA prepared above. Specifically, pHSB0P, pHSB1AP, pHSB3AP, pHSB7AP, pHSB10A, pHSB30A, pHSB35AP, or pHSB40AP were introduced into Lactiplantybacillus plantarum WCFS1 strain using the same method as in Example 2, and the colony-forming units (CFUs) were measured.

[0104] Genomic DNA was prepared from eight positive colonies using the Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka Corporation). Using the prepared genomic DNA mixture as a template, a DNA fragment of approximately 3.7 kb was amplified using primers <SEQ ID NO: 24> and <SEQ ID NO: 25>. ldh-out-fw (SEQ ID NO: 24) CCAGTCGTAAAGGGTAGTCCGCCATC ldh-out-rv (SEQ ID NO: 25) TACCGGGCTTTCCAGTAAACGATTC PCR reaction conditions: 2.0 μL template DNA solution, 0.2 μL KOD FX Neo (Toyobo), 5.0 μL 2× PCR Buffer for KOD FX Neo (Toyobo), 2.0 μL each of 2 mM dNTP Mix, 0.4 μL each of 10 μM forward and reverse primers, sterile water 0.4 μL was mixed and preheated at 94°C for 2 minutes using a thermal cycler. This was followed by thermal denaturation at 98°C for 10 seconds, annealing at an appropriate temperature for 30 seconds, and extension reaction at 68°C for 30 seconds per kb plus a 30-second buffer time. This process was repeated for 25 to 35 cycles, after which the mixture was stored at 4°C.

[0105] Subsequently, the PCR reaction mixture was subjected to electrophoresis on a 1.0% agarose gel to excise approximately 3.7 kb of DNA, and the DNA was recovered from the agarose gel. The QIAquick Gel Extraction Kit (manufactured by QIAGEN) was used to recover the DNA from the agarose. After DNA recovery, a sequencing reaction was performed. The sequencing reaction mixture was prepared by mixing 40-100 ng of template DNA, 3.75 μL of sequencing buffer, 1 μL of 3.2 pmol primer, and 0.5 μL of Big Dye, and adjusting the volume to 20 μL with sterile water. <Sequence ID: 26> was used as the primer. ldh_mutant_fw (Sequence ID: 26) AGTTCGCCGATGTTCGCGTGATAAC.

[0106] Cycle sequencing was performed using the SuperDye v3.1 Cycle Sequence Kit (AdvancedSeq), and analysis was performed using the SeqStudio® genetic analyzer (Thermo Fisher Scientific). Among the positive colonies, it was found that there were WT colonies showing wild-type nucleotide sequences, pure mutant colonies showing mutant nucleotide sequences derived from homologous arms, and mixed mutant colonies containing both wild-type and mutant nucleotide sequences.

[0107] The HDR rate, which indicates the relative abundance of pure mutant in positive colonies, was determined by ICE assay (as described in Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data. BioRxiv, 251082, August 2019). The genome editing efficiency of each base-addition gRNA was evaluated as the product of the HDR rate and the number of positive colonies. The HDR rate used was the mean value of eight randomly selected colonies. The results are shown in Figure 2.

[0108] As is clear from the results shown in Figure 2, when sgRNA without adenine was introduced, the abundance of pure mutants was 0%. On the other hand, when sgRNA with at least one nucleotide added was used, the abundance of pure mutants increased, and mutation introduction by genome editing was observed. Furthermore, it was revealed that adding 3 to 40 adenine nucleotides significantly improved the efficiency of mutation introduction by genome editing compared to adding one adenine nucleotide.

[0109] (Example 6) Isolation of pure mutant from mixed mutant colonies Although genome editing of the lactiplantybacillus plantarum WCFS1 strain was made possible by the above method, the appearance of mixed mutant colonies (mix mutants) containing both mutants and wild-type (WT) cells was observed in the obtained colonies. An attempt was made to obtain colonies composed solely of mutant cells (pure mutants) from these colonies.

[0110] Specifically, to isolate pure mutant from mixed mutant, the obtained mixed mutant was streaked onto MRS agar medium containing 10 μg / mL erythromycin and incubated at 37°C for 20 hours. Four colonies were randomly selected, and their HDR rates were determined using the same method as in Example 5 (shown in Table 1 below under "Method-1").

[0111] Alternatively, to isolate pure mutant from mixed mutant, the obtained mixed mutant was subcultured in MRS liquid medium containing 10 μg / mL erythromycin and cultured at 37°C for 20 hours. The culture medium was then transferred to sterile MRS medium for 10 hours. ６ Ten μL of the 1:1 diluted suspension was spread onto MRS agar containing 10 μg / mL of erythromycin and incubated at 37°C for 20 hours. Eight colonies were randomly selected, and their HDR rates were determined using the same method as in Example 5 (shown in Table 1 below under "Method-2").

[0112]

[0113] As is clear from the results shown in Table 1, in Method 1, the survival of the mixed mutant was observed even after subculturing. On the other hand, it became clear that the pure mutant could be isolated by Method 2, and this method increased the frequency of DNA breaks, suggesting that strains with wild-type base sequences were killed.

[0114] (Example 7) Construction of a plasmid expressing gRNA with a modified identical consecutive nucleotide sequence The purpose was to evaluate the effect of gRNA with a nucleotide other than adenine added to the 5' end, and in the same manner as in Example 4, a plasmid encoding a spacer sequence with 10 consecutive uracil, guanine, or cytosine added to the 5' end was constructed. Specifically, oligo DNA was annealed in the same manner as in Example 1, except that <SEQ ID NO: 27> and <SEQ ID NO: 28>, <SEQ ID NO: 29> and <SEQ ID NO: 30>, <SEQ ID NO: 31> and <SEQ ID NO: 32>, <SEQ ID NO: 33> and <SEQ ID NO: 34>, <SEQ ID NO: 35> and <SEQ ID NO: 36>, and <SEQ ID NO: 37> and <SEQ ID NO: 38> were used.ldh-10T-sense (Sequence ID: 27) TTCTTTTTTTTTTTTTCCAGTTCGCGAATGTTCGCTG ldh-10T-antisense (Sequence ID: 28) AAACAGCGCACATTCGCGAACTGGGAAAAAAAAAA ldh-30T-sense (Sequence ID: 29) TTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCAGTTCGCGAATGTTCGCTG ldh-30T-antisense (Sequence ID: 30) AAACAGCGACATCGCGAACTGGGAAAA ldh-30G-antisense (Sequence number: 34) AAACAGCGACATCGCGAACTGGGACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCA ldh-10C_sense (Sequence number: 35) TTCTCCCCCCCCCCCCCTCCAGTTCGCGAATGTTCGCTAG ldh-10C_antisense (Sequence number: 36) AAACTAGCGGACATCGCGAACTGGGAGGGGGGGGGGGA ldh-30C_sense (Sequence number: 37) TTCTCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCAGTTCGCGATGTCGCTAG ldh-30C_antisense (SEQ ID NO: 38) AAAACTAGCGACATCGCGAACTGGAGGGGGGGGGGGGGGGGGGGGGGA.

[0115] pHSB06XP was cleaved with BspQI and linked with an annealing oligo in the same manner as in Example 1 to produce pHSB10TP, pHSB30TP, pHSB10GP, pHSB30GP, pHSB10CP, and pHSB30CP.

[0116] (Example 8) Effect of the type of nucleotide added on genome editing efficiency Using the nucleotide-added gRNA prepared above, we attempted to modify the base sequence in the ldh gene of Lactiplantybacillus plantarum WCFS1 strain.

[0117] Specifically, pHSB0P, pHSB10TP, pHSB30TP, pHSB10GP, pHSB30GP, pHSB10CP, or pHSB30CP were introduced into Lactiplantybacillus plantarum WCFS1 strain using the same method as in Example 2, and the colony-forming units (CFUs) were measured.

[0118] Using the same method as in Example 5, the genome editing efficiency was determined when using pHSB06XP, pHSB0P, pHSB10TP, pHSB30TP, pHSB10GP, pHSB30GP, pHSB10CP, or pHSB30CP. As is clear from the results shown in Figure 3, when the number of identical nucleotides added consecutively was set to 10 or 30, adenine addition showed the highest genome editing efficiency compared to other nucleotides. On the other hand, cytosine addition did not result in genome editing, suggesting that it tends to be difficult to suppress DNA cleavage activity.

[0119] (Example 9) Construction of a gRNA containing an additional sequence with one nucleotide substitution The purpose was to evaluate the effect of a gRNA with a sequence that does not have consecutive identical nucleotides added to the 5' end. Similar to Example 4, a plasmid was constructed that encoded a spacer sequence in which one nucleotide in an additional sequence of 10 consecutive adenines was substituted with another nucleotide. Specifically, for gRNA in which a sequence of 10 consecutive adenines was added to the 5' end of a spacer sequence, oligo DNA was annealed in the same manner as in Example 1, except that <Sequence ID: 39> and <Sequence ID: 40>, <Sequence ID: 41> and <Sequence ID: 42>, <Sequence ID: 43> and <Sequence ID: 44>, <Sequence ID: 45> and <Sequence ID: 46>, <Sequence ID: 47> and <Sequence ID: 48>, <Sequence ID: 49> and <Sequence ID: 50>, <Sequence ID: 51> and <Sequence ID: 52>, <Sequence ID: 53> and <Sequence ID: 54>, and <Sequence ID: 55> and <Sequence ID: 56> were used to replace the 5, 6, or 7 positions of the sequence with one of the nucleotides of thymine, guanine, or cytosine. ldh-10A-5T-sense (Sequence number: 39) TTCTAAAAATAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-5T-antisense (Sequence number: 40) AAACAGCGCACATCCGCGAACTGGGAATTTTTATTTTTTA ldh-10A-5G-sense (Sequence number: 41) TTCTAAAAAGAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-5G-antisense (Sequence number: 42) AAACAGCGCACATCCGCGAACTGGGAATTTTTCTTTTTTA ldh-10A-5C-sense (Sequence number: 43) TTCTAAAAACAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-5C-antisense (Sequence number: 44) AAAACAGCGACATTCGCGAACTGGGAATTTTTGTTTTTTA ldh-10A-6T-sense (Sequence number: 45) TTCTAAAAATAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-6T-antisense (Sequence number: 46) AAAACAGCGACATTCGCGAACTGGGAATTTTTTTATTTTA ldh-10A-6G-sense (Sequence number: 47)TTCTAAAAAAAGAAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-6G-antisense (Sequence number: 48) AAACAGCGCACATTCGCGAACTGGGAATTTTTTCTTTTTA ldh-10A-6C-sense (Sequence number: 49) TTCTAAAAAAAACAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-6C-antisense (Sequence number: 50) AAACAGCGCACATTCGCGAACTGGGAATTTTTTTGTTTTA ldh-10A-7T-sense (Sequence number: 51) TTCTAAAAAAAATAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-7T-antisense (Sequence number: 52) AAACAGCGCACATTCGCGAACTGGGAATTTTTTTATTTTA ldh-10A-7G-sense (Sequence number: 53) TTCTAAAAAAAAGAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-7G-antisense (Sequence number: 54) AAACAGCGCACATTCGCGAACTGGGAATTTTTTTTTTTTA ldh-10A-7C-sense (Sequence number: 55) TTCTAAAAAAAACAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-7C-antisense (Sequence number: 56) AAACAGCGAACATGCCGGAACTGGGAATTTTTTTGTTTTA.

[0120] Then, pHSB06XP was cut with BspQI and linked with the annealing oligo in the same manner as in Example 1 to produce pHSB10A5TP, pHSB10A5GP, pHSB10A5CP, pHSB10A6TP, pHSB10A6GP, pHSB10A6CP, pHSB10A7TP, pHSB10A7GP, or pHSB10A7CP.

[0121] (Example 10) Effect of gRNA containing an added sequence with one nucleotide substitution on genome editing Using the nucleotide-added gRNA prepared above, we attempted to modify the base sequence in the ldh gene of Lactiplantybacillus plantarum WCFS1 strain. Specifically, using the same method as in Example 5, we determined the genome editing efficiency when using pHSB10AP, pHSB10A5TP, pHSB10A5GP, pHSB10A5CP, pHSB10A6TP, pHSB10A6GP, pHSB10A6CP, pHSB10A7TP, pHSB10A7GP, and pHSB10A7CP. As a result, as shown in Figure 4, genome editing was possible even when the continuous adenine sequence was interrupted by other nucleotides.

[0122] (Example 11) Construction of a plasmid encoding a gRNA containing an additional sequence substituted with other nucleotides In order to clarify the effect of the type of nucleotide in the sequence added to the 5' end of the gRNA, oligo DNA was annealed in the same manner as in Example 1, except that <SEQ ID NO: 57> and <SEQ ID NO: 58>, <SEQ ID NO: 59> and <SEQ ID NO: 60>, <SEQ ID NO: 61> and <SEQ ID NO: 62>, and <SEQ ID NO: 63> and <SEQ ID NO: 64> were used to replace positions 1, 3, 9, and 10 of the additional sequence, which consists of 10 consecutive adenines at the 5' end, with uracil. Note that the positions in the additional sequence are indicated when the 5' end of the sequence is considered as position 1 (the same applies hereinafter).

[0123] Furthermore, the oligoDNA was annealed in the same manner as in Example 1, except that <SEQ ID NO: 65> and <SEQ ID NO: 66>, and <SEQ ID NO: 67> and <SEQ ID NO: 68> were used to replace all adenines at odd positions (positions 1, 3, 5, 7, and 9) or even positions (positions 2, 4, 6, 8, and 10) with uracil in the aforementioned additional sequences. ldh-10A-1T-sense (Sequence number: 57) TTCTTTAAAAAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-1T-antisense (Sequence number: 58) AAACAGCGCACATCCGCGAACTGGGAATTTTTTTTTTA ldh-10A-3T-sense (Sequence number: 59) TTCTAATAAAAAAATCCCAGTTCGCGAATGTTCGCTG ldh-10A-3T-antisense (Sequence number: 60) AAACAGCGCACATCCGCGAACTGGGAATTTTTTTTTTA ldh-10A-9T-sense (Sequence number: 61) TTCTAAAAAAAATATCCCAGTTCGCGAATGTTCGCTG ldh-10A-9T-antisense (Sequence number: 62) AAACAGCGCACATTCGCGAACTGGGAATTTTTTTTTTATA ldh-10A-10T-sense (Sequence number: 63) TTCTAAAAAAAATTCCAGTTCGCGAATGTTCGCTG ldh-10A-10T-antisense (Sequence number: 64) AAACAGCGCACATTCGCGAACTGGGAATTTTTTTTTTTTAA ldh-10A-oddT-sense (Sequence number: 65) TTCTTATATATATATCCCAGTTCGCGAATGTTCGCTG ldh-10A-oddT-antisense (Sequence number: 66) AAACAGCGCACATTCGCGAACTGGGAATATATATATATA ldh-10A-evenT-sense (Sequence number: 67) TTCTAATATATATATTCCAGTTCGCGAATGTTCGCTG ldh-10A-evenT-antisense (Sequence number: 68) AAACAGCGCACATTCGCGAACTGGGAATATATATATAA.

[0124] Then, pHSB06XP was cut with BspQI and linked with annealing oligo in the same manner as in Example 1 to produce pHSB10A1TP, pHSB10A3TP, pHSB10A9TP, pHSB10A10TP, pHSB10AoddTP, and pHSB10AevenTP.

[0125] (Example 12) Effects of gRNA containing an additional sequence substituted with another nucleotide on genome editing Using the plasmid prepared in Example 11, the genome editing efficiency when a continuous sequence of the same nucleotide was substituted with another nucleotide was evaluated. Specifically, the genome editing efficiency was determined using pHSB10AP, pHSB10A1TP, pHSB10A3TP, pHSB10A9TP, pHSB10A10TP, pHSB10AoddTP, or pHSB10AevenTP using the same method as in Example 5. As a result, as shown in Figure 5, it became clear that genome editing is possible even when one of the continuous sequences of the same nucleotide is interrupted by substitution with another nucleotide. Furthermore, it was suggested that genome editing is induced even when 50% of the additional sequence is substituted with another nucleotide.

[0126] Therefore, it was suggested that genome editing can be induced not only by adding a base to the 5' end of a gRNA, but also by adding a base to a sequence consisting of multiple types of nucleotides.

[0127] (Example 13) Genome editing of Lactiplantybacillus plantarum Rec strain with enhanced repair activity We also attempted genome editing of Lactiplantybacillus plantarum Rec strain with enhanced repair activity using gRNA with a nucleotide added to the 5' end as described above. When this strain is cultured in the presence of a synthetic peptide (IP-673), SppK on the cell membrane is activated, and the phosphate group of SppK is transferred to the regulator (SppR). Then, the phosphorylated SppR turns on the recE / T promoter (PsppA), thereby enhancing the repair activity.

[0128] First, the Lactiplantibacillus plantarum Rec strain was cultured in MRS medium containing 10 μg / mL chloramphenicol at 37°C for 20 hours. Then, this medium was further cultured in fresh MRS medium containing 0.75 M sorbitol, 2% glycine, and 10 μg / mL chloramphenicol at 37°C until the turbidity was 0.3 to 0.6. Synthetic peptide (IP-673, MAGNSSNFIHKIKQIFTHR) was added to this medium to a final concentration of 100 ng / mL, and the culture was continued at 37°C for another 30 minutes. The genome editing efficiency was determined using pHSB06XP, pHSB0P, pHSB10TP, pHSB30TP, pHSB10GP, pHSB30GP, pHSB10CP, and pHSB30CP, using the same method as in Example 5, except that the Lactiplantybacillus plantarum WCFS1 strain was replaced with the Lactiplantybacillus plantarum Rec strain.

[0129] As a result, as shown in Figure 6, colonies were not formed when gRNA without nucleotides was used, similar to Example 3, indicating that DNA cleavage is lethally toxic in the *Lactipruntilabacillus plantarum* Rec strain. On the other hand, genome editing was not possible with gRNA with cytosine added to the *Lactipruntilabacillus plantarum* WCFS1 strain (Figure 3), but genome editing was possible in the *Lactipruntilabacillus plantarum* Rec strain. This suggests that the toxicity caused by DNA cleavage was mitigated by enhancing repair activity.

[0130] (Example 14) Construction of a plasmid encoding a native bimolecule gRNA Similar to the single-molecule gRNA (sgRNA) shown in the above example, a bimolecule gRNA (a combination of crRNA and tracrRNA) was constructed by adding a nucleotide to the 5' end of the spacer sequence, and genome editing in prokaryotic cells using this plasmid was evaluated.

[0131] First, a plasmid expressing native bimolecule gRNA was constructed. Specifically, using an expression system for crRNA and tracrRNA encoded by pCas9 (Addgene #42876), a plasmid containing two cassettes whose expression is regulated by a PslpA promoter sequence was synthesized so that these RNAs are transcribed separately. In the first expression cassette, in order to express crRNA, two direct repeats derived from pCas9, a site for inserting a spacer sequence located between them (a site with a BsaI recognition sequence), the downstream sequence of the direct repeats, and a sequence derived from the downstream sequence of the sgRNA scaffold of pHSB06X are arranged downstream of the PslpA promoter sequence of pHSB06X. In the second expression cassette, tracrRNA derived from pCas9 and its downstream sequence 200bp are arranged, and their expression is regulated by a different PslpA promoter sequence than that described above. In this plasmid expressing bimolecular gRNA, a second expression cassette is located downstream of the first expression cassette. Furthermore, an ApaI recognition sequence is added to the 5' end of the former, and a SalI recognition sequence is added to the 3' end of the latter. A plasmid expressing this native bimolecular gRNA was then synthesized, and the plasmid was transformed into E. coli and amplified.

[0132] Next, in order to transfer the expression cassette to pHSB06X, the plasmid was cut with ApaI and SalI to prepare a fragment of approximately 1000 bp containing the expression cassette. pHSB06X was also cut with ApaI and SalI to prepare a fragment of approximately 12 kb. Restriction enzyme digestion was performed by mixing DNA solution (400 ng in the case of plasmid), 15 U of restriction enzyme stock solution, 1.0 μL of 10× buffer, 1.0 μL of 10× BSA if necessary, and sterile water to a total volume of 10 μL, and incubating in a 37°C incubator for 1 hour to overnight.

[0133] These two fragments were linked by a ligation reaction to produce a plasmid (pHSB06X-dR1) encoding the native bimolecule gRNA expression cassette. The ligation reaction was performed by mixing 1.0 μL of DNA solution containing the plasmid, 1.0 μL of DNA solution containing the expression cassette, and 2.0 μL of 2×Ligation Mix (manufactured by NIPPON GENE), and incubating in a 16°C incubator for 1 hour.

[0134] pHSB06X-dR1 was introduced into E. coli XL10-Gold by the Hanahan method to obtain the transformed strain E. coli XL10-Gold (pHSB06X-dR1), and the plasmid was recovered. For plasmid recovery, first, 200 μL of Resussion Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) 500 μL was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate containing the plasmid recovered in this manner was stored.

[0135] Next, 0.5 μL each of oligoDNA (100 μM) containing the sense and antisense strands of a spacer sequence targeting the lactate dehydrogenase gene (ldh) of Lactiplantybacillus plantarum WCFS1 strain, 1.0 μL of 10× annealing buffer, and 8.0 μL of sterile water were mixed. Using a thermal cycler, the mixture was incubated at 95°C for 5 minutes, and then the temperature was lowered to 25°C over 90 minutes using the slope function to anneal the two oligoDNAs. The annealed oligoDNA combinations were <SEQ ID NO: 69> and <SEQ ID NO: 70>, and <SEQ ID NO: 71> and <SEQ ID NO: 72>. Furthermore, the 10x annealing buffer was prepared by mixing 2 mL of 1 M Tris acetate (pH 7.8), 1 mL of 1 M magnesium acetate, 2.5 mL of 4 M potassium glutamate, 0.2 mL of 50 mM NAD, 1 mL of 1 M ammonium sulfate, and 3.3 mL of distilled water. dR1-ldh-sense (Sequence number: 69) AAACTCCCAGTTCGCGAATGTCGCTAG dR1-ldh-antisense (Sequence number: 70) AAAACTAGCGGAACATCGCGGAACTGGA dR1-ldh-10A-sense (Sequence number: 71) AAAAAAAAAAAATCCCAGTTCGCGAATGTCGCTAG dR1-ldh-10A-antisense (Sequence number: 72) AAAACTAGCGGAACATCGCGGAACTGGGAATTTTTTTTTTTTT.

[0136] pHSB06X-dR1 was digested with BsaI to obtain a fragment of approximately 12kb. Restriction enzyme digestion was performed by mixing DNA solution (400ng in the case of plasmids), 15U of restriction enzyme stock solution, 1.0μL of 10× buffer, 1.0μL of 10× BSA if necessary, and sterile water to a total volume of 10μL, and incubating in a 37°C incubator for 1 hour to overnight. The digested pHSB06X-dR1 and annealing oligonucleotides were ligated to produce pHSB0-dR1 and pHSB10A-dR1. The ligation reaction was performed by mixing 0.4μL of vector-side DNA solution, 0.6μL of insert-side DNA solution, and 1.0μL of 2× Ligation Mix (manufactured by NIPPON GENE) and incubating in a 16°C incubator for 1 hour.

[0137] pHSB0-dR1 and pHSB10A-dR1 were introduced into E. coli XL10-Gold using the Hanahan method to obtain transformed strains E. coli XL10-Gold (pHSB0-dR1) and E. coli XL10-Gold (pHSB10A-dR1), and the plasmids were recovered. For plasmid recovery, first, 200 μL of Resuscitation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, and the mixture was centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifugating, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, the supernatant of the centrifuged lysate was applied to the column, and it was centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. 500 μL of Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied to the column, and it was centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. 750 μL of Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column, and it was centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. With nothing applied to the column, it was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate containing the plasmid recovered in this manner was stored.

[0138] pHSB06X-dR1, pHSB0-dR1, and pHSB10A-dR1 were digested with ApaI to prepare fragments of approximately 12 kb. Restriction enzyme digestion was performed by mixing DNA solution (400 ng in the case of plasmids), 15 U of restriction enzyme stock solution, 1.0 μL of 10x buffer, 1.0 μL of 10x BSA if necessary, and sterile water to a total volume of 10 μL, and incubating in a 37°C incubator for 1 hour to overnight.

[0139] Using the genomic DNA of Lactiplantybacillus plantarum WCFS1 strain as a template, approximately 1 kb of DNA fragments were amplified by PCR using primers <SEQ ID NO: 73> and <SEQ ID NO: 6>, and <SEQ ID NO: 7> and <SEQ ID NO: 74> to amplify two gRNA homologous arms-1 and-2. In this process, as in Example 1, nucleotides 603 to 608 of the ldh ORF (SEQ ID NO: 9) were modified from TAAGG to GTGAT. dR-ldh-1-fw (Sequence number: 73) CTTTTTCTAAACTAGATGTTTGCCTAAACCCAACGACATCATTTC ldh-1-rv (Sequence number: 6) CAGAAAACGCCCTTGTTTATCAGCCGACATC ldh-2-fw (Sequence number: 7) AACAAGGCGTTTCTGAACGAAAGAATTTTAGC dR-ldh-2-rv (Sequence number: 74) TAAAAGCCTTTGTTAAAGTGGGCGTTTTTCAGTTTTTGTTCGTTTG.

[0140] The PCR reaction conditions were as follows: 2.0 μL of template DNA solution, 0.2 μL of KOD FX Neo (Toyobo), 5.0 μL of 2× PCR Buffer for KOD FX Neo (Toyobo), 2.0 μL of 2 mM each of dNTP Mix, 0.4 μL each of 10 μM forward and reverse primers, and 0.4 μL of sterile water were mixed. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, then denatured at 98°C for 10 seconds, annealed at an appropriate temperature for 30 seconds, and extended at 68°C for 30 seconds per kb plus a 30-second buffer time. This process was repeated for 25-35 cycles, after which the mixture was stored at 4°C.

[0141] pHSB06XP-dR1, pHSB0P-dR1, and pHSB10AP-dR1 were prepared by inserting PCR reaction products (approximately 1 kb each) into the ApaI cleavage sites of pHSB06X-dR1, pHSB0-dR1, and pHSB10A-dR1, respectively, via infusion reaction. The infusion reaction solution was based on 0.4 μL of 5×In-Fusion Mix (TaKaRa), 0.8 μL of vector DNA solution, and 0.4 μL of insert DNA solution. The mixtures were incubated at 50°C for 15 minutes using a thermal cycler or incubator.

[0142] pHSB06X-dR1, pHSB0-dR1, or pHSB10A-dR1 were introduced into E. coli XL10-Gold by the Hanahan method to obtain transformed E. coli XL10-Gold strains (pHSB0XP, pHSB0P), and the plasmids were recovered. For plasmid recovery, first, 200 μL of Resussion Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) containing RNase A, cooled at 4°C, was added, and the precipitate was suspended by vortexing. Then, 200 μL of Lysis Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μL of Neutralization / Binding Buffer (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added and mixed by inversion, then centrifuged at 4°C and 13,000 rpm for 10 minutes. While the lysate was centrifuging, the column was prepared by setting it in a collection tube, applying 500 μL of Column Preparation Solution (GenElute Plasmid Miniprep Kit, Sigma-Aldrich) to the column, and centrifuging at 4°C and 13,000 rpm for 1 minute. The filtrate was discarded, and the supernatant of the centrifuged lysate was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, then the filtrate was discarded. Wash Solution 1 (GenElute plasmid miniprep kit, Sigma-Aldrich) 500 μL was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. Wash Solution 2 (GenElute plasmid miniprep kit, Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 4°C and 13,000 rpm for 1 minute, and the filtrate was discarded. A column with nothing applied was centrifuged at 4°C and 13,000 rpm for 1 minute, and the membrane was dried. The column was transferred to a 1.5 mL collection tube, 40 μL of Elution Buffer (GenElute plasmid miniprep kit, Sigma-Aldrich) was applied, and the column was centrifuged at 4°C and 13,000 rpm for 1 minute.The filtrate was returned to the column and centrifuged at 4°C and 13,000 rpm for 1 minute. The filtrate containing the plasmid recovered in this manner was stored.

[0143] (Example 15) Genome editing efficiency of nucleotide-added bimolecular gRNA The genome editing efficiency of nucleotide-added bimolecular gRNA was evaluated using the plasmid prepared in Example 14. Specifically, the genome editing efficiency was determined using pHSB06XP-dR1, pHSB0P-dR1, and pHSB10AP-dR1 using the same method as in Example 5. As shown in Figure 8, it was revealed that genome editing is possible even with nucleotide-added bimolecular gRNA. Furthermore, it was revealed that the genome editing efficiency was higher compared to bimolecular gRNA without nucleotide addition.

[0144] As described above, the present invention makes it possible to efficiently produce genome-edited prokaryotic cells. Therefore, by utilizing such prokaryotic cells as a site for producing useful substances, the present invention will contribute to various industrial fields such as medicine, food, energy, and the environment.

Claims

1. A method for producing genome-edited prokaryotic cells, comprising the steps of introducing a gRNA having at least one nucleotide attached to the 5' end of a spacer sequence, or a DNA construct for expressing said gRNA, and a Cas protein, or a DNA construct for expressing said protein, into prokaryotic cells.

2. The manufacturing method according to claim 1, wherein an oligonucleotide consisting of at least three nucleotides is attached to the 5' end of the spacer sequence.

3. The manufacturing method according to claim 1, wherein an oligonucleotide consisting of 3 to 40 nucleotides is attached to the 5' end of the spacer sequence.

4. The manufacturing method according to claim 1, wherein an oligonucleotide consisting of at least 10 identical nucleotides, or an oligonucleotide in which 50% or less of the oligonucleotides are replaced with other nucleotides, is attached to the 5' end of the spacer sequence.

5. The manufacturing method according to claim 4, wherein the same nucleotide is adenine.

6. The manufacturing method according to any one of claims 1 to 5, wherein the Cas protein is a Cas9 protein.

7. The manufacturing method according to any one of claims 1 to 5, further comprising the steps of: culturing prokaryotic cells into which the DNA construct for expressing the gRNA and the Cas protein or a DNA construct for expressing the protein have been introduced in liquid under conditions corresponding to the selection marker gene; and culturing the liquid-cultured prokaryotic cells in solid form under conditions corresponding to the selection marker gene to select genome-edited prokaryotic cells.

8. A DNA construct for encoding a gRNA in which at least one nucleotide is attached to the 5' end of a spacer sequence, and for expressing said gRNA in a prokaryotic cell.

9. The DNA construct according to claim 8, wherein an oligonucleotide consisting of at least three nucleotides is attached to the 5' end of the spacer sequence.

10. The DNA construct according to claim 8, wherein an oligonucleotide consisting of 3 to 40 nucleotides is attached to the 5' end of the spacer sequence.

11. The DNA construct according to claim 8, wherein an oligonucleotide consisting of at least 10 identical nucleotides, or an oligonucleotide in which 50% or less of the oligonucleotides are replaced by other nucleotides, is attached to the 5' end of the spacer sequence.

12. The DNA construct according to claim 11, wherein the same nucleotide is adenine.

13. The DNA construct according to any one of claims 8 to 11, further comprising a DNA construct that encodes a Cas protein and expresses said protein.

14. The DNA construct according to claim 13, wherein the Cas protein is the Cas9 protein.

15. The DNA construct according to any one of claims 8 to 11, further comprising a selection marker gene.

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