JP2013017473A - Quick and highly efficient genome modification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: a genome modification method excellent in quickness, easiness, selectivity and efficiency and applicable to automation; and a nucleic acid construct to be used in the method.SOLUTION: The nucleic acid construct to be used for the modification of a genome in a cell contains a gene sequence encoding a nucleoside kinase, preferably thymidine kinase as a gene sequence for selecting a cell in which a genome is modified by the nucleic acid construct. There is further provided a method for modifying the genome in a cell by the use of the nucleic acid construct.

Description

  The present invention relates to a nucleic acid construct used for modifying a genome in a cell and a method for modifying a genome in a cell comprising using the nucleic acid construct. More particularly, the present invention is used for modification of an intracellular genome, including a gene sequence encoding a nucleoside kinase, preferably thymidine kinase, as a gene sequence for selecting cells whose genome has been modified by a nucleic acid construct. Nucleic acid constructs, as well as methods for modifying the genome in a cell comprising using the nucleic acid constructs.

  Gene expression control systems are extremely important as basic tools for protein production, metabolic engineering, and synthetic biology. Biotechnology using a gene expression control system is used in various fields such as mass production of useful proteins, metabolic engineering, and whole cell biosensors. Regardless of whether it is a natural product or an artificially created one, there is a need for a technique for producing a gene expression control system having desired characteristics on demand and quickly.

  Recently, as a technique for arbitrarily rewriting the genome of a cell, a technique for improving the efficiency of gene recombination using the λRED system has been reported (Non-patent Document 1).

  If you simply want to knock-out a gene, you can simply replace the target gene with a selector gene using a homologous recombination system. Cells having genomes with replaced genes can be selected by using a positive selector.

  On the other hand, when it is desired to repeatedly knock out a plurality of gene loci, it is necessary to “extract” the selector cassette containing the selector gene introduced in the initial genome sequence editing operation. This is because if the positive selector is still introduced at the first editing site, it will not be possible to select recombinant clones using the same selection principle in the next genome sequence editing operation. If the selector gene is simply “removed”, it is possible to remove the selector gene by P1 transduction. However, in addition to the selector gene, the selector cassette contains an FRT sequence (FLT-recombination target sequence) that joins the genomic gene and the selector gene, so this method removes the selector gene. FRT sequence remains. That is, every time a knockout operation is performed, this FRT sequence remains in the genome (FIG. 1 and Non-Patent Document 2). In this case, when a gene located at a close locus is knocked out, recombination may occur due to an unexpected combination of FRT sequences. Therefore, after completion of the knockout operation, it is necessary to select a cell in which the desired recombination has occurred, and there is a problem in the speed and ease of the operation.

  In addition, it is not desirable that the selector cassette remains even when the genome is rewritten with an accuracy of one base level.

  In order to rewrite the genome with high accuracy, at least the following two steps are required: (1) introduction of a selector gene into a target locus (locus); and (2) insertion of a replacement sequence following the step (1). In step (1), positive selection is performed by the incorporated selector gene. In step (2), since the integrated selector gene is replaced with a replacement sequence and lost, negative selection is performed to concentrate clones in which the selector gene is lost. For example, when rewriting a genome using a drug resistance gene as a selector gene, at least the following two steps are performed: (1) The drug resistance gene is incorporated at the target position of the genome and cells are selected by drug resistance (positive) (Selection); Thereafter, (2) the gene containing the target information is replaced with the drug resistance gene, and the cells are selected based on drug sensitivity (negative selection). More specifically, a tetracycline resistance gene is used as a drug resistance gene capable of positive selection and negative selection, and a method for modifying a genome including the above two steps is performed by combining with this gene recombination technique. However, this method requires several days for selection in order to perform positive selection and negative selection based on the growth rate difference of cells depending on the presence or absence of a tetracycline resistance gene in a selection medium, and false positives contained in the final product. The colony retains the ability to grow.

  In large-scale genome editing and / or processing, it is necessary to rewrite various loci repeatedly or in parallel. Therefore, development of a genome modification method that can support automation using robotics and a selector system used in the method are expected (Non-patent Document 3).

  To date, several selector systems have been reported (Table 1). However, each selector system has its merits and demerits, and a selector system excellent in quickness, ease, selectivity, efficiency, and automation has not been developed yet.

K. A. Datsenko and B. L. Wanner: Proc Natl Acad Sci U S A, 97, 6640 (2000) F. Buchholzand M. Bishop: Biotechniques, 31, 906 (2001) H. H. Wang, F. J. Isaacs, P. A. Carr, Z. Z. Sun, G. Xu, C. R. Forest and G. M. Church: Nature, 460, 894 (2009) J. E. Karlinsey: Methods Enzymol, 421, 199 (2007) H. Mizoguchi, K. Tanaka-Masuda and H. Mori: Biosci Biotechnol Biochem, 71, 2905 (2007) K. C. Murphy, K. G. Campellone and A. R. Poteete: Gene, 246, 321 (2000) M. Hashimoto, T. Ichimura, H. Mizoguchi, K. Tanaka, K. Fujimitsu, K. Keyamura, T. Ote, T. Yamakawa, Y. Yamazaki, H. Mori, T. Katayama and J. Kato: Mol Microbiol , 55, 137 (2005) R. Heermann, T. Zeppenfeld and K. Jung: Microb Cell Fact, 7, 14 (2008) Y. Tashiro, H. Fukutomi, K. Terakubo, K. Saito and D. Umeno: Nucleic Acids Res, 39, e12 (2011)

  In the conventional genetic engineering techniques that have been performed to arbitrarily rewrite the genome of a cell, as described above, there is room for improvement in the selector to be used, and quickness, ease, selectivity, efficiency, and There was a problem with automation.

  An object of the present invention is to provide a method for modifying a genome which is excellent in rapidity, ease, selectivity and efficiency, and can be applied to automation, and a nucleic acid construct used therefor.

In order to solve the above-mentioned problems, the present inventors have paid attention to a selection marker used for selection of a cell whose genome has been modified by a nucleic acid construct, and have conducted intensive research on searching for a useful selection marker. An important property as a selection marker is that it has a dual selector function. It is desirable in the target gene writing operation that one selector gene has both a positive selection function and a negative selection function. This is because, by first performing positive selection, the inactive mutation of the negative selector, which is the largest cause of false positive in negative selection, can be removed. Every cell accumulates replication errors every 10 ^9-10 bases. Many genome engineering of selecting recombinant clones from the cell, reaches ¹⁰⁹ size selection for cell pools. It is calculated that cells having a mutation that inactivates the negative selector gene are present in a pool of this size by about ~ 100. The probability of random amino acid substitutions eradicating the function of the enzyme gene is 20-50%, so for a protein consisting of 300 amino acids, the 60-150 amino acid locus, ie the 180-450 locus, is lost once and the negative selector is lost. Make it live. In fact, this adds an inactivating mutation to the regulator site of the gene. An example of a selectable marker having this property is tetracycline excretion protein (TetA). Although its utility has not been shown, since TetA has both positive and negative selection functions, it is possible to remove or dilute inactivated negative selectors from the cell pool (FIG. 2). Moreover, the property desired for the selection marker is the quickness of the selection operation. For example, the above-described negative selection using TetA needs to be performed by stationary culture on a selected solid medium for a very long time of about 1-3 days. In addition, known selectors other than TetA can also be used for cell growth rates. Since this is a selection technique based on the difference between the two, it takes 1-2 days for each selection operation. Furthermore, a property desired for the selection marker is that a selection operation in a liquid is possible. This condition is an important advantage in automation and the like. Furthermore, as a property of the selection marker, it is preferable that its function is dependent on the conditions of the selection operation. Since the negative selector is basically a toxic gene, there is a fundamental problem in maintaining stability in the cell. Therefore, it is preferred that the condition be “conditional” that is only exerted in the presence of a chemical or physical initiator that exhibits its toxicity.

  The present inventors have developed a cell expressing a thymidine kinase (HSVTK) derived from human herpes simplexvirus (HSVTK) as a mutagenic nucleoside such as 6- (β-D-2-deoxyribo-furanosyl) -3,4- Cell death occurs in the presence of dihydro-8H-pyrimido [4,5-c] [1,2] oxazin-7-one (hereinafter abbreviated as dP), whereas cells that do not express HSVTK Has already been found (Non-patent Document 9). By using HSVTK as a negative selector, the genome modification method according to the present invention, which is excellent in rapidity, ease, selectivity, and efficiency and can be applied to automation, has been completed.

That is, the present invention relates to:
1. A method of modifying the genome of a cell,
(1) A nucleic acid construct used for modification of a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. Introducing a nucleic acid construct to be used for genome modification into a cell;
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying a genome in a cell, comprising:
2. A method of modifying the genome of a cell,
(1) A nucleic acid construct used for modification of a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct Introducing into the cell a nucleic acid construct used for modification of the genome in the cell;
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying a genome in a cell, comprising:
3. In addition to the gene sequence encoding thymidine kinase, the genome in the cell of the above 1 or 2 comprising a gene sequence encoding a drug resistance protein as a gene sequence for selecting a cell having a modified genome. How to modify,
4. The method of modifying the intracellular genome of the above 3, wherein the drug resistant protein is chloramphenicol acetyltransferase,
5. A method for altering the genome of a cell,
(1) A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. And a step of introducing a nucleic acid construct used for modification of the genome in the cell, comprising a gene sequence encoding chloramphenicol acetyltransferase as a gene sequence for selecting a cell whose genome has been further modified. ,
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying a genome in a cell, comprising:
6. A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. A nucleic acid construct used for the modification of
7. A nucleic acid construct used for modification of an intracellular genome according to the above 6, wherein the gene sequence encoding thymidine kinase is a gene sequence encoding human herpesvirus-derived thymidine kinase,
8. In addition to the gene sequence encoding thymidine kinase, the gene sequence encoding the drug resistance protein as a gene sequence for selecting cells having a modified genome, and Nucleic acid constructs used for modification,
9. A nucleic acid construct used for modification of an intracellular genome according to the above 8, wherein the drug resistance protein is chloramphenicol acetyltransferase;
10. A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct, A nucleic acid construct used for modifying the genome in the cell, further comprising a gene sequence encoding chloramphenicol acetyltransferase as a gene sequence for selecting cells whose genome has been modified;
11. A nucleic acid construct used for modifying a genome in a cell, comprising DNA represented by the base sequence described in any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence listing;
12. Use of the nucleic acid construct according to 11. above, wherein the cell is a microorganism, in modifying the genome in the cell;
13. Use of the nucleic acid construct according to the above 12., wherein the cell is Escherichia coli or cyanobacteria, in modifying the genome in the cell.

  According to the present invention, a nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct, and the nucleic acid construct Can be used to provide a method of modifying the genome in a cell.

  Unlike the conventional method, the genome modification method according to the present invention uses a negative selection method using HSVTK and a mutagenic nucleoside, for example, dP. In this method, when the HSVtk gene is in an activated state, In addition, almost 100% selectivity is realized by a mechanism that loses the proliferation ability, and the selection operation is completed in about 5 minutes because it is a selection method that does not depend on cell proliferation. In other words, the present invention provides a genome modification method that overcomes the problem of long selection time and false positive colonies contained in the final product that retain the growth ability, which was a disadvantage of the method using a drug resistance gene such as a tetracycline resistance gene. Is possible.

  As described above, according to the present invention, it is possible to provide a genome modification method that can improve the speed, ease, selectivity, and efficiency, which are problems of the conventional genome modification methods, and can be applied to automation.

It is a figure explaining the genome deletion method (nonpatent literature 2) using Flp / Cre system. It is a figure explaining the generation mechanism of the false positive by the inactivation of HSVtk gene, and its prescription. It is a selector plasmid map. HSVTK acts as a positive selector and a negative selector (System 1). (Example 1) It is a selector plasmid map. HSVtk and cat are located in tandem and downstream of Plac (System 2). (Example 1) It is a selector plasmid map. HSVtk and cat are located in tandem downstream of PT5 / lac (System 3). (Example 1) It is a figure which shows the outline | summary of the gene recombination experiment using System 1, 2, and 3 (System-1, -2, and -3). A selector gene is knocked in at the target site of the wild-type genome in the cell (indicated as wt in the figure), and a cell having a genome in which the selector gene is knocked in by positive selection (indicated as knock-in in the figure) Next, the gene (gfp _uv ) encoding the green fluorescent protein and the selector gene are replaced (replacement in the figure), and the selector gene is replaced by negative selection (represented as replaced in the figure) Select cells with (Example 2) It is a figure which shows the experimental outline and result of dT / HSVTK selection for recombinant isolation | separation. Panel (A) shows the experimental outline. HSVtk cassettes with homology arms to the reading frame of lacZ were electroporated into cells and allowed to stand overnight on a TK selection plate to form colonies. Panel (B) shows the number of colonies that have grown on the selective medium. (Example 2) Overview of the experimental procedure (panel (A)) and recombination scheme (panel (B) where a selector cassette containing the HSVtk gene and the cat gene is introduced into the lacZ locus, and then these selector genes are replaced with the gene encoding the target protein. )). (Example 2) It is a figure explaining the mechanism of "another" recombination reaction which may occur. (Example 2) It is a figure which shows the uptake activity of dP by HSVTK introduce | transduced into the genome. Panel (A) shows the outline of the operation, and panel (B) shows the decrease in colony forming units (CFU) by dP treatment. (Example 2) It is a figure which shows the result of the search of the selection conditions of HSVTK + strain | stump | stock in SOB culture medium. (Example 2) It is a figure which shows the viability (viability) change of the HSVTK possession stock | strain by the short time dP process in a liquid medium. (Example 2) It is a figure which shows the gene introduction | transduction scheme (panel (A)) of HSVtk-cat cassette to a lacZ locus, and an experimental operation outline (panel (B)). (Example 3) It is a figure which shows concentration of the Cm resistant clone using a CAT activity. (Example 3) In the HSVtk-cat cassette introduction experiment, it is a figure which shows the result of the genotype analysis of the colon_bacillus | E._coli clone which has grown on Cm (+) plate. Panel (A) shows the experimental design of genotype analysis. The site to which the primer binds is indicated by an arrow, and the site to be amplified is indicated by a dotted line. Panels (B) and (C) show the results of genotyping by gel electrophoresis of DNA fragments PCR amplified from the genomic DNA of each clone. Samples (Sample) 1-3 is, LacZ (-), a Cm ^R, clones that showed the phenotype dP ^S, samples ^{4-6, LacZ (-), Cm R} , shows the phenotype of dP ^R Clone. C1 is MG1655, and C2 is MG1655ΔlacZ :: HSVtk-cat. Furthermore, Sample 7 is a LacZ (+), Cm ^R, clones that showed the phenotype dP ^R. (Example 3) It is a schematic diagram which shows the genotype of the HSVtk site | part in 3 clones which have lost dP sensitivity among the HSVtk-cat cassettes introduce | transduced into the genome. (Example 3) It is a figure which shows the scheme (panel (A)) of gene recombination in the exclusion of HSVtk-cat introduce | transduced into the genome, and experimental operation outline (panel (B)). (Example 3) It is a figure which shows the enrichment of the clone which shows LacZ activity using HSVTK / dP system. (Example 3) In the reintroduction experiment of lacZ cassette, it is a figure which shows the result of the genotype analysis of the clone which grew on dP (+) plate. Panel (A) shows the experimental design of the reintroduction experiment. The site to which the primer binds is indicated by an arrow, and the site to be amplified is indicated by a dotted line. Panels (B) and (C) show the results of genotyping by gel electrophoresis of DNA fragments PCR amplified from the genomic DNA of each clone. C1 is MG1655, sample 1-4 is a clone showing the phenotype of LacZ (-), dP ^R , Cm ^R , sample 5-8 is a clone showing the phenotype of LacZ (+), dP ^R , Cm ^S , C2 is a clone showing the phenotypes of MG1655ΔlacZ :: HSVtk-cat and samples 9-15 are LacZ (−), dP ^R and Cm ^S. (Example 3) It is a schematic diagram which shows the genotype of the HSVtk site | part in the clone which shows dP resistance among HSVtk-cat cassettes which were not removed from the genome. (Example 3) It is a figure which shows that the genome recombinant was able to be concentrated in a short time by negative selection in liquid phase operation by HSVTK / dP system. (Example 4) In HSVTK / dP system, it is a figure which shows that dP does not affect the gene mutation frequency of the cell which does not have an HSVtk gene. The left panel shows the outline of the experimental procedure, and the right panel shows the measurement results of the gene mutation frequency. (Example 5) In HSVTK / dP system, it is a figure which shows that HSVTK expressed in the cell does not affect gene mutation frequency. The left panel shows the outline of the experimental operation, and the upper right panel and the lower right panel show the measurement results of cell growth rate and gene mutation frequency, respectively. (Example 5) It is a figure which shows the recombination scheme (panel (A)) and experimental operation outline (panel (B)) in the experiment for introducing the P _λ -gfpmut3.1 cassette into the lacZ locus. (Example 6) It is a diagram showing the concentration of genomic recombinants by dP in replacement experiments to _P λ -gfpmut3.1 cassette of the lacZ gene on the genome. (Example 6) In the experiment of replacing the lacZ gene on the genome with the P _λ -gfpmut3.1 cassette, it is a diagram showing the results of genotype analysis of Cm ^S clones grown on dP (+) plates. Panel (A) shows the experimental design of genotype analysis. The site to which the primer binds is indicated by an arrow, and the site to be amplified is indicated by a dotted line. Panel (B) shows the results of genotyping by gel electrophoresis of DNA fragments PCR amplified from the genomic DNA of each clone. Samples 1-8 LacZ (-), Cm ^S, clones that showed the phenotype dP ^R, C1 is MG1655, C2 is MG1655ΔlacZ :: HSVtk-ca. (Example 6)

  The present invention relates to a nucleic acid construct used for modifying a genome in a cell and a method for modifying a genome in a cell comprising using the nucleic acid construct.

  In the present invention, the term “genome” means the entire nucleic acid sequence of a chromosome contained in a cell constituting an organism or a DNA constituting an entire chromosome.

  The term “genome modification” or “genome modification” means that a part of a nucleic acid sequence in the genome is recombined with a foreign nucleic acid sequence, that is, a genome having a nucleic acid sequence different from the genome before the modification is created. That means. For example, knocking out a specific gene sequence in the genome, replacing a part of the nucleic acid sequence in the genome with a gene encoding the target protein, inserting a gene encoding the target protein in the genome, and a promoter And control sequences, and partial rewriting of proteins.

  The term “nucleic acid construct” refers to a construct comprising a nucleic acid molecule (eg, DNA, RNA, DNA / RNA). As long as the nucleic acid construct used in the present invention is a nucleic acid construct used for modification of an intracellular genome, any nucleic acid construct can be used, and conventionally used nucleic acid constructs for genome modification that have been conventionally used. Can be used.

  The nucleic acid construct according to the present invention is characterized by a gene encoding a selection marker for selecting a cell whose genome has been modified by the nucleic acid construct. That is, in the present invention, the nucleic acid construct includes a gene sequence encoding a nucleoside kinase, preferably thymidine kinase (hereinafter sometimes abbreviated as TK) as a gene sequence encoding a selection marker.

  The term “selection marker” is used herein interchangeably with the term “selector” and refers to an indicator for selecting cells whose genome has been modified by a nucleic acid construct. More specifically, the “selector” is a protein encoded by a gene sequence constituting a part of a nucleic acid construct, and refers to an index for selecting a cell whose genome has been modified by the nucleic acid construct.

  The selector is roughly divided into a positive selector and a negative selector. “Positive selector” refers to a cell that cannot survive unless the selector is expressed in the presence of a selective agent. On the other hand, “negative selector” refers to those in which the expression of the selector is lethal to cells in the presence of a selective agent, and those that cause growth inhibition. A selector having both a positive selector function and a negative selector function is referred to as a “dual selector”. The dual selector can exhibit the function of a positive selector or the function of a negative selector, for example, depending on the type of the selective agent used.

  As the selector, any protein can be used as long as it is resistant to a substance that causes cell death in a short period of time, or is a protein involved in cell death caused by the substance or a protein that enhances sensitivity to the substance. Specific examples of such substances include gene denaturing agents, alkylating agents, organic solvents, ultraviolet / radiation, heat, acids / alkalis, oxidizing agents, and the like. Specific examples of the selector include nucleoside kinases such as thymidine kinase, drug resistant proteins, and alkylated DNA repair enzymes. The selector is not limited to these examples, and any selector can be used as long as it is involved in cell death or avoidance of cell death.

  The term “selective agent” is an agent used in combination with a selector to select cells whose genome has been modified, a substance capable of inducing cell death under the expression of the selector, preferably a compound, or the gene sequence Means a substance capable of inducing cell death under non-expression, preferably a drug containing the compound.

  The term “positive selection” means selecting cells in which the selector gene is expressed. In other words, the term “positive selection” means selection by removing cells that do not express the selector gene under conditions where the cells survive if the selector gene is expressed. For positive selection, a positive selector gene is placed in the nucleic acid construct to be introduced into the cell. Cells that do not express the positive selector gene are removed under conditions where the cells survive if the positive selector gene is expressed. Substances that cause cell death in a short period of time include alkylating agents, organic solvents, UV / radiation, heat, acids / alkalis, oxidizing agents, protein synthesis inhibitors, and the like. In addition, a protein synthesis inhibitor, a gene conferring resistance to these can be used as a positive selector gene.

  The term “negative selection” means selecting cells that do not express the selector gene. In other words, the term “negative selection” means selection by removing cells expressing the selector gene under conditions where the cells survive if the selector gene is not expressed. In order to perform negative selection, a negative selector gene is placed in a nucleic acid construct to be introduced into a cell. Cells expressing the negative selector gene are removed under conditions that cause cell death if the negative selector gene is expressed. Substances that cause cell death in a short period of time include gene denaturants, alkylating agents, organic solvents, UV / radiation, heat, acids / alkalis, oxidizing agents, protein synthesis inhibitors, and the like. A gene involved in cell death caused by these or a gene that increases sensitivity to these can be used as a negative selector gene.

  The term “dual selection” means performing a combination of positive selection and negative selection. In dual selection, either positive selection or negative selection may be performed first, but positive selection is preferably performed first. Dual selection may be performed only once or multiple times. Preferably, the dual selection is performed a plurality of times in succession. By performing the treatment a plurality of times, cells having a desired genomic modification can be selectively obtained.

  The nucleic acid construct used for modifying the genome in the cell according to the present invention contains a gene sequence encoding a nucleoside kinase, preferably thymidine kinase, as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. It is characterized by. In addition to the gene sequence encoding nucleoside kinase, the nucleic acid construct according to the present invention can include a further selector gene sequence, for example, a gene sequence encoding a drug resistance protein and a gene sequence encoding an alkylated DNA repair enzyme.

  As a nucleic acid construct used for modification of an intracellular genome according to the present invention, a nucleic acid construct comprising a DNA represented by the nucleotide sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence listing Can be preferably exemplified.

  Nucleoside kinases refer to enzymes that catalyze the phosphorylation of nucleosides by adenosine triphosphate (ATP). Nucleoside kinases form part of the salvage pathway of nucleotide synthesis and play an important role as regulators of DNA synthesis. Examples of the nucleoside kinase include thymidine kinase, adenosine kinase, guanosine kinase, and deoxycytidine kinase.

  A preferable example of the nucleoside kinase used in the present invention is thymidine kinase. The thymidine kinase is not particularly limited, and examples thereof include thymidine kinase derived from mammalian cells and viruses, and human herpes virus (human herpes simplex virus) thymidine kinase (HSVTK) can be exemplified more preferably.

  Thymidine kinase is an enzyme that catalyzes phosphorylation of deoxythymidine and plays an important role as a regulator of DNA synthesis. Intracellular supply of deoxythymidine triphosphate (dTTP), a direct precursor of DNA synthesis, is mediated by the de novo and salvage pathways. The de novo pathway synthesizes dTTP via deoxythymidine monophosphate (dTMP) synthesis, which is known to stop when 5-fluorouracil is added (SS Cohen, JG Flaks, HD Barner, MR Loeb and MR J. Lichtenstein: Proc Natl Acad Sci U SA, 44, 1004 (1958). E. Yagil and A. Rosner: J Bacteriol, 108, 760 (1971).). 5-Fluorouracil and its derivatives are metabolized intracellularly to become 5-fluoro-2'-deoxyuridine monophosphate (5FdUMP). 5FdUMP is an inhibitor of dTMP synthase (ThyA), and its presence inhibits intracellular dTMP synthesis. Under this circumstance, cell growth relies on the salvage pathway to synthesize dTTP using exogenous deoxythymidine (dT). If the cells have TK, they can synthesize dTMP from dT via the salvage pathway, but can survive without TK. When TK is introduced into a TK-deficient cell line, its salvage pathway is restored. Utilizing this mechanism, active function selection methods for thymidine kinase and related enzyme activities have been studied (ME Black, TG Newcomb, HM Wilson and LA Loeb: Proc NatlAcad Sci USA, 93, 3525 (1996). ).).

  Positive selection can be performed by using thymidine kinase as the selector and an inhibitor of the de novo synthetic pathway of deoxythymidine triphosphate (dTTP) as the selection agent. In this case, since cell growth depends on the salvage pathway, positive selection is performed in the presence of dT. An inhibitor of deoxythymidine triphosphate (dTTP) de novo synthesis pathway is not particularly limited as long as it is a compound that can inhibit dTTP production by inhibiting the de novo synthesis pathway. For example, it is involved in the de novo synthesis pathway. Mention may be made of enzyme inhibitors. Specifically, 5-fluorouracil and derivatives thereof can be preferably exemplified. More specifically, 5-fluoro-2′-deoxyuridine (5FdU) can be exemplified. The concentration of 5-fluorouracil and its derivative may be any concentration that can inhibit the de novo synthesis pathway of dTTP, and can be determined by simple repeated experiments. For example, 5FdU is used at a concentration of 10 μg / mL-50 μg / mL, more preferably 20 μg / mL. The incubation time between the dTTP de novo synthesis pathway inhibitor and the cells is not particularly limited, and can be determined by simple repeated experiments. In selection using an inhibitor of the de novo synthesis pathway of dTTP, it takes time to proliferate viable cells, and therefore, time corresponding to the growth rate of the cells to be used is required. For example, when selection is performed using an E. coli strain in the presence of 5FdU, the incubation is performed for 6 hours to 20 hours, preferably 20 hours.

  On the other hand, it is known that various nucleoside analogs are activated in cells by nucleotide metabolizing enzymes such as thymidine kinase and cause cell death. Thymidine kinase has long been studied as a suicide gene for gene therapy. (ME Black and LA Loeb: Biochemistry, 32, 11618 (1993). DKDube, ME Black, KM Munir and LA Loeb: Gene, 137, 41 (1993)).

  Examples of compounds capable of inducing cell death under the expression of thymidine kinase include mutagenic nucleosides. Mutagenic nucleosides are incorporated into the genome via the thymidine salvage pathway, causing gene mutations and inducing cell death.

Negative selection can be performed by using thymidine kinase as a selector and using, for example, a mutagenic nucleoside as a selection agent. The mutagenic nucleoside is not particularly limited as long as it induces cell death by mutating the gene when incorporated into the gene, and may be a naturally occurring mutagenic nucleoside, which is artificially It may be produced. Specifically, the artificial nucleoside 6- (β-D-2-deoxyribo-furanosyl) -3,4-dihydro-8H-pyrimido [4,5-c] [1,2] oxazin-7-one ( A preferred example is dP). Like other nucleosides, dP is incorporated into the genome via the thymidine salvage pathway. Many other toxic nucleosides are chain terminators of chromosomal DNA synthesis (K. Negishi, D. Maehara, S. Nakamura, D. Loakes, L. Worth, Jr., RM Schaaper, K. Seio, M. Sekine and T. Negishi: Nucleic Acids Res Suppl, 221 (2001). Therefore, cells that express thymidine kinase are induced to die by the addition of dP. However, in cells lacking thymidine kinase, no death is induced by the addition of dP. The genotoxicity of dP is low, and only after giving 37 μM dP, only 80% of the E. coli cell population die (K. Negishi, D. Loakes and RMSchaaper: Genetics, 161, 1363 (2002)). . However, cells that express thymidine kinase do not physically destroy chromosomes due to the addition of dP, but lose their ability to grow in a short period of 5 to 15 minutes because genetic information is irreversibly degraded. . The concentration of the mutagenic nucleoside is not particularly limited as long as it is a concentration that causes mutation in the gene and induces cell death when incorporated into the gene, and can be determined by simple repeated experiments. For example, dP is used at a concentration of 50 nM-1 μM, more preferably 100 nM. The cell concentration upon treatment with a mutagenic nucleoside is not particularly limited and can be determined by simple repeated experiments. For example, 10 ⁵ cells / mL to 10 ⁹ cells / mL, more preferably 10 ⁶ cells / mL to 10 ⁸ cells / mL, and even more preferably about 10 ⁷ cells / mL are appropriate. In addition, since cells in the logarithmic growth phase are more sensitive to the drug, it is preferable to use cells in the logarithmic growth phase. The incubation time between the mutagenic nucleoside and the cell is not particularly limited, and can be determined by simple repeated experiments. Since cell death caused by mutagenic nucleoside mutation is induced in a very short time, for example, when dP is used, incubation of dP with cells is performed for 5 minutes to 12 hours, preferably 5 minutes to 60 minutes. More preferably, it may be 5 minutes to 30 minutes, and more preferably 30 minutes. As a medium to be used when cells are treated with a mutagenic nucleoside, a standard medium can be used. For example, in the case of E. coli, examples include LB medium and M9-glucose medium.

  Thymidine kinase can be used as a dual selector as described above. Nucleoside kinases other than thymidine kinase can also be used as a positive selector when used in combination with an agent that inhibits the de novo pathway, and compounds that can induce cell death under the expression of the nucleoside kinase to be used, such as suitable compounds When used in combination with a mutagenic nucleoside, it can be used as a negative selector.

  A drug-resistant protein can be preferably exemplified as the selector. Drug-resistant protein means a protein that can confer resistance to a drug on a cell and avoid cell death in the presence of a drug that causes cell death.

  Drug resistance proteins can therefore be used as positive selectors when used in combination with drugs that cause cell death. The genes encoding drug resistance proteins include chloramphenicol (Cm) resistance gene, kanamycin (Kan) resistance gene, tetracycline (Tet) resistance gene, ampicillin (Amp) resistance gene, neomycin (Neo) resistance gene, and gentamicin ( Gen) resistance gene can be exemplified. An example of the chloramphenicol resistance gene is a chloramphenicol acetyltransferase (cat) gene. When a protein encoded by a drug resistance gene is used as a selector, it is used in combination with a drug that induces cell death when the protein is not expressed. The operation of selecting transformants with drug resistant proteins can be performed under conditions well known to those skilled in the art.

  An example of the selector is an alkylated DNA correcting enzyme. An alkylated DNA modifying enzyme is an enzyme that acts on and cleaves alkylated nucleotides in alkylated DNA. The deoxyribose strand after being cleaved by the alkylated DNA modifying enzyme is further cleaved by AP endonuclease (APE1), and then the DNA at the cleaved site is repaired by DNA polymerase and ligase based on the information of the complementary strand. . When alkylation occurs in genomic DNA, the alkylated nucleotides cause DNA replication inhibition and genetic mutation, resulting in cell death. For example, when cells are treated with the alkylating agent methanesulfonic acid (MMS), MMS crosses the cell membrane and methylates the 3rd position of adenine in the genomic DNA. Methylated adenine causes cell death due to inhibition of DNA replication and gene mutation.

Alkylating DNA repair enzymes can therefore be used as positive selectors when used in combination with alkylating agents (CY Chen, HH Guo, D. Shah, A. Blank, LD Samson and LALoeb: DNA Repair (Amst), 7, 1731 (2008).). Examples of the alkylating DNA correcting enzyme include alkyl adenine DNA glycosidase (AAG) and O6-methylguanine-DNA transferase (MGMT). Alternatively, examples of gene sequences encoding 3-alkyladenine repair enzymes include those derived from E. coli such as AlkA (glycosylase) and AlkB (alkyltransferase). When an alkylated DNA modifying enzyme is used as a selector, it is used in combination with an alkylating agent. Alkylating agents include compounds with molecular structures called alkyl groups such as methyl, ethyl, propyl, etc., and a function to alkylate DNA, that is, a function that acts on DNA to change to a polymer having an alkyl group Means a drug having The alkylating agent is not particularly limited as long as it has an action of alkylating DNA, and any known alkylating agent can be used. Specifically, methanesulfonic acid (MMS) can be preferably exemplified as the alkylating agent. MMS crosses the cell membrane and methylates the 3rd position of adenine in the genomic DNA. Methylated adenine causes cell death due to inhibition of DNA replication and gene mutation. In addition, examples of preferable alkylating agents that can be used include methyl iodide, ethylmethanesulfonic acid (EMS), and N-methyl-N′-nitro-nitrosoguanidine (MNNG). The concentration of the alkylating agent is not particularly limited as long as it has a function of alkylating DNA, and can be determined by simple repeated experiments. For example, MMS is used at a concentration of 0.1% -0.4%, preferably 0.2% -0.3%, more preferably 0.2%, in other words 10 mM-50 mM, preferably 20 mM-40 mM, more preferably 20 mM. Since cell concentration at the time of treatment with alkylating agents, the cell death due to too low of whether alkylating DNA repair enzymes regardless alkylating agent is caused, for example, 10 ⁶ cells / ml to 10 ⁸ cells / mL , more preferably 10 ⁶ cells / ml to 10 ⁷ cells / mL, still more preferably it is suitable about 10 ⁷ cells / mL. In addition, since cells in the logarithmic growth phase are more sensitive to the drug, it is preferable to use cells in the logarithmic growth phase. The incubation time between the alkylating agent and the cells is not particularly limited, and can be determined by simple repeated experiments. Since cell death due to alkylation of DNA by an alkylating agent is induced in a short time, for example, when MMS is used, incubation of MMS with cells is 15 minutes to 45 minutes, preferably 15 minutes to 30 minutes. More preferably, it may be 30 minutes. Conditions for treating alkylation of DNA in cells with an alkylating agent are determined by a combination of the concentration of the alkylating agent, the cell concentration, and the incubation time. Specifically, when MMS is used as the alkylating agent, the number of cells is suitably 10 ⁶ cells / mL to 10 ⁷ cells / mL if the MMS concentration is about 0.2%. Furthermore, when the MMS concentration is about 0.2% and the number of cells is 10 ⁷ cells / mL, the incubation time is suitably 5 minutes to 45 minutes, for example, 30 minutes. The medium used when treating cells with the alkylating agent is preferably a minimal medium composed of only essential substances, for example, M9 medium in the case of Escherichia coli, in order to sufficiently obtain the action of the alkylating agent.

  In addition to the gene sequence encoding the selection marker, the nucleic acid construct can be combined with a gene sequence carrying information on replication and control by a method known per se. Examples of such gene sequences include control sequences operably linked to the gene sequences, such as promoters and enhancers, replication origins, ribosome binding sequences, terminators, signal sequences, and splicing signals. It can be illustrated. One or more kinds of gene sequences selected from these can be incorporated into the nucleic acid construct. For example, a promoter is appropriately selected depending on the type of host cell to be used. Promoter refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a nucleic acid sequence that initiates transcription upon binding of RNA polymerase. When a bacterium is used as a host, any promoter can be used as long as it can be expressed in a host cell such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as λPR promoter, λPL promoter, trp promoter, and lac promoter can be exemplified. An artificially modified promoter such as the trc promoter may be used. When yeast is used as a host, the promoter is not particularly limited as long as it can be expressed in yeast, and any promoter may be used. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like can be exemplified. When an animal cell is used as a host, it is preferable that the recombinant vector is autonomously replicable in the cell, and at the same time is composed of a promoter, an RNA splice site, a target gene, a polyadenylation site, and a transcription termination sequence. Moreover, the replication origin may be included if desired. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like can be used, and a cytomegalovirus early gene promoter and the like may be used.

  The target gene can be further incorporated into the nucleic acid construct according to the present invention so that the target gene is expressed. By introducing the nucleic acid construct into which the target gene is further incorporated into the cell and modifying the genome, the target gene can be expressed in the cell. On the other hand, a desired genome can be knocked out by introducing the nucleic acid construct into which a target gene is not incorporated into a cell and modifying the genome.

  As a method for incorporating a target gene into a nucleic acid construct, a genetic engineering technique known per se can be applied. For example, a method is used in which the target gene is treated with an appropriate restriction enzyme, cleaved at a specific site, then mixed with a similarly treated nucleic acid construct, and religated with ligase. Alternatively, a target gene can be incorporated into a nucleic acid construct by ligating a suitable linker to the target gene and inserting it into a multicloning site of a vector suitable for the purpose.

  The nucleic acid construct according to the present invention can be an expression vector. An expression vector is DNA that carries an external gene to a host cell, in other words, vector DNA that can express a target gene in the host cell. The vector DNA is not particularly limited as long as it can replicate in the host, and is appropriately selected depending on the type of host and intended use. In addition to the vector DNA obtained by extracting naturally occurring DNA, the vector DNA may be a vector DNA in which a part of the DNA other than the part necessary for replication is missing. Representative vector DNAs include, for example, plasmid, bacteriophage and virus-derived vector DNA. Examples of plasmid DNA include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, and the like. Examples of bacteriophage DNA include λ phage. Examples of virus-derived vector DNA include vectors derived from animal viruses such as retrovirus, vaccinia virus, adenovirus, papovavirus, SV40, fowlpox virus, and pseudorabies virus, or vectors derived from insect viruses such as baculovirus. Can do. Other examples include vector DNA derived from a transposon, an insertion element, and a yeast chromosome element. Alternatively, vector DNA prepared by combining these, for example, vector DNA (cosmid, phagemid, etc.) prepared by combining plasmid and bacteriophage genetic elements can be exemplified.

  The nucleic acid construct according to the present invention can be used in modification of an intracellular genome. The modification of the genome in the cell is preferably performed in vitro.

  By using the nucleic acid construct according to the present invention, a method for modifying a genome in a cell can be carried out quickly, selectively and efficiently.

The method for modifying an intracellular genome according to the present invention includes the following steps:
(1) a step of introducing the nucleic acid construct according to the present invention into a cell,
(2) culturing the cells obtained in (1) above, and
(3) A step of selecting a cell having a modified genome after the step (2).

More specifically, the method for modifying an intracellular genome according to the present invention may be a method including the following steps:
(a) A nucleic acid construct (first nucleic acid construct) used for modifying a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. A step of introducing into the cell,
(b) a step of performing positive selection after the step (a) and selecting a cell whose genome has been modified by the first nucleic acid construct,
(c) introducing the nucleic acid construct containing the target gene (second nucleic acid construct) into the cell obtained in (b) above,
(d) A step of performing negative selection after the step (c) and selecting a cell in which the first nucleic acid construct is replaced by the second nucleic acid construct.

In addition, the method for modifying an intracellular genome according to the present invention may be a method including the following steps:
(a) a nucleic acid construct used for modifying a genome in a cell (first sequence), comprising a gene sequence encoding thymidine kinase and a drug resistance gene sequence as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. A nucleic acid construct) in a cell,
(b) a step of performing positive selection after the step (a) and selecting a cell whose genome has been modified by the first nucleic acid construct,
(c) introducing the nucleic acid construct containing the target gene (second nucleic acid construct) into the cell obtained in (b) above,
(d) A step of performing negative selection after the step (c) and selecting a cell in which the first nucleic acid construct is replaced by the second nucleic acid construct.

  Positive selection can be performed by introducing and culturing a nucleic acid construct containing a gene sequence encoding a positive selector into cells, and culturing all or part of the obtained cells in the presence of a selection agent. Cells expressing the positive selector survive in culture in the presence of the selective agent, whereas cells not expressing the positive selector cause cell death in culture in the presence of the selective agent. Therefore, cells that survived by positive selection can be determined to be cells whose genome has been modified by the nucleic acid construct, and cells whose genome has been modified can be selected.

  Negative selection can be performed by introducing a nucleic acid construct containing a gene sequence encoding a negative selector into cells and culturing, and culturing a part of the obtained cells in the presence of a selection agent. Cells that do not express the negative selector survive in culture in the presence of the selective agent, whereas cells that express the negative selector cause cell death in culture in the presence of the selective agent. Therefore, a cell that caused cell death by negative selection can be determined to be a cell whose genome has been modified by the nucleic acid construct, and a cell whose genome has been modified can be selected.

  Positive selection and negative selection can each be performed alone or in combination. When performing a combination of positive selection and negative selection, either may be performed first, but preferably positive selection is performed first. The selection may be performed only once or may be performed a plurality of times. Preferably, it is appropriate to perform the selection again on the selected cells a plurality of times in succession. By performing the treatment a plurality of times, cells having a desired genomic modification can be selectively obtained.

  An inevitable problem with negative selection is the inactivation of the selector by spontaneous mutation. It is inevitable that random mutations occur in the selector at a certain frequency due to spontaneous mutation in the cell. Therefore, cells with inactivated negative selectors survive negative selection, which is the main cause of false positives without recombination with the cassette to be introduced (top row of FIG. 2). The HSVtk gene has an open reading frame that functions as both a positive selector and a negative selector depending on conditions. That is, when HSVTK in a selection cassette is inactivated, cells into which the selection cassette has been introduced cannot survive positive selection under conditions where HSVTK functions as a positive selector. Therefore, if positive selection with HSVTK is performed immediately before transformation, cells having a genome in which HSVTK has been inactivated can be efficiently removed or diluted (lower part of FIG. 2). Therefore, it is preferable to modify the genome after positive selection with HSVTK.

  The nucleic acid construct containing the target gene used in the genome modification method can be prepared by using a method for constructing a targeting vector used for gene transfer into the genome by homologous recombination. The method for constructing the targeting vector is a technique well known in the technical field related to the present invention.

  The term “cell” is a structural and functional basic unit of an organism, and means a living body having a nucleic acid molecule that carries genetic information and is composed of a membrane structure that isolates the outside world and its internal cytoplasm. The present invention is applicable to both prokaryotic cells and isolated eukaryotic cells, preferably to microorganisms such as bacteria, more preferably to E. coli and cyanobacteria, and more preferably to E. coli.

  The term “cell death” in the present invention means a state in which cell functions such as proliferative ability are lost. For example, when cells such as E. coli are cultured on a solid medium, the state in which the ability to grow from a single cell and form a cell colony of a certain size or larger that can be counted visually is lost. In the invention, it is called cell death. The term “cell death” includes active cell death (apoptosis) that actively removes unwanted cells and damaged cells caused by physiological or pathological factors, and passive cell death by reaction to external factors. (Necrosis) may also be included. On the other hand, the term “live cell” means a cell that normally retains functions as a cell such as proliferative ability. For example, when a cell such as E. coli is cultured on a solid medium, it means a cell having the ability to grow from a single cell and form a cell colony having a certain size or larger that can be visually counted.

  When a nucleic acid construct according to the present invention, such as a nucleic acid construct containing an HSVtk gene, is introduced into Escherichia coli, the addition of dP induces cell death in a short period of 5 to 15 minutes and induces cell death, and the presence of 5FdU and dT. Underneath it could grow. In addition, when a nucleic acid construct according to the present invention, for example, a nucleic acid construct containing an HSVtk gene, is introduced into cyanobacteria, the addition of dP causes loss of proliferation ability in a short time and cell death is induced, and 5FdU and dT It was able to grow in the presence. Thus, E. coli and cyanobacteria effectively perform negative selection using dP and positive selection using 5FdU and dT by introducing the nucleic acid construct according to the present invention, for example, a nucleic acid construct containing the HSVtk gene. be able to. Therefore, the present invention can be preferably applied to microorganisms, and the microorganisms can be confirmed to have a cytocidal effect due to the use of TK and dP, and are preferably applicable to Escherichia coli and cyanobacteria, more preferably Escherichia coli.

  The method for introducing the nucleic acid construct into the cell is not particularly limited as long as the nucleic acid construct is introduced into the cell and further allows the modification of the genome in the nucleus, and any known method appropriately selected depending on the cell species. Can also be used. For example, an electroporation method, a calcium phosphate method, a lipofection method, etc. can be illustrated.

  As the conditions for culturing the cells, known conditions appropriately selected according to the cell species are used. Culturing can be performed in either a liquid medium or a solid medium. In addition, since the growth rate varies depending on the cell species, the period and temperature for culturing the cells are selected by selecting appropriate conditions for reaching the desired number of cells from known conditions.

  Selection of cells with a modified genome is performed using a selector and a selection agent contained in the nucleic acid construct used for the modification of the genome. Specifically, cells cultured with the introduction of the nucleic acid construct according to the present invention are cultured in the presence of a selective agent, and the genome is modified using cell death caused by expression of the selector or avoidance of cell death as an index. Cell selection. The cells cultured in the presence of the selection agent may be all or part of the cells cultured by introducing the nucleic acid construct.

  Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited by the following examples.

  A selector plasmid was produced. In the presence of 5FdU, the de novo synthesis pathway of thymidine is stopped and cell death occurs in various cell types. This thymidine-deficient death can be avoided if dT given from the outside can be phosphorylated by thymidine kinase activity. Cells expressing HSVK selectively grow in a medium containing 5FdU and dT (Non-patent Document 9). This mechanism can be used to select the knocked-in selection cassette.

  First, the HSVtk gene as a selector was cassetted alone (FIG. 3-A) or together with the cat gene (positive selector) (FIGS. 3-B and 3-C) and subcloned into a plasmid.

  The resulting selector plasmids are referred to as System 1, System 2, and System 3 (FIGS. 3-A, 3-B, and 3-C).

  System 1 is a selector plasmid for using HSVTK as a dual selector and is called pTrc-HSVtk (FIG. 3-A, Non-Patent Document 9). It was prepared by inserting the HSVtk gene into pTrcHis2-TOPO. The HSVtk gene can be replaced with NcoI-HindIII. It was confirmed that HSVtk in this selector plasmid functions as a dP kinase (negative selector) and functions as a positive selector in TK selection.

  System 2 is a selector plasmid designed for positive selection by CAT and negative selection by HSVTK, and is called pMW-HSVtk_cat (FIG. 3-B). The HSVtk gene and the cat gene were inserted downstream of the lac promoter. Cells transfected with this selector plasmid may exhibit dP sensitivity and Cm resistance in the presence of isopropyl-β-D (-)-thiogalactopyranoside (Isopropyl-β-D (-)-thiogalactopyranoside (IPTG)). confirmed.

  System 3 begins replication of pLTSUB-202 (provided by Dr. R. Weiss: S. Basu, R. Mehreja, S. Thiberge, MTChen and R. Weiss: Proc Natl Acad Sci USA, 101, 6355 (2004)) A plasmid created by amplifying the dot and kanR gene by polymerase chain reaction (PCR) and combining the HSVtk and cat operons of pMW-HSVtk_cat with XhoI and HindIII, and is called pLT-HSVtk_cat (FIG. 3-C) . The expression of HSVtk gene and cat gene is regulated by PT5 / lac promoter. The cat gene can be excised with XbaI-HindIII, and the HSVtk gene can be excised with ClaI-XbaI. It was confirmed that HSVTK in this selector plasmid functions as dP kinase (negative selector) and also functions as a positive selector in Cm selection.

  Using the systems 1 to 3 prepared in Example 1, the genomic gene was modified. An outline of the gene editing experiment is shown in FIG. PCR was performed using a plasmid as a template and an oligonucleotide consisting of about 40 nucleosides that perfectly matched the target genomic site as a primer (Non-patent Document 1). This was electroporated into Escherichia coli into which plasmid pKD46 encoding λRed recombination was introduced, and positive selection was performed to complete introduction of the selector cassette into the genome. Subsequently, the gene sequence to be knocked in was amplified by PCR in the form having the homology arm used in the above step, and electroporated again into the E. coli clone obtained as described above. About 1 μM of dP was added to the obtained Escherichia coli clone to perform negative selection, or the obtained Escherichia coli clone was left on an agar medium containing dP to perform negative selection, and the selector was replaced with the sequence to be introduced.

  Next, the function as a selector of the system 1 and the system 2 was confirmed. The thymidine kinase-deficient E. coli strain JW1226 used was the KEIO collection (T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, KA Datsenko, M. Tomita, BL Wanner and H. Mori: Mol Syst Biol, 2, 2006 0008 (2006).) Unless otherwise specified, LB medium (manufactured by Invitrogen) containing 2% (w / v) LB broth was used as the culture medium. Cells were grown at 37 ° C. in glass test tubes.

(1) Function as a selector of system 1 (Ptrc-HSVtk) The function as a selector of system 1 (Ptrc-HSVtk) was measured as follows (panel (A) in FIG. 5). First, a selector fragment (100 ng) was added to competent cells (corresponding to 20 μL) of E. coli strain JW1226 (Δtdk strain) into which pKD46 had been introduced, and the mixture was left on ice for 10 minutes. Then, it was placed in a 2 mm cuvette (Bio-Rad) and electroporated. Thereafter, 1 mL of SOC medium was added, transferred to a test tube, and cultured with shaking at 37 ° C. and 200 rpm for 1 hour. Subsequently, 400 μL of each TK selection plate (containing IPTG of 0 or 100 μM) was inoculated and incubated at 30 ° C. overnight to form colonies, and colony forming units (CFU) were calculated. As a negative control, the same operation was performed using water instead of the selector fragment.

  The number of colonies formed was almost the same between those transformed with the selector fragment added and those using water instead of the DNA fragment (panel (B) in FIG. 5). This indicates that transformants are not significantly less than E. coli that survived TK selection. In fact, the number of colonies formed appears to increase by several tens of percent in the presence of IPTG. This increase is considered to be an Escherichia coli strain in which the expression of HSVTK under Ptrc is enhanced and its activity survives TK selection. In addition, as described later, the HSVtk-introduced strain can be selectively amplified by culturing in dT / 5FdU medium (FIG. 9), clearly showing that HSVtk can be used as a positive selector.

(2) Gene introduction using system 2 (Plac-HSVtk_cat) System 2 (Plac-HSVtk_cat) was used to introduce genes (panels (A) and (B) in FIG. 6). In System 2, CAT functions as a positive selector and HSVTK functions as a negative selector.

  (2-1) First, HSVtk_CAT was introduced into the lacZ locus (upper panel of panel (A) in FIG. 6). Specifically, a selector fragment (200 ng) was added to a competent cell (corresponding to 50 μL) of E. coli strain MG1655 into which pKD46 had been introduced, and left on ice for 10 minutes. Next, it was placed in a 2 mm cuvette (Bio-rad) and electroporated. Thereafter, 1 mL of SOC medium was added, transferred to a test tube, and cultured with shaking at 30 ° C. and 200 rpm for 1 hour. Subsequently, 100 μL of each plate was inoculated and statically cultured overnight at 30 ° C. in the presence of chloramphenicol.

  When the transformant was inoculated on the plate, 7 positive colonies were obtained. All of them showed resistance to Cm and sensitivity to dP. From this, it was judged that gene transfer was performed in all seven colonies.

  On the other hand, the introduction of the selector gene was confirmed by measuring the function of the lacZ gene that is destroyed by the introduction of the selector gene. For the measurement of the function of the lacZ gene, colonies were formed on X-gal plates, and the determination was made based on the color of the colonies. As a result, three of the seven colonies were white and thus determined to be lacZ-, and the remaining four colonies were determined to be lacZ + because they were blue (Table 2).

  Since the selection cassette of System 2 is dotted with partial sequences of the lac operon present in the plasmid, homologous recombination by another mechanism as shown in FIG. 7 can be established. According to the results of the colony PCR, three of the four colonies showing blue color corresponded to this.

  (2-2) Next, the HSVtk_cat gene on the genome was replaced with the green fluorescent protein (gfp) gene (bottom panel (A) in FIG. 6). Specifically, a selector fragment (200 ng) containing the GFP gene was added to the above-mentioned competent cell of E. coli that had been introduced with HSVtk_CAT into the lacZ locus (equivalent to 50 μL), and left on ice for 10 minutes. Next, it was placed in a 2 mm cuvette (Bio-rad) and electroporated. Thereafter, 1 mL of SOC medium was added, transferred to a test tube, and cultured with shaking at 30 ° C. and 200 rpm for 8 hours. Subsequently, 100 μL of each plate was inoculated and statically cultured overnight at 30 ° C. in the presence of dP.

  As a result, about 10,000 transformants were obtained. Of these, 7 transformants were confirmed to emit green fluorescence. Since all of these seven transformants had Cm resistance, it was determined that the template plasmid used to make the gfp cassette was contaminated.

  Most of the transformants were non-fluorescent. The fact that many of them did not replace the HSVtk_cat gene with the gfp gene is clear from the fact that they had lost dP sensitivity but retained Cm resistance.

  Four out of 120 clones gave Cm-sensitive (Cm Sensitive) and dP-resistant (dP Resistant) results indicating the absence of the cassette (Table 3).

(2-3) Confirmation of function of system 2 (Plac-HSVtk_cat) as a negative selector The function of system 2 (Plac-HSVtK_cat) as a negative selector was measured (panel (A) in FIG. 8). It was confirmed whether cells introduced with the Plac-HSVtK_cat cassette into the genome were efficiently killed by the addition of dP.

First, E. coli strain MG1655 into which pKD46 had been introduced was cultured at 30 ° C. in an LB medium (LB-carb medium) containing carbenicillin. Next, 20 μL of the culture solution was inoculated into LB medium (2 mL) and cultured at 30 ° C. Four hours later, LB-carb medium plates with dP or LB-carb medium plates without dP (both containing 100 μM IPTG) were inoculated. The cells were cultured at 30 ° C. to form colonies, and CFU was calculated (OD ₆₀₀ = 1.4).

As a result of negative selection by addition of dP, the concentration rate was approximately 2 × 10 ⁵ (approximate panel (B) in FIG. 8). From this result, it was shown that the HSVtk gene in the Plac-HSVtk_cat cassette introduced into the genome functions to induce cell death in negative selection by addition of dP. On the other hand, gene transfer into the genome of the cell, if starting from ¹⁰⁹ cells, given from the concentration ratio, considered cell death is not induced is about 5,000 cells. If so, in order to avoid contamination with false positive cells, a higher recombination efficiency is required, for example an efficiency of obtaining 10,000 or more recombinants per transformant. Cells in which this cell death is not induced are presumed to have inactivated mutations in the genomic HSVtk gene before dP treatment. Such false positive cells can be diluted or removed by positive selection utilizing the HSVtk gene in the Plac-HSVtk_cat cassette introduced into the genome.

(2-4) Removal by false selection of false positive cells generated by negative selection An inevitable problem with negative selection is the inactivation of the selector by spontaneous mutation. Regardless of the SacB or HSVTK system, it is inevitable that random mutations will occur in this selector with a certain frequency due to spontaneous mutation in the cell. There are tens to hundreds of gene mutations that disrupt protein functions, such as enzyme function. Thus, in ¹⁰⁹ cells also population, HSVTK genomic inactivated are included considerable number. Once inactivated, the negative selector does not kill the host. Therefore, cells with inactivated negative selectors survive negative selection, which is the main cause of false positives without recombination with the cassette to be introduced (top row of FIG. 2).

  The HSVtk gene has an open reading frame that functions as both a positive selector and a negative selector depending on conditions. That is, when HSVTK in a selection cassette is inactivated, cells into which the selection cassette has been introduced cannot survive positive selection under conditions where HSVTK functions as a positive selector. Therefore, if positive selection with HSVTK is performed immediately before transformation, cells having a genome in which HSVTK has been inactivated can be efficiently removed or diluted (lower part of FIG. 2).

In order to examine whether false positives can be reduced by performing positive selection with HSVTK immediately before transformation, 5FdU / dT selection was performed in SOB medium using an E. coli strain into which the Plac-HSVtk_cat cassette was introduced into the genome. Specifically, JW1226 (Δtdk strain) introduced with pTrc-HSVtk or pTrc-gfp _UV was cultured overnight in 2 mL LB-carb medium in a test tube. Next, 500 μL of SOB medium added with a combination of 5FdU (0 to 100 μg / mL) and dT (0 to 100 μg / mL) was prepared in a 48-well deep well plate and inoculated to be diluted to 1/1000. . Thereafter, the cells were cultured with shaking at 1,000 rpm at 37 ° C., and the OD ₆₀₀ value was measured timely.

  As shown in FIG. 9, it was revealed that in the presence of a sufficient 5FdU concentration (20 μg / mL or more), only an E. coli strain expressing HSVTK (HSVTK + strain) can be concentrated. In addition, the higher the dT concentration, the better the growth of the HSVTK + strain. If electroporation / dP selection is performed on cells that have become competent cells under these conditions, the number of false positives is drastically reduced.

(2-4) Rate of cell death in negative selection using System 2 (Plac-HSVtk_cat) Negative selection using TetRA, which is a conventional method, requires two days of selective culture and There is a problem that the size is extremely small. Therefore, the time required for negative selection using HSVTK was confirmed.

First, a dP-sensitive Escherichia coli strain (MG1655ΔlacZ :: Plac-HSVtk_cat) containing HSVTK was prepared. Subsequently, it was cultured at 37 ° C. in 15 mL of LB medium (LB-Cm-IPTG medium) containing IPTG and chloramphenicol (OD ₆₀₀ = about 0.08). Thereafter, a portion of the culture was taken 5, 15, 30, and 60 minutes after dP was added, and a prescribed amount was plated on an LB plate. Subsequently, the plate was cultured overnight at 37 ° C., and the number of colonies formed per medium volume (CFU) was counted.

  As shown in FIG. 10, the viability of the cells into which HSVTK was introduced into the genome was reduced by about 4-6 orders of magnitude. This is because HSVTK phosphorylates dP, resulting in rapid accumulation of genetic mutations in the genome, resulting in cell death. In this example, a promoter called Plac, which is not so high in transcription efficiency, was used. However, if a stronger promoter, such as PT5 / lac, is placed upstream of the selector cassette HSVtk, it is expected that faster and more efficient selection will be possible. The This dP treatment was completely non-toxic to cells that did not have HSVtk in their genome.

  Using the system 2 (see FIG. 4) prepared in Example 1, the genomic gene was modified. PCR was performed using a plasmid as a template and an oligonucleotide consisting of about 40 nucleosides that perfectly matched the target genomic site as a primer (Non-patent Document 1). This was electroporated into Escherichia coli into which plasmid pKD46 encoding λRed recombination was introduced, and positive selection was performed to complete introduction of the selector cassette into the genome. Subsequently, the gene sequence to be knocked in was amplified by PCR in the form having the homology arm used in the above step, and electroporated again into the E. coli clone obtained as described above. About 1 μM of dP was added to the obtained Escherichia coli clone to perform negative selection, and the selector was replaced with the sequence to be introduced.

(1) Introduction of HSVtk-cat cassette into lacZ domain using CAT
Using CAT activity as a positive selection marker, an experiment was conducted in which the lacZ gene was replaced with the HSVtk-cat cassette (System 2) (panel (A) in FIG. 11). The operation outline is shown in the panel (B) of FIG. First, an HSVtk-cat cassette (PCR product, 150 ng) was electroporated into a competent cell (40 μL) of Escherichia coli MG1655 into which pKD46 had been introduced, using a 1 mm cuvette (manufactured by Bio-Rad). 1 mL of LB-IPTG (0.1 mM) was added and cultured with shaking in a test tube for 4 hours (37 ° C., 200 rpm). A part of this culture solution was inoculated in an LB agar medium (IPTG 0.1 mM, X-gal 40 μg / mL) with / without Cm (30 μg / mL), followed by stationary culture at 37 ° C. overnight.

On the Cm (−) plate, ˜10 ⁹ colonies were obtained per transformation. On the other hand, approximately 10 ⁴ colonies were observed per transformation on the Cm (+) plate. In other words, the efficiency of genomic recombination is estimated as 10 ^{4/10 9} = 10 about ^-5.

  Of the ~ 65 colonies that grew on the Cm (-) plate, all appeared blue on the X-gal plate (ie, showed LacZ activity). On the other hand, 99.7% of the colonies growing on Cm (+) showed white color on the X-gal plate (that is, lost LacZ activity) (FIG. 12).

When 92 LacZ (-) clones on the Cm (+) plate were randomly selected and drug resistance was examined, 89 of them showed resistance to Cm and sensitivity to dP (Table 4). From this, it was judged that in these 89 clones, the introduced HSVtk-cat cassette was replaced with the lacZ gene at the target site as designed in the panel (A) of FIG. In Table 4, dP ^R , dP ^S , Cm ^S , and Cm ^R indicate dP resistance, dP sensitivity, Cm sensitivity, and Cm resistance, respectively.

  Ninety-two LacZ (−) clones were randomly picked and liquid cultured. One LacZ (−) clone was liquid cultured. These were stamp-inoculated on a dP plate and a Cm plate, and the growth was examined based on the presence or absence of colony formation.

  Three clones were randomly selected from colonies showing white color on the Cm (+) plate, and the sequence around the target locus of the genome was examined. Specifically, PCR was performed with a pair of primers that annealed upstream and downstream of the target site (Targetsite) (panel (A) in FIG. 13). The primer sequences used for PCR are shown in Table 5. In Table 5, the base sequences of the primers indicated by the names of F1 to F5 are shown in SEQ ID NOs: 4 to 8, respectively. Moreover, the base sequences of the primers indicated by the names R1 to R5 in Table 5 are shown in SEQ ID NOs: 9 to 13, respectively.

  In all three selected clones, it was confirmed that the HSVtk-cat cassette was introduced at the correct locus (panel (B) in FIG. 13, sample 1-3).

  About 3% of white clones growing on Cm (+) plates did not show dP sensitivity, ie, had dP resistance. In order to examine these identities, three clones were randomly selected and the sequence around the lacZ of the genome was confirmed by PCR. HSVtk-cat gene was introduced into the correct locus in all three clones. (Panel (B) in FIG. 13, Sample 4-6). Analysis of the base sequence of the gene of this PCR product revealed non-synonymous base substitution mutations (with amino acid mutations) such as R → D, R → H, and Y → C within the reading frame of the HSVtk gene. (Figure 14). From this, it was speculated that colonies that had grown on the dP (+) plate despite the LacZ (-) phenotype were inactivated due to harmful mutations in the genomic HSVtk gene .

  Only one blue colony (LacZ (+) clone) has grown on the Cm (+) plate. When this drug was examined for drug resistance, it showed resistance to both Cm and dP (Table 4). From this, it was determined that the replacement of the HSVtk-cat cassette with the lacZ gene did not occur as per the design shown in panel (A) of FIG. In fact, when the sequence around the lacZ gene in the genome was confirmed by PCR, it was confirmed that the lacZ gene was not removed from the blue colonies (LacZ (+) clones) grown on the Cm (+) plate ( Panel (C) in FIG. 13, sample 7).

(2) Reintroduction of lacZ gene fragment using dP kinase activity
Using HSVTK dP kinase activity as a negative selection marker, an experiment was conducted in which the HSVtk-cat cassette (system 2) was replaced with the lacZ gene (panel (A) in FIG. 15). The operation outline is shown in the panel (B) of FIG. First, a lacZ cassette (PCR product, ˜250 ng) was electroporated into a competent cell (40 μL) of E. coli MG1655ΔlacZ :: Plac-HSVtk-cat into which pKD46 was introduced using a 1 mm cuvette (manufactured by Bio-Rad). . LB (1 mL) was added, and cultured with shaking in a test tube for 2 hours (37 ° C., 200 rpm). A part of this culture solution was inoculated in an LB agar medium (IPTG 0.1 mM, X-gal 40 μg / mL) with / without dP (1 microM), and statically cultured at 42 ° C. overnight.

In the dP (−) plate, 10 ⁹ colonies were obtained per transformation. 99.96% of them showed blue color on X-gal plates (showing LacZ (+) activity). 0.04% was white.

On the dP (+) plate there were approximately 10 ⁴ colonies per transformation. Again, the transformation efficiency is estimated to be around ^10-5 . 84% of them showed a blue color on the X-gal plate (ie, showed LacZ activity) (FIG. 16).

By randomly selecting 46 LacZ (+) clones on the dP (+) plate, inoculating the stamp on the dP and Cm plates, and measuring their viability (presence of colony formation) When drug resistance was examined, all of them showed dP resistance and lost Cm resistance (Table 6). In Table 6, dP ^R , dP ^S , Cm ^S , and Cm ^R indicate dP resistance, dP sensitivity, Cm sensitivity, and Cm resistance, respectively.

  From this, it was judged that in all 46 clones, the introduced lacZ cassette was replaced with the HSVtk-cat cassette at the target site as designed in the panel (A) of FIG.

  To confirm that the introduced lacZ cassette replaced the HSVtk-cat cassette in the LacZ (+) clone on the dP (+) plate, four colonies from the blue color on the dP (+) plate were used. A clone was randomly selected, and the base sequence around the target sequence for introduction of the lacZ gene in the genome was confirmed by PCR. Specifically, PCR was performed by pairing a primer that anneals upstream and downstream of the target sequence with a primer that anneals inside the HSVtk-cat or lacZ gene (panel (A) in FIG. 17). In PCR, the primers shown in Table 5 were used. In all four cases, it was confirmed that the lacZ gene fragment was introduced into the correct locus (panel (B) in FIG. 17, sample 1-4).

  Next, the identity of the white colony (LacZ (−) clone) that had come on the dP plate was examined. First, 46 white colonies were selected and examined for drug resistance, all of which showed dP resistance. That is, all of these lost HSVTK gene activity. However, 30 clones of these showed Cm resistance (Table 6). From this, it is presumed that these white colonies (LacZ (−) clones) have lost the function of the HSVtk gene although the HSVtk-cat cassette has not been removed.

  In fact, when a PCR test was performed, white colonies (lacZ (-) clones) that had grown on the dP (+) plate were confirmed to have no HSVtk-cat cassette removed (panel (B) in FIG. )), Sample 5-8). When the HSVtk gene part of the PCR product was sequenced, amino acid residue deletions or non-synonymous base substitution mutations (including amino acid mutations) such as Y → C and R → H were found in each of the three clones. (FIG. 18, Sample 5-7). In addition, there was a clone in which no gene mutation occurred in the HSVtk gene despite showing dP resistance (FIG. 18, sample 8). The reason why this clone is dP resistant is unknown.

As described above, in the genome recombination experiment in which the HSVtk-cat cassette is replaced with the lacZ cassette, the genome recombination frequency is about 10 ⁻⁵ . By growing on dP (+) plates, the correct genomic recombinants could be concentrated to about 81% of the total. On the other hand, 19% of the “growing” colonies were not correct genomic recombinants, and many of them were inactivated clones with mutations in the genomic HSVtk gene. It was found that due to loss of dP kinase activity, the HSVtk-cat cassette was spared from dP selection without being removed.

  On the other hand, among the white colonies (LacZ (−) clones) that had grown on the dP (+) plate, 7 clones that showed dP resistance and lost resistance to Cm were also found. It is speculated that the functions of both HSVtk and cat genes have been lost.

  When an actual PCR test was performed, it was confirmed that one of these seven clones did not have the HSVtk-cat cassette removed (panel (C) in FIG. 17, sample 9). When the base sequence of the cat gene of this clone was examined, no base substitution mutation occurred. From this, it was found that this clone lost its Cm resistance even though it retained the activity of the cat gene. However, it is unclear why this clone lost Cm resistance. On the other hand, in the remaining 6 clones, the lacZ gene was not introduced even though the HSVtk-cat cassette was removed (panel (C) in FIG. 17, sample 10-15). The reason for removal of the HSVtk-cat cassette is unknown.

  Research needs to modify bacterial genomes repeatedly are increasing. In order to modify the bacterial genome over and over again, it is required that the method of genome recombination can be performed only by 1) liquid phase operation and 2) its operation time is short. Therefore, the negative selection method using HSVTK / dP was investigated 1) that the genomic recombinants could be concentrated by liquid phase operation, and 2) the operation time required for the enrichment of the genomic recombinants. Specifically, the examination was performed as follows.

  First, a lacZ cassette (PCR product: 500 ng) was electroporated into a competent cell (corresponding to 50 μL) of E. coli MG1655Δ :: HSVtk-cat into which pKD46 had been introduced, using a 1 mm cuvette (manufactured by Bio-Rad). LB (1 mL) was added, and cultured with shaking in a test tube for 3 hours (37 ° C., 200 rpm). 100 μL of this culture solution was added to 10 mL of LB-IPTG (0.1 mM) -dP (1000 nM) and cultured with shaking (37 ° C., 200 rpm). After 0.5, 1, 2, 3, 4, 6, and 8 hours, a part of the culture solution was inoculated into an LB-IPTG-X-gal plate and cultured at 42 ° C. overnight.

Immediately after dP treatment, genomic recombinants showing a blue color on X-gal plates were obtained with an efficiency of ˜10 ⁴ per transformation (˜0.01% of total cells). After 4 hours of treatment with 1000 nM dP, the genomic recombinants were enriched 6000 times, reaching ~ 60% of the total cells (Figure 19). From this, it was confirmed that the HSVTK / dP system enables 1) genome recombinants to be negatively selected by liquid phase operation, and 2) completes the enrichment of genome recombinants in as little as 4 hours. It could be confirmed.

  The effect of the HSVTK / dP system on the frequency of genetic mutations in cells was examined. Since dP is a mutagenic nucleoside, random gene mutations can occur in the genome if it is incorporated into the genome. If the phosphorylation of dP occurs due to the effects of various kinases in the cell, significant randomness is also observed in genomic recombinants that do not have the HSVtk gene (negative selectable marker) when cells are treated with dP. Mutations will accumulate. Therefore, we estimated how much dP affects the gene mutation frequency of cells without HSVtk. In addition, there are trace amounts of naturally occurring mutagenic nucleosides that are damaged by oxidation (such as 8-oxoguanine), deamination (such as uracil), and alkylation (such as 3-methyladenine). Exists. If HSVTK is expressed and phosphorylated and incorporated into genomic DNA (or because of secondary reasons such as imbalance in nucleic acid metabolism), there is a concern that the frequency of genetic mutations in cells will increase without giving dP. is there. Thus, the effect of HSVTK expression on the frequency of cellular gene mutations was estimated. Specifically, the following experiment was conducted.

(1) Effect of dP treatment on gene mutation frequency of cells without HSVtk gene First, E. coli MG1655 introduced with pKD46 was cultured in LB-Amp medium. 30 μL of the culture solution was inoculated into 30 mL of LB medium. 4 mL of this culture solution was fractionated, and dP was added so as to be 0-10 ³ nM. Dispense each culture broth into 1.2 mL tubes, shake culture (37 ° C, 200 rpm) for 6 hours, and part of the culture broth rifampicin (hereinafter abbreviated as rif) 50 μg / mL Each was inoculated on an agar medium containing / not containing and incubated at 37 ° C. overnight (left panel in FIG. 20). CFU was calculated from the obtained colonies, and the gene mutation frequency defined by the following mathematical formula (1) was calculated.

At a dP concentration of 100 nM or less, no significant increase in gene mutation frequency could be confirmed (right panel in FIG. 20). On the other hand, at the dP concentration of 1000 nM, the gene mutation frequency increased about 10 times compared to the case of the dP concentration of 0 nM. At this time, a base substitution mutation occurs in the genome with a probability of 2 × 10 ⁸ bp. Since the genome of Escherichia coli is approximately 4 Mbps, the probability that a genetic mutation occurs anywhere in the genome is 8%. That is, approximately 8% of the cell population is a clone having a genetic mutation somewhere in the genome. From this, it was found that after a series of genome recombination operations, it is unlikely that an E. coli clone having a genetic mutation caused by dP treatment was isolated.

(2) Effects of HSVTK expressed in cells on gene mutation frequency
E. coli MG1655 and MG1655ΔlacZ :: HSVtk-cat into which pKD46 was introduced were cultured overnight in LB-Amp medium. After inoculating 25 μL of the culture solution into 25 mL of LB medium (IPTG 0.1 mM, Arabinose 10 mM), shaking culture (37 ° C., 200 rpm), and containing rif (50 μg / mL) after 0, 4, and 8 hours / Each strain was inoculated into an agar medium not containing it, and cultured at 37 ° C. CFU was calculated from the obtained colonies, and the gene mutation frequency was defined by the above mathematical formula (1) and calculated.

  The cell growth rate of both cells expressing HSVTK and cells not expressing HSVTK did not change (upper right panel in FIG. 21). At this time, the gene mutation frequency was not changed at all after 8 hours of culture (lower right panel in FIG. 21). Therefore, we concluded that the HSVtk gene is an excellent negative selection marker that does not cause genomic destabilization in the absence of dP and has mutagenic properties only in the presence of dP.

The introduction of P _λ -gfpmut3.1 into the lacZ region by sequential positive / negative selection in liquid medium was examined.

CAT activity as positive selection marker, using dP kinase activity HSVTK as a negative selection marker, a lacZ gene region is replaced by _P λ -gfpmut3.1 cassette (panel of FIG. 22 (A)). The operation outline is shown in the panel (B) of FIG. First, competent cells (40 μL) of E. coli MG1655 into which pKD46 had been introduced were electroporated with an HSVtk-cat cassette (PCR product˜150 ng) using a 1 mm cuvette (Bio-Rad). 1 mL of LB-IPTG (0.1 mM) was added and cultured with shaking in a test tube for 3 hours (37 ° C., 200 rpm). Next, 20 μL of the culture solution diluted 100 times was inoculated into 2 mL of LB-Amp-Cm (30 μg / mL) -IPTG (0.1 mM) medium, and cultured with shaking in a test tube for 25 hours (30 ° C., 200 rpm). did. 20 μL of this culture solution was inoculated into 2 mL of LB-Amp-Cm (30 μg / mL) -IPTG (0.1 mM) medium, and cultured with shaking (30 ° C., 200 rpm) for 12 hours in a test tube. 20 μL of the obtained culture solution was inoculated into 20 mL of LB-Amp-Arab (10 mM) -Cm (30 μg / mL) -IPTG (0.1 mM), medium, and shake culture in a test tube for 6 hours (30 ° C., 200 rpm )did. The culture broth was collected by centrifugation and washed twice with 10 mL of sterilized water and 10 wt / v% glycerol solution, respectively, and then added with a 20 wt / v% glycerol solution to produce a competent cell. This competent cell (40 μL) was electroporated with a P _λ -gfpmut3.1 cassette using a 1 mm cuvette (manufactured by Bio-Rad). LB (1 mL) was added, and cultured with shaking in a test tube for 3 hours (37 ° C., 200 rpm). Finally, each was inoculated in an LB agar medium (containing IPTG 0.1 mM, X-gal 40 μg / mL) with / without dP (1 μM), followed by stationary culture at 37 ° C. overnight.

On the dP (+) plate, approximately 10 ⁴ colonies were seen per transformation. 19% of them showed a blue color on the X-gal plate. That is, it showed LacZ activity (FIG. 23). No colonies showing green fluorescence were observed. On the other hand, 10 ⁹ colonies were obtained per transformation on the dP (−) plate. 99.993% of them showed blue color on the X-gal plate (showing LacZ (+) activity).

When 94 LacZ (-) clones on the dP (+) plate were randomly selected and examined for drug resistance, all of them showed dP resistance. Of these, 63 clones lost Cm resistance. The colonies of these 64 clones showed green fluorescence. From these, it was judged that in 63 clones, the introduced P _λ -gfpmut3.1 cassette was replaced with the HSVtk-cat cassette at the target site as designed in the panel (A) of FIG. .

In order to confirm that the P _λ -gfpmut3.1 cassette was replaced with the HSVtk-cat cassette at the target site, 8 clones out of 63 clones that showed LacZ (-), Cm ^S phenotype Was randomly selected, and the base sequence around the target sequence of the P _λ -gfpmut3.1 cassette introduced into the genome was confirmed by PCR. Specifically, PCR was performed by pairing the upstream and downstream of the target sequence with a primer that annealed to the inside of the P _λ -gfpmut3.1 cassette (panel (A) in FIG. 24). In PCR, the primers shown in Table 5 were used. In seven of the eight clones, it was confirmed that _P λ -gfpmut3.1 cassette is introduced into the right locus (panel of FIG. 24 (B), samples 1-3 and 5-8) . The remaining two clones, although HSVtk-cat cassette is _removed, P λ -gfpmut3.1 cassette is found to have not been introduced as designed as shown in panel FIG. 22 (A) (Fig. 24 Panel (B), sample 4). The reason for this is unknown.

  In basic research and industry, techniques for rewriting bacterial genomes are widely used. According to the present invention, free modification of the genome can be performed efficiently in a short time. In the present genome modification method, false-positive products are not generated in principle, so that a person skilled in the art can easily and efficiently carry out modification of the target genome. Furthermore, this genome modification method can be applied to large-scale parallelization by robotics, and a significant cost reduction such as time and labor costs can be expected. In addition, since it does not select the species that can be used, it is a very versatile basic technology that enables genome modification in a wide variety of cells.

  The main applications of the present invention are preferably the following three applications in bioscience / biotechnology: 1) Applications to basic research and applied research using genomic engineering. To analyze the role of a specific gene on the genome, change the promoter strength by precise substitution with single-base writing accuracy (promoter strength modulation), or incorporate a new externally operable promoter to express the gene It is possible to manipulate the timing arbitrarily (temporal localization control). Furthermore, it enables the investigation and analysis of spatial localization by fusion of fluorescent probes, and the elucidation of the mechanism of spatial localization control of arbitrary genes by fusion with membranes and organelle localization protein / tag. In addition, as an example of application to pharmaceutical research, elucidation of the pathogenic expression mechanism by recombination of a gene producing a highly toxic substance in the genome of a pathogenic bacterium with a reporter gene or a detoxified gene. 2) Use for mass production of useful proteins. Research and industry that make cells useful for humans in large quantities are becoming increasingly popular. However, when a large amount of substance is made, there are many cases in which a plurality of genes responsible for the defense reaction inherent in the cell interfere with it. The expression level (promoter site rewriting) and in situ rewriting of enzyme activity and function (introduction of useful mutations into the reading frame) can be transformed into a useful protein producer that is easy to use; and 3) Use for DNA genome marking. As in the applications 1) and 2) above, the demand for technology that rewrites genomes into cells useful for research and industry will continue to increase. Along with this, the introduction of barcode-like technology that writes water marking to cells to be managed in factories and research institutes, that is, a label such as a serial number, into the genome is attracting attention. According to the present invention, since an arbitrary sequence can be incorporated at an arbitrary site on the genome, the present invention can be said to be an optimal technique for water marking.

  In the above three applications in bioscience / biotechnology, genome rewriting technology is an indispensable technology. The present invention provides a genome engineering method having high writing accuracy, high writing speed, high writing efficiency, and easy operability, and has a great ripple effect in various fields.

SEQ ID NO: 1: A designed expression plasmid designated pTrc-HSVtk, in which the human herpesvirus thymidine kinase (HSVtk) gene is located downstream of the trc promoter.
SEQ ID NO: 2: A designed expression plasmid called pMW-HSVtk_cat, in which a human herpesvirus thymidine kinase (HSVtk) gene and a chloramphenicol acetyltransferase (cat) gene are arranged downstream of the lac promoter.
SEQ ID NO: 3: A designed expression plasmid called pLT-HSVtk_cat in which the human herpesvirus thymidine kinase (HSVtk) gene and the chloramphenicol acetyltransferase (cat) gene are arranged downstream of the PT5 / lac promoter.
SEQ ID NO 4: Oligonucleotide designed for primer.
SEQ ID NO: 5: oligonucleotide designed for primer.
SEQ ID NO: 6: oligonucleotide designed for primer.
SEQ ID NO: 7: oligonucleotide designed for primer.
SEQ ID NO: 8: oligonucleotide designed for primer.
SEQ ID NO: 9: oligonucleotide designed for primer.
SEQ ID NO: 10: oligonucleotide designed for primer.
SEQ ID NO: 11: oligonucleotide designed for primer.
SEQ ID NO: 12: oligonucleotide designed for primer.
SEQ ID NO: 13: oligonucleotide designed for primer.

Claims (13)

A method for modifying a genome in a cell,
(1) A nucleic acid construct used for modification of a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. Introducing a nucleic acid construct to be used for genome modification into a cell;
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying an intracellular genome. A method for modifying a genome in a cell,
(1) A nucleic acid construct used for modification of a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct Introducing into the cell a nucleic acid construct used for modification of the genome in the cell;
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying an intracellular genome. 3. In addition to the gene sequence encoding thymidine kinase, the gene sequence encoding a drug resistance protein is included as a gene sequence for selecting cells whose genome has been modified. how to. 4. The method for modifying an intracellular genome according to claim 3, wherein the drug resistance protein is chloramphenicol acetyltransferase. A method for modifying a genome in a cell,
(1) A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. And a step of introducing a nucleic acid construct used for modification of the genome in the cell, comprising a gene sequence encoding chloramphenicol acetyltransferase as a gene sequence for selecting a cell whose genome has been further modified. ,
(2) culturing the cells obtained in (1) above, and
(3) After the step (2), a step of selecting a cell whose genome has been modified,
A method for modifying an intracellular genome. A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct. Nucleic acid constructs used in 7. The nucleic acid construct used for modification of an intracellular genome according to claim 6, wherein the gene sequence encoding thymidine kinase is a gene sequence encoding human herpesvirus-derived thymidine kinase. The modification of the genome in a cell according to claim 6 or 7, comprising a gene sequence encoding a drug resistance protein as a gene sequence for selecting a cell having a modified genome in addition to the gene sequence encoding thymidine kinase. Nucleic acid constructs used in 9. The nucleic acid construct used for modification of an intracellular genome according to claim 8, wherein the drug resistance protein is chloramphenicol acetyltransferase. A nucleic acid construct used for modifying a genome in a cell, comprising a gene sequence encoding human herpesvirus-derived thymidine kinase as a gene sequence for selecting a cell whose genome has been modified by the nucleic acid construct, A nucleic acid construct used for modification of a genome in a cell, comprising a gene sequence encoding chloramphenicol acetyltransferase as a gene sequence for selecting a cell in which is modified. A nucleic acid construct used for modifying a genome in a cell, comprising a DNA represented by the nucleotide sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence listing. Use of a nucleic acid construct according to claim 11 in the modification of the genome in a cell, wherein the cell is a microorganism. Use of the nucleic acid construct according to claim 12, wherein the cell is Escherichia coli or cyanobacteria, in modification of an intracellular genome.