JP2019154244A - Compositions for accumulating effector proteins to target genes and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for accumulating an effector protein in a target gene having a high effect of accumulating an effector protein in the target gene. A composition for accumulating an effector protein in a target gene includes a guide RNA that specifically binds to the target gene in a first region, a Cas protein, and a protein that binds to a second region of the guide RNA. , A fusion with a peptide that serves as a tag for binding a plurality of first effector proteins, and a plurality of the above-mentioned first effector proteins. [Selection diagram] Fig. 1

Description

  The present invention relates to a composition and kit for accumulating an effector protein in a target gene, and a method for producing a cell or mammal in which the effector protein is accumulated in a target gene.

  A technique is known in which an effector protein is accumulated in a target gene to alter / modify the activity or the like of the target gene.

  For example, in the field of gene expression activation technology, a transcriptional activator is allowed to act site-specifically by fusing a transcriptional activator to dCas9 protein inactivated nuclease activity of Cas9 used for genome editing. Thus, systems that activate the expression of target genes have been developed.

  In the first generation artificial gene expression enhancing system described above, there is a problem that only one molecule of transcriptional activator acts on one target site and there is often no sufficient gene expression enhancing effect. On the other hand, a second generation artificial gene expression enhancing system that allows a plurality of transcriptional activators to act on one target site has been developed (see FIG. 1A).

  For example, Non-Patent Document 1 reports a SAM (Synergistic Activation Mediator) system (hereinafter simply referred to as SAM). In SAM, a transcription activator fused with MS2 coat protein is called into the target site by adding a motif recognized by MS2 coat protein to the loop part of sgRNA. As a result, the synergistic effect of the transcriptional activator called at the target site and the transcriptional activator directly fused to dCas9 has a gene expression enhancing effect compared to the first generation artificial gene expression enhancing system. It is higher (see FIG. 1B).

  Non-Patent Document 2 reports a SunTag system (hereinafter simply referred to as SunTag). In SunTag, a plurality of antigens called SunTag are fused to dCas9, whereby transcription activators fused with an antibody scFv that recognizes the antigen are accumulated at the target site. As a result, the gene expression enhancing effect is higher than that of the first generation artificial gene expression enhancing system.

  Non-Patent Document 3 reports that when SAM and SunTag are compared, SAM is relatively superior, but there is no significant difference. Further, even when SAM and SunTag are combined, a stronger gene expression enhancing effect than the effect of each of them alone is not observed.

  In addition to the gene expression activation technique described above, a technique has been developed that alters / modifies the expression of a target gene or the like according to the purpose by using another effector protein instead of a transcriptional activator.

  For example, Non-Patent Document 4 reports a system for efficiently labeling a specific chromosomal region using GFP as an effector protein.

  Non-Patent Document 5 reports a system that increases the efficiency of DNA demethylation using TET1 as an effector protein.

  Non-Patent Document 6 reports a system that improves the efficiency of base substitution using deaminase as an effector protein.

  Non-Patent Document 7 reports a system that increases the efficiency of histone acetylation using p300cd as an effector protein.

Konermann S et al. (2015) "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex", Nature, 517, pp.583-588 Tanenbaum ME et al. (2014) "A protein-tagging system for signal amplification in gene expression and fluorescence imaging", Cell, 159, pp.635-646 Chavez A et al. (2016) "Comparison of Cas9 activators in multiple species", Nat Methods, 13, pp.563-567 Fu Y et al. (2016) "CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci", Nat Commun, 7, pp.11707 Morita S et al. (2016) "Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions", Nat Biotechnol, 34, pp.1060-1065 Hess GT et al. (2016) "Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells", Nat Methods, 13, pp.1036-1042 Liu P et al. (2018) "CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency", Cell Stem Cell, in press

  However, in the conventional system, the effector protein is not sufficiently accumulated in the target gene, and further development of the next generation system is desired.

  An object of one embodiment of the present invention is to provide a composition for accumulating an effector protein in a target gene having a high effect of accumulating the effector protein in the target gene.

  As a result of intensive investigations to achieve the above object, the present inventors fused a peptide serving as a tag for binding a plurality of first effector proteins to a protein that binds to the second region of the guide RNA. By using this fusion, we succeeded in constructing a third generation system having a higher effect of accumulating effector proteins in the target gene than in the second generation system, and completed the present invention. That is, one embodiment of the present invention includes the following configuration.

<1> (A) a polynucleotide comprising a base sequence encoding a guide RNA that specifically binds to a target gene in the first region, or an expression vector comprising the polynucleotide,
(B) a polynucleotide comprising a base sequence encoding Cas protein or an expression vector comprising the polynucleotide,
(C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide A composition for accumulating an effector protein in a target gene, comprising: an expression vector comprising: (D) a polynucleotide comprising a base sequence encoding the first effector protein or an expression vector comprising the polynucleotide.

<2> (A ′) a guide RNA that specifically binds to the target gene in the first region,
(B ′) Cas protein,
(C ′) a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, and (D ′) a plurality of the first effectors protein,
A composition for accumulating an effector protein in a target gene.

  <3> A composition for accumulating an effector protein in a target gene according to <1> or <2>, wherein the Cas protein is fused with a second effector protein.

  <4> A composition for accumulating an effector protein on a target gene according to any one of <1> to <3>, wherein the Cas protein is dCas9.

<5> The composition for enhancing expression of a target gene according to <4>, wherein the dCas9 is encoded by a polynucleotide having a base sequence represented by any of the following (a1) to (a3):
(A1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1,
(A2) A polynucleotide comprising a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 1, and the protein expressed from the polynucleotide is the guide RNA of (A) above, (C) a fusion, and a polynucleotide having an activity of accumulating an effector protein in a target gene in cooperation with the first effector protein of (D), and (a3) a base sequence represented by SEQ ID NO: 1 A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary base sequence, and the protein expressed from the polynucleotide is a guide RNA of (A) above, a fusion of (C) above, And the effector in the target gene in cooperation with the first effector protein of (D) above A polynucleotide having an activity of accumulating proteins.

  <6> A composition for accumulating an effector protein in a target gene according to any one of <1> to <5>, wherein the second region of the guide RNA has a stem-loop structure.

<7> The effector protein is added to the target gene according to <6>, wherein the second region of the guide RNA is encoded by a polynucleotide having a base sequence represented by any of the following (b1) to (b3): Composition for accumulation:
(B1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2,
(B2) A polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2, and RNA transcribed from the polynucleotide binds to the fusion of (C) above. (B3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2, and from the polynucleotide A polynucleotide in which transcribed RNA has an activity of binding to the fusion of (C) above.

  <8> A composition for accumulating an effector protein in a target gene according to any one of <1> to <7>, wherein the protein that binds to the second region of the guide RNA is an MS2 coat protein.

<9> The MS2 coat protein is encoded by a polynucleotide having a base sequence represented by any of the following (c1) to (c3), and for integrating the effector protein in the target gene according to <8> Composition:
(C1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3,
(C2) a polynucleotide comprising a nucleotide sequence having a nucleotide sequence of 90% or more with the nucleotide sequence represented by SEQ ID NO: 3, and a protein expressed from the polynucleotide is the number of the guide RNA of the above (A) A polynucleotide having an activity of binding to the region 2, and (c3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3, and The polynucleotide in which the protein expressed from the polynucleotide has an activity of binding to the second region of the guide RNA of (A).

<10> The first effector protein comprises an antigen or an antibody,
The composition for accumulating an effector protein in the target gene according to any one of <1> to <9>, wherein the peptide serving as the tag comprises an antibody or antigen for binding to the antigen or antibody.

  <11> The composition for accumulating effector proteins in the target gene according to <10>, wherein the peptide serving as the tag comprises 4 to 24 antigens.

<12> The first effector protein comprises an antibody,
The composition for accumulating an effector protein in the target gene according to <10>, wherein the antibody is scFv.

  <13> A composition for accumulating an effector protein in a target gene according to <10>, which has a linker between antigens or antibodies provided in the peptide serving as the tag.

  <14> A composition for accumulating an effector protein in a target gene according to <13>, wherein the linker is 5 to 60 amino acid residues.

  <15> The composition for accumulating an effector protein in the target gene according to <3>, wherein the second effector protein is VP64.

  <16> A composition for accumulating an effector protein on a target gene according to any one of <1> to <15>, wherein the first effector protein is a transcriptional activator.

<17> (A) a polynucleotide comprising a base sequence encoding a guide RNA that specifically binds to a target gene in the first region, or an expression vector comprising the polynucleotide,
(B) a polynucleotide comprising a base sequence encoding Cas protein or an expression vector comprising the polynucleotide,
(C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide A kit for accumulating an effector protein in a target gene, comprising: an expression vector comprising: (D) a polynucleotide comprising a base sequence encoding the first effector protein or an expression vector comprising the polynucleotide.

  <18> The composition according to any one of <1> to <16>, the polynucleotide provided in the kit according to <17>, or the expression vector provided in the kit according to <17> to a cell in vitro. A method for producing a cell in which an effector protein is accumulated in a target gene, comprising a step of introducing.

  <19> The composition according to any one of <1> to <16>, the polynucleotide provided in the kit according to <17>, or the expression vector provided in the kit according to <17> A method for producing a mammal (excluding humans) in which an effector protein is accumulated in a target gene, which comprises a step of introducing into a target gene (excluding humans).

  According to one embodiment of the present invention, it is possible to provide a composition for enhancing the expression of a target gene that has a high effect of enhancing the expression of the target gene.

(A) is a diagram schematically showing a first generation artificial gene expression enhancing system in which VP64 is fused to dCas9. (B) is a schematic diagram of a second generation artificial gene expression enhancing system in which VP64 is directly fused to dCas9, a stem loop that binds MS2 coat protein to sgRNA, and a transcriptional activator is fused to MS2 coat protein. FIG. (C) VP64 is directly fused to dCas9, a stem loop that MS2 binds to sgRNA, 22sTag is fused to MS2 coat protein, and the first transcriptional activator is fused to scFv that binds 22sTag. It is a figure which shows typically the 3rd generation artificial gene expression enhancement system which concerns on one Embodiment of this invention. (A) is a figure which shows typically the structure of MS2-4x22sTag used in the Example. (B) is a figure which shows typically the structure of MS2-8 * 22sTag used in the Example. (C) is a figure which shows typically the structure of MS2-16 * 22sTag used in the Example. (D) is a figure which shows typically the structure of MS2-24x22sTag used in the Example. (A) is a figure which shows typically the structure of sgRNA2.0 and dCas9-VP64 all-in-one expression vector used in the Example. (B) is a diagram schematically showing the structure of an expression vector of MS2-n × 22sTag (n = 4, 8, 16, 24) used in the examples. (C) is a diagram schematically showing the structure of an expression vector for scFv-Effect used in the examples. It is the result of the agarose gel electrophoresis which performed the expression vector of MS2-nx22sTag (n = 4, 8, 16, 24) treated with NotI performed in the Example. (A) is a figure which shows typically the area | region corresponding to the promoter of CDH1 gene of five sgRNA used in the Example. (B) is a figure which shows typically the structure of the luciferase reporter vector used in the Example. It is a figure which shows the result of the assay performed in the Example in the presence / absence of three vectors of dCas9-VP64, MS2-4 × 22sTag, and scFv-p65-HSF1. It is a figure which shows the result of the assay in the 1st generation, the 2nd generation, and the 3rd generation artificial gene expression enhancement system at the time of using each 5 sgRNA and 5 total sgRNA performed in the Example. The total amount of the expression vector of the constructed system using dCas9-VP64, MS2-n × 22sTag (n = 4, 8, 16, 24) and p65-HSF1 or VPR in the Examples is 100 ng. It is a figure which shows the result of the assay which targeted the CDH1 gene at a certain time. It is a figure which shows the result of the mRNA expression induction | guidance | derivation evaluation with respect to CDH1 gene using dCas9-VP64, MS2-nx22sTag (n = 4, 8), and p65-HSF1 or VPR performed in the Example. . The left column is a diagram showing the results of detection of E-cadherin in HCT116 cells by immunostaining performed in the examples. The middle row and the right row are diagrams showing the results of detection of E-cadherin in MIA-PaCa2 cells by immunostaining performed in Examples. It is a figure which shows the result of the detection of E-cadherin in the MIA-PaCa2 cell by the immunostaining performed in the Example. It is a figure which shows the result of the detection of E-cadherin in HCT116 cell and MIA-PaCa2 cell by Western blotting performed in the Example. FIG. 5 (a) is a diagram specifically showing the sequence containing the promoter region of the CDH1 gene and the sequence of the region corresponding to the promoter of the CDH1 gene of five sgRNAs used in the examples. The total amount of the expression vector of the constructed system using dCas9-VP64, MS2-n × 22sTag (n = 4, 8, 16, 24) and p65-HSF1 or VPR, as determined in the Examples, was 50 ng and It is a figure which shows the result of the assay which targeted CDH1 gene when it is 100 ng. It is a figure which shows the result of the detection of E-cadherin in HCT116 cell and MIA-PaCa2 cell by Western blotting performed in the Example. It is a figure which shows the result of the detection of E-cadherin in the MIA-PaCa2 cell by the immunostaining performed in the Example. It is a figure which shows the result of the detection of E-cadherin in the MIA-PaCa2 cell by the immunostaining performed in the Example. (A) is a figure which shows typically the area | region corresponding to the promoter of a RANKL gene of two sgRNA used in the Example. (B) is a figure which shows typically the structure of the luciferase reporter vector used in the Example. The total amount of the expression vector of the constructed system using dCas9-VP64, MS2-n × 22sTag (n = 4, 8, 16, 24) and p65-HSF1 or VPR in the Examples is 100 ng. It is a figure which shows the result of the assay which targeted the RANKL gene at a certain time. It is a figure which shows the result of the mRNA expression induction | guidance | derivation evaluation with respect to the RANKL gene using dCas9-VP64, MS2-n * 22sTag (n = 4, 8), and p65-HSF1 or VPR performed in the Example. FIG. 18 (a) is a diagram specifically showing the sequence containing the promoter region of the RANKL gene and the sequence of the region corresponding to the promoter of the RANKL gene of two sgRNAs used in the examples. The total amount of the expression vector of the constructed system using dCas9-VP64, MS2-n × 22sTag (n = 4, 8, 16, 24) and p65-HSF1 or VPR, as determined in the Examples, was 50 ng and It is a figure which shows the result of the assay which targeted the RANKL gene when it is 100 ng. DCas9-VP64 or dCas9-n × 22sTag (n = 4, 8), MS2-VP64, MS2-n × 22sTag (n = 4, 8), MS2-p65-HSF1, or MS2- It is a figure which shows the result of the assay which targeted RANKL gene using VPR and scFv-VP64, scFv-p65-HSF1, or scFv-VPR.

  Hereinafter, although an example of an embodiment of the invention is explained in detail, the present invention is not limited to these.

  Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more and B or less”.

  As used herein, the term “gene” refers to a polymer of nucleotides and is used interchangeably with the terms “polynucleotide”, “nucleic acid” or “nucleic acid molecule”. A gene can exist in the form of DNA (eg, cDNA or genomic DNA) or in the form of RNA (eg, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). The gene may be chemically synthesized. Further, in the present specification, when “polynucleotide” is described, it may be DNA or RNA.

  As used herein, the term “protein” is used interchangeably with “peptide” or “polypeptide”.

In this specification, the one-letter code or three-letter code defined by IUPAC and IUB is used as appropriate for the base and amino acid.

[1. (Composition for accumulating effector protein in target gene)
A composition for accumulating an effector protein in a target gene according to an embodiment of the present invention (hereinafter referred to as “a composition according to an embodiment of the present invention” as appropriate) is (A) a target gene, A polynucleotide comprising a base sequence encoding a guide RNA that specifically binds in the first region or an expression vector comprising the polynucleotide; (B) a polynucleotide comprising a base sequence encoding a Cas protein or the polynucleotide An expression vector, (C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide An expression vector comprising a polynucleotide, and (D) the first effector protein Including expression vectors, comprising the polynucleotide or the polynucleotide comprising a nucleotide sequence encoding.

  In the present specification, a system that artificially accumulates effector proteins in a target gene using the composition according to one embodiment of the present invention may be referred to as a third generation system. In the present specification, a system that artificially enhances the expression of a target gene using the composition according to one embodiment of the present invention may be referred to as a third generation artificial gene expression enhancing system.

A third generation system according to an embodiment of the present invention is a method for binding a plurality of guide RNA (also referred to as sgRNA), Cas protein, a protein that binds to the second region of the guide RNA, and a plurality of first effector proteins. A fusion with a peptide serving as a tag and the first effector protein function in cooperation, thereby producing an effect of accumulating the effector protein in the target gene. The mechanism for accumulating the effector protein in the target gene will be described in more detail as follows.
1. The guide RNA and Cas protein bind to form a complex.
2. The complex binds to the target gene in the first region of the guide RNA.
3. The second region of the guide RNA is a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins to the second region of the guide RNA. Proteins that bind to the region bind.
4). The first effector protein binds to the peptide that becomes the tag of the fusion. The first effector protein is accumulated in the target gene.

  The “target gene” to which the guide RNA binds includes not only a target gene but also a sequence (for example, a promoter sequence or an enhancer sequence) that controls expression of the gene.

  The guide RNA has a first region that specifically binds to a target gene and a second region that binds to a specific protein. The guide RNA is designed such that the first region has a sequence complementary to the sequence of a part of the target gene to be bound, and the second region has a sequence to which a specific protein binds. The

  In the composition according to one embodiment of the present invention, the second region of the guide RNA has a stem-loop structure. Thereby, the protein that recognizes the stem loop structure can bind to the second region of the guide RNA. The second region of the guide RNA only needs to have at least one stem loop structure, but more preferably has a plurality of stem loop structures. When the second region of the guide RNA has a plurality of stem loop structures, the number of proteins that bind to the stem loop structures can be increased. Thereby, more effector proteins can be accumulated in the target gene.

  The guide RNA may be one type or a combination of multiple types. In the third generation system according to an embodiment of the present invention, even with one type of guide RNA, the effector protein can be accumulated in the target gene as compared with the first generation and second generation systems. . When a plurality of types of guide RNAs are used, it is more preferable that there are a plurality of types of guide RNAs because more effector proteins can be accumulated in the target gene than when a single type of guide RNA is used.

  The Cas protein is a protein that is a component of the CRISPR-Cas system used in genome editing technology, and forms a complex by binding to a guide RNA. The complex binds to the target gene in the first region of the guide RNA.

  The Cas protein may be a nuclease-inactive Cas protein or a nuclease-active Cas protein, and is preferably a nuclease-inactive Cas protein. The nuclease-inactive Cas protein is a Cas protein that has been modified so that DNA cleavage is not caused by inactivating the nuclease activity while maintaining the binding ability to the guide RNA.

  Examples of the Cas protein include Cas12a and Cas9. Examples of the nuclease-inactive Cas protein include, for example, the above-mentioned Cas protein nuclease-inactive type (that is, nuclease-inactive Cas12a, nuclease-inactive Cas9 (hereinafter, appropriately referred to as “dCas9”)). Etc. When the Cas protein is a nuclease-active Cas protein, a Cas protein having the same function as the nuclease-inactive Cas protein can be obtained by appropriately adjusting the length of the guide RNA.

  In the composition according to one embodiment of the present invention, the Cas protein is preferably dCas9.

  In the composition according to an embodiment of the present invention, the dCas protein is fused with a second effector protein. Thereby, in addition to the first effector protein, the second effector protein can also be accumulated in the target gene.

  Although the said 2nd effector protein will not be specifically limited if it has the target function, It is preferable that molecular weight is 1-60 kDa, and it is more preferable that it is 5-10 kDa.

  In the composition according to one embodiment of the present invention, the second effector protein is preferably VP64. Since the molecular weight of VP64 is as small as 5.5 kDa, in addition to the first effector protein in the target gene, VP64 is added to the target gene while suppressing the enlargement of the molecular size of the fusion when forming a fusion with the Cas protein. Can be integrated. Since VP64 is a transcriptional activator, the expression of the target gene can be further enhanced.

  In the composition according to one embodiment of the present invention, the protein that binds to the second region of the guide RNA is not particularly limited as long as it can bind to the second region of the guide RNA, but it is an MS2 coat protein. preferable. Since the MS2 coat protein recognizes the stem loop structure of the second region of RNA, it can bind to the second region of RNA.

  The first effector protein is not particularly limited as long as it has a desired function, but the molecular weight is preferably 1 to 60 kDa, and more preferably 32 to 56 kDa.

  The first effector protein and the second effector protein are preferably those having the same function, but may have different functions.

The first effector protein and the second effector protein are not particularly limited as long as they are proteins that accumulate in a target gene and modify / modify the expression or the like of the target gene.
Examples of the first effector protein include a transcriptional activator, a fluorescent protein (eg, GFP), a DNA demethylation factor (eg, TET1), a histone acetylation factor (eg, p300cd), and a deaminase. Can be mentioned.

  When the effector protein is a transcriptional activator, the expression of the target gene in which the effector protein is accumulated is enhanced. Therefore, the composition according to one embodiment of the present invention can be a composition for enhancing the expression of a target gene. When the effector protein is a fluorescent protein, the target gene region in which the effector protein is accumulated is labeled. Therefore, the composition according to one embodiment of the present invention can be a composition for labeling a region of a target gene. Further, when the effector protein is a DNA demethylation factor, the efficiency of demethylation of the target gene in which the effector protein is accumulated is increased. Therefore, the composition according to one embodiment of the present invention can be a composition for promoting demethylation of a target gene. Further, when the effector protein is a histone acetylation factor, the efficiency of histone acetylation around the target gene in which the effector protein is accumulated is enhanced. Therefore, the composition according to an embodiment of the present invention can be a composition for promoting acetylation of histones around a target gene. Further, when the effector protein is deaminase, the efficiency of base substitution around the target gene in which the effector protein is accumulated is increased. Therefore, the composition according to one embodiment of the present invention can be a composition for promoting base substitution around the target gene.

  In the composition according to an embodiment of the present invention, the first effector protein is preferably a transcriptional activator, and includes a group consisting of p65-HSF1, VPR, and VP64 (all of which are transcriptional activators). More preferably, it is at least one selected.

  In the composition according to an embodiment of the present invention, the first effector protein includes an antigen or an antibody, and the peptide serving as the tag includes an antibody or an antigen for binding to the antigen or the antibody. Preferably, the first effector protein includes an antigen, and the peptide serving as the tag includes an antibody for binding to the antigen. Thereby, even when the first effector protein has no binding force with the peptide serving as a tag, the binding between the antigen or antibody provided in the first effector protein and the antibody or antigen provided in the peptide serving as a tag Thus, the first effector protein can be bound to the peptide serving as a tag. As a result, the first effector protein can be accumulated in the target gene.

  When the peptide serving as the tag includes an antigen, the antigen is preferably GCN4. A peptide having an amino acid sequence in which the amino acid sequence of GCN4 is arranged in tandem may be referred to as SunTag.

  In the composition according to an embodiment of the present invention, the peptide serving as the tag preferably includes 4 to 24 antigens, and more preferably includes 8 antigens. Thereby, the above-mentioned number of first effector proteins comprising an antibody that binds to the antigen can be bound.

  It is preferable that the composition which concerns on one Embodiment of this invention has a linker between the antigen or antibodies with which the peptide used as the said tag is equipped. The linker is preferably 5 to 60 amino acid residues, more preferably 22 to 51 amino acid residues, and preferably 22 amino acid residues. There may be a linker between the protein that binds to the second region of the guide RNA and the antigen or antibody. Since the fusion protein of the protein that binds to the second region of the guide RNA and the peptide serving as the tag has the linker, the first effector protein to be bound can be bound to the antigen at an optimal interval.

  In the composition according to an embodiment of the present invention, the first effector protein preferably includes an antibody, and the antibody is preferably scFv (single chain antibody).

[1-1 Polynucleotide]
[Sequence encoding dCas9]
In the composition according to an embodiment of the present invention, preferably, the dCas9 is encoded by a polynucleotide having a base sequence represented by any of the following (a1) to (a3):
(A1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1,
(A2) A polynucleotide comprising a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 1, and the protein expressed from the polynucleotide is the guide RNA of (A) above, (C) a fusion, and a polynucleotide having an activity of accumulating an effector protein in a target gene in cooperation with the first effector protein of (D), and (a3) a base sequence represented by SEQ ID NO: 1 A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary base sequence, and the protein expressed from the polynucleotide is a guide RNA of (A) above, a fusion of (C) above, And the effector in the target gene in cooperation with the first effector protein of (D) above A polynucleotide having an activity of accumulating proteins.

(A1) is
1. By artificially modifying the base sequence encoding wild-type Cas9, a polynucleotide that maintains the ability to bind to guide RNA and inactivates nuclease activity to prevent DNA cleavage (SEQ ID NO: 1).

(A2)
2. It has a certain sequence identity with (a1).
3. It encodes a protein having the same function as the protein encoded by (a1).
A polynucleotide that satisfies both of the conditions.
Hereinafter, each of 2 and 3 will be described.

(About 2)
The sequence identity of the base sequence is at least 90% or more, more preferably 95% or more (for example, 95%, 96%, etc.) in the entire base sequence (or a region encoding a part necessary for the function of dCas9). 97%, 98%, 99% or more).

  The identity of the base sequence can be determined using a program such as BLASTN ([Altschul SF (1990) “Basic local alignment search tool”, Journal of Molecular Biology, Vol. 215 (Issue 3), pp. .403-410]). An example of parameters for analyzing a base sequence by BLASTN is a setting of score = 100 and wordlength = 12. Specific methods for performing analysis by BLASTN are known to those skilled in the art. In order to align the base sequences to be compared with each other in an optimal state, addition or deletion (such as a gap) may be allowed.

(About 3)
Whether or not “has a function equivalent to the protein encoded by (a1)” is expressed from a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1 in (a2) Whether or not the obtained protein (dCas9) has the activity of accumulating the effector protein in the target gene in cooperation with the guide RNA of (A), the fusion of (C), and the first effector protein of (D) This can be determined by testing. Such testing can be performed using routine methods in the art.

  Here, the activity of accumulating the effector protein in the above-mentioned gene means that the amount of the effector protein accumulated in the target gene in the cell into which the nucleotide is introduced is the amount of the effector protein in the cell into which the gene is not introduced, preferably the first generation And greater than the amount of the effector protein in the cells to which the second generation system was applied.

  The third generation system according to an embodiment of the present invention is preferably a third generation artificial gene expression enhancing system. The third generation artificial gene expression enhancing system according to an embodiment of the present invention preferably enhances the expression of a target gene. Examples of the method for confirming the expression level of mRNA transcribed from the target gene include qPCR described in Examples. Moreover, since the expression level of the protein encoded by the target gene is also enhanced as a result of the transcriptional activation of the target gene, the expression level of the protein may be confirmed. Examples of the method for confirming the expression level of the protein include reporter assay, immunostaining, and Western blotting described in Examples.

(A3)
4). Hybridization is performed under stringent conditions with a polynucleotide comprising a base sequence complementary to (a1).
5). It encodes a protein having the same function as the protein encoded by (a1).
A polynucleotide that satisfies both of the conditions. Hereinafter, 4 will be described. Since the explanation about 5 is the same as the above (about 3), it is omitted.

(About 4)
In this specification, “stringent conditions” means that two polynucleotide strands form a double-stranded polynucleotide specific to the base sequence, but a non-specific double-stranded polynucleotide is formed. The condition that does not. In other words, “hybridizes under stringent conditions” means that the temperature is 15 ° C. lower, preferably 10 ° C. lower than the melting temperature (Tm value) of nucleic acids having high sequence identity (for example, perfectly matched hybrids). It can also be said that the conditions allow hybridization in a temperature range, more preferably in a temperature range up to 5 ° C.

An example of stringent conditions is as follows. First, in a buffer solution (pH 7.2) composed of 0.25M Na ₂ HPO ₄ , 7% SDS, 1 mM EDTA, 1 × Denhardt solution, at 60 to 68 ° C. (preferably 65 ° C., more preferably 68 ° C.). , 16-24 hours, 2 kinds of polynucleotides are hybridized. Thereafter, washing is performed for 2 minutes at 60 to 68 ° C. (preferably 65 ° C., more preferably 68 ° C.) in a buffer solution (pH 7.2) composed of 20 mM Na ₂ HPO ₄ , 1% SDS and 1 mM EDTA. Do it once.

Other examples include the following methods. First, high containing 25% formamide (50% formamide under more severe conditions), 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes (pH 7.0), 10 × Denhardt's solution, 20 μg / mL denatured salmon sperm DNA After performing prehybridization overnight at 42 ° C. in a hybridization solution, a labeled probe is added and incubated overnight at 42 ° C. to hybridize two types of polynucleotides. Next, cleaning is performed under any of the following conditions.
(Normal conditions) Wash at about 37 ° C. using 1 × SSC and 0.1% SDS as washing solutions.
(Severe conditions) Wash at about 42 ° C. as 0.5 × SSC and 0.1% SDS cleaning solution.
(More severe conditions) Wash at about 65 ° C. using 0.2 × SSC and 0.1% SDS as washing liquid.

  As the hybridization washing conditions become more severe, hybridization with higher specificity is achieved. The combination of the above SSC, SDS and temperature conditions is merely an example. It is possible to achieve the same stringency as described above by appropriately combining the above-mentioned factors that determine the stringency of hybridization, or other factors (for example, probe concentration, probe length, hybridization reaction time, etc.). it can. This is described, for example, in [Joseph Sambrook & David W. Russell, “Molecular cloning: a laboratory manual 3rd Ed.”, New York: Cold Spring Harbor Laboratory Press, 2001].

  Hereinafter, the polynucleotide comprising the base sequence represented by any of (a1) to (a3) is also collectively referred to as the polynucleotide (a).

[Sequence encoding the second region of the guide RNA]
In the composition according to one embodiment of the present invention, preferably, the second region of the guide RNA is encoded by a polynucleotide having a base sequence represented by any of the following (b1) to (b3):
(B1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2,
(B2) A polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2, and RNA transcribed from the polynucleotide binds to the fusion of (C) above. (B3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2, and from the polynucleotide A polynucleotide in which transcribed RNA has an activity of binding to the fusion of (C) above.

(B1)
1. For example, a polynucleotide (SEQ ID NO: 2) encoding RNA comprising two stem loop structures to which MS2 coat protein binds.

(B2)
2. It has a certain sequence identity with (b1).
3. It encodes the second region of the guide RNA having the same function as the second region of the guide RNA encoded by (b1).
A polynucleotide that satisfies both of the conditions. 2 is the same as (a2). Hereinafter, 3 will be described.

(About 3)
Whether or not “has a function equivalent to that of the second region of the guide RNA encoded by (b1)” is determined by determining whether the nucleotide sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2 in (b2) It can be determined by testing whether RNA transcribed from the polynucleotide consisting of has the activity of binding to the fusion of (C) above. Such testing can be performed using routine methods in the art.

(B3)
4). It hybridizes with a polynucleotide comprising a base sequence complementary to (b1) under stringent conditions.
5). It encodes the second region of the guide RNA having the same function as the second region of the guide RNA encoded by (b1).
A polynucleotide that satisfies both of the conditions. The explanation about 4 is the same as (about 4) in (a3), and the explanation about 5 is the same as the above (about 3), and will be omitted.

  Hereinafter, the polynucleotide having the base sequence represented by any of (b1) to (b3) is also collectively referred to as the polynucleotide (b).

[Sequence encoding MS2 coat protein]
In the composition according to one embodiment of the present invention, preferably, the MS2 coat protein is encoded by a polynucleotide having a base sequence represented by any of the following (c1) to (c3):
(C1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3,
(C2) a polynucleotide comprising a nucleotide sequence having a nucleotide sequence of 90% or more with the nucleotide sequence represented by SEQ ID NO: 3, and a protein expressed from the polynucleotide is the number of the guide RNA of the above (A) A polynucleotide having an activity of binding to the region 2, and (c3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3, and The polynucleotide in which the protein expressed from the polynucleotide has an activity of binding to the second region of the guide RNA of (A).

(C1)
1. It is a polynucleotide (sequence number 3) which consists of a base sequence which codes MS2 coat protein which is an example of the protein couple | bonded with the 2nd area | region of guide RNA.

(C2)
2. It has a certain sequence identity with (c1).
3. It encodes a protein having the same function as the protein encoded by (c1).
A polynucleotide that satisfies both of the conditions. 2 is the same as (a2). Hereinafter, 3 will be described.

(About 3)
Whether or not “has a function equivalent to the protein encoded by (c1)” is expressed from a polynucleotide comprising a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 3 of (c2) It can be determined by testing whether or not the obtained protein has an activity of binding to the second region of the guide RNA of (A). Such testing can be performed using routine methods in the art.

(C3)
4). Hybridization is performed under stringent conditions with a polynucleotide comprising a base sequence complementary to (c1).
5). It encodes a protein having the same function as the protein encoded by (c1).
A polynucleotide that satisfies both of the conditions. The explanation about 4 is the same as (about 4) in (a3), and the explanation about 5 is the same as the above (about 3), and will be omitted.

  Hereinafter, the polynucleotide having the base sequence represented by any one of (c1) to (c3) is collectively referred to as the polynucleotide (c).

[Sequence encoding GCN4]
In the composition according to an embodiment of the present invention, preferably, the GCN4 is encoded by a polynucleotide having a base sequence represented by any of the following (d1) to (d3):
(D1) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 4 to 7,
(D2) a polynucleotide comprising a nucleotide sequence having a nucleotide sequence of 90% or more of the nucleotide sequence represented by any of SEQ ID NOs: 4 to 7 and comprising a peptide expressed from the polynucleotide (C) A polynucleotide having the activity of accumulating an effector protein in a target gene in cooperation with the guide RNA of (A), the Cas protein of (B), and the first effector protein of (D) And (d3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 4 to 7, and expressed from the polynucleotide The fusion of (C) containing a peptide comprises the guide RNA of (A), the Cas tag of (B) And a polynucleotide having an activity to accumulate the effector protein in the target gene in cooperation with the first effector protein of (D) above.

  In addition, all the proteins (GCN4) translated from the polynucleotide which consists of a base sequence shown by either sequence number 4-7 have the same amino acid sequence.

(D1) is
1. It is a polynucleotide (any one of SEQ ID NOs: 4 to 7) having a base sequence encoding GCN4, which is an example of an antigen provided in a peptide serving as a tag.

(D2) is
2. It has a certain sequence identity with (d1).
3. It is a polynucleotide that satisfies both conditions of encoding a protein having the same function as the protein encoded by (d1). 2 is the same as (a2). Hereinafter, 3 will be described.

  Whether or not “has a function equivalent to the protein encoded by (d1)” is determined from the base sequence having 90% or more sequence identity with the base sequence represented by any of SEQ ID NOs: 4 to 7 in (d2) A fusion product of (C) containing a peptide expressed from a polynucleotide comprising the target RNA in cooperation with the guide RNA of (A), the Cas protein of (B), and the first effector protein of (D) It can be determined by testing whether or not the gene has an activity of accumulating effector proteins. Such testing can be performed using routine methods in the art.

(D3)
4). Hybridization is performed under stringent conditions with a polynucleotide comprising a base sequence complementary to (d1).
5). It encodes a protein having the same function as the protein encoded by (d1).
A polynucleotide that satisfies both of the conditions. In the following, the explanation about 4 is the same as (about 4) of (a3), and the explanation about 5 is the same as the above (about 3), and will be omitted.

  Hereinafter, the polynucleotide comprising the base sequence represented by any of (d1) to (d3) is also collectively referred to as the polynucleotide (d).

[Linker sequence of peptide to be tagged]
The linker sequence of the peptide serving as the tag is not particularly limited as long as the antigen or antibody of the peptide serving as the tag does not interfere with the function of binding to the antibody or antigen provided in the first effector protein.

In the composition according to one embodiment of the present invention, preferably, the linker is encoded by a polynucleotide having a base sequence represented by any of the following (e1) to (e3):
(E1) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 8 to 11,
(E2) a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NOs: 8 to 11, and (e3) a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NOs: 8 to 11 A polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising

  Hereinafter, the polynucleotide having the base sequence represented by any one of (e1) to (e3) is collectively referred to as the polynucleotide (e).

  In addition, all the linkers translated from the polynucleotide which consists of a base sequence shown by either of sequence number 8-11 have the same amino acid sequence.

[Sequence encoding scFv]
An antibody will not be specifically limited if it couple | bonds with the antigen with which the peptide used as a tag is equipped.

In the composition according to one embodiment of the present invention, for example, when the antigen included in the peptide serving as a tag is GCN4, the scFv is preferably based on the base sequence represented by any of the following (f1) to (f3): Is encoded by the polynucleotide:
(F1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 12,
(F2) A polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 12, and a protein expressed from the polynucleotide binds to an antigen provided in the peptide serving as a tag And (f3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 12, and from the polynucleotide A polynucleotide in which an expressed protein has an activity of binding to an antigen included in a peptide serving as a tag.

(F1) is
1. It is a polynucleotide (SEQ ID NO: 12) consisting of a base sequence encoding scFv designed to bind to GCN4.

(F2) is
2. It has a certain sequence identity with (f1).
3. It is a polynucleotide satisfying both conditions of encoding a protein having the same function as the protein encoded by (f1). 2 is the same as (a2). Hereinafter, 3 will be described.

(About 3)
Whether or not “has a function equivalent to the protein encoded by (f1)” is expressed from a polynucleotide comprising a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 12 of (f2) It can be determined by testing whether or not the obtained protein has an activity of binding to an antigen included in the peptide serving as a tag. Such testing can be performed using routine methods in the art.

(F3)
4). Hybridization is performed under stringent conditions with a polynucleotide comprising a base sequence complementary to (f1).
5). It encodes a protein having the same function as the protein encoded by (f1).
A polynucleotide that satisfies both of the conditions. In the following, the explanation about 4 is the same as (about 4) of (a3), and the explanation about 5 is the same as the above (about 3), and will be omitted.

  Hereinafter, the polynucleotide having the base sequence represented by any one of (f1) to (f3) is collectively referred to as the polynucleotide (f).

[1-2 Expression Vector Containing Polynucleotide]
The composition according to an embodiment of the present invention may include the above-described nucleotide, or may include an expression vector including the polynucleotide. That is, the composition according to one embodiment of the present invention includes an expression vector containing the polynucleotide described above.

  In the composition according to one embodiment of the present invention, the polynucleotide contained in the expression vector is the same as that shown in [1-1].

  The expression vector contained in the composition according to an embodiment of the present invention can be any of various commonly used vectors as a substrate vector, and is appropriately selected according to the cell to be introduced or the introduction method. sell. Specifically, plasmids, phages, cosmids and the like can be used. The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected.

  Examples of the expression vectors described above include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors and viral vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements and viruses (baculo And vectors derived from viruses, papovaviruses, vaccinia viruses, adenoviruses, tripox viruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, and the like, and combinations thereof (cosmids, phagemids, etc.).

  The expression vector contained in the composition according to one embodiment of the present invention preferably further includes sites for transcription initiation and transcription termination, and a ribosome binding site in the transcription region. The coding portion of the mature transcript in the vector will contain a transcription start codon AUG at the beginning of the polypeptide to be translated and a stop codon appropriately located at the end.

  In order to express RNA or protein, the expression vector contained in the composition according to one embodiment of the present invention may contain a promoter sequence. The promoter sequence may be appropriately selected according to the type of host cell.

  The expression vector contained in the composition according to one embodiment of the present invention may contain a sequence for enhancing transcription from DNA. In one embodiment, the sequence for enhancing transcription from the DNA is an enhancer sequence. Examples of the enhancer include SV40 enhancer (which is located 100 to 270 bp downstream of the origin of replication), cytomegalovirus early promoter enhancer, polyoma enhancer and adenovirus enhancer located downstream of the origin of replication. Is mentioned.

  The expression vector contained in the composition according to one embodiment of the present invention may contain a sequence for stabilizing the transcribed RNA. In one embodiment, the sequence for stabilizing the transcribed RNA is a poly A addition sequence (polyadenylation sequence, polyA). Examples of poly A addition sequences include growth hormone gene-derived poly A addition sequences, bovine growth hormone gene-derived poly A addition sequences, human growth hormone gene-derived poly A addition sequences, SV40 virus-derived poly A addition sequences, human or An example is a poly A addition sequence derived from a rabbit β-globin gene.

  The structure of the expression vector contained in the composition according to one embodiment of the present invention can be appropriately set. For example, matters such as “where to place a base sequence encoding RNA or protein in a substrate vector”, “what kind of sequence to place before and after the base sequence encoding RNA or protein” Can be appropriately set depending on the purpose.

  In addition, the number of polynucleotides comprising a base sequence encoding RNA or protein incorporated in the same vector is as long as the function of the third generation artificial gene expression enhancing system can be exhibited in the host cell into which the expression vector is introduced. However, there is no particular limitation. For example, a design of “mounting the above-mentioned polynucleotides (a) to (f) in one (identical) vector” is possible. In addition, a design of “mounting the polynucleotides (a) to (e) on one type (the same) vector and mounting the polynucleotide (f) on another vector” is possible. However, when the polynucleotides (c) and (d) are continuously mounted on one (identical) vector and the polynucleotide (e) is included, the polynucleotides (d) and (e) are It is continuously mounted on one type of (identical) vector. In addition, “continuously” is intended to express as a fusion without expressing as a separate protein, and if the fusion can be expressed, a sequence for forming a further linker between the two You may mount the polynucleotide which has. Furthermore, a design that “each polynucleotide is mounted on four different vectors” is possible. From the viewpoint of stabilizing the guide RNA, the polynucleotides (a) and (b) are preferably mounted on one type (the same) vector. From the viewpoint of expression efficiency and the like, a vector containing the polynucleotides (a) and (b), a vector containing the polypeptides (c) and (d) (and (e)), and a polypeptide (f) For the vector containing a peptide, a method of loading on three different types of vectors is used.

  In addition, for the purpose of adjusting the expression level, a plurality of polynucleotides comprising base sequences encoding the same RNA or protein may be mounted in the same vector. For example, it is possible to design such that “the polynucleotide (a) is arranged in two places in one (identical) vector”.

  Note that the above-described method for mounting a polypeptide on a vector is merely an example, and can be appropriately designed and changed by those skilled in the art according to the purpose and the like.

  The expression vector contained in the composition according to one embodiment of the present invention described above can be prepared by a known technique. Examples of such methods include the methods described in various manuals in addition to the methods described in the implementation manual attached to the kit for preparing the vector.

[1-3 RNA and protein]
The composition according to an embodiment of the present invention includes (A ′) a guide RNA that specifically binds to a target gene in a first region, (B ′) a Cas protein, (C ′) a second of the above guide RNA. A fusion of a protein that binds to a region and a peptide that serves as a tag for binding a plurality of first effector proteins, and (D ′) a plurality of the first effector proteins.

  The composition which concerns on one Embodiment of this invention may contain RNA and protein which are encoded by the polynucleotide as described in said [1-1].

[2. Kit for collecting effector protein in target gene]
A kit for accumulating an effector protein in a target gene according to an embodiment of the present invention (hereinafter referred to as “kit according to an embodiment of the present invention” as appropriate) includes (A) a first A polynucleotide comprising a base sequence encoding a guide RNA that specifically binds in a region or an expression vector comprising the polynucleotide; (B) a polynucleotide comprising a base sequence encoding a Cas protein or an expression vector comprising the polynucleotide; (C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide An expression vector, and (D) the first effector protein Comprising an expression vector comprising the polynucleotide or the polynucleotide consisting of the nucleotide sequence over de.

  In this embodiment, [1. Items described in “Composition for Accumulating Effector Protein in Target Gene]” will not be repeated.

  A “kit” according to an embodiment of the present invention is intended to be a package including a container (for example, a bottle, a plate, a tube, a dish, or the like) containing a specific material. The kit according to one embodiment of the present invention may be in a form in which each material contained therein is present independently, or a form in which a plurality of materials are mixed (for example, in the form of a composition). It may be. The kit preferably includes instructions for using each material.

[3. Method for producing cells and mammals (excluding humans) in which effector proteins are accumulated in the target gene]
According to one embodiment of the present invention, a method for producing a cell in which an effector protein is accumulated in a target gene includes the above-described composition, the polynucleotide provided in the above-described kit, or the expression vector provided in the above-described kit. Including the step of introducing. In addition, the method for producing a mammal (excluding humans) in which an effector protein is accumulated in a target gene according to an embodiment of the present invention includes the above-described composition, the polynucleotide provided in the above-described kit, or the above-described kit. A step of introducing the provided expression vector into a mammal (excluding humans).

  The cell or mammal obtained by the production method according to one embodiment of the present invention has a modified or modified expression or the like of the target gene as a result of accumulation of the effector protein in the target gene within the cell or mammal. Yes. That is, in one embodiment of the present invention, the expression of the target gene and the like can be appropriately modified / modified by selecting an effector protein according to the purpose. The effector protein is as described above.

  In this embodiment, [1. Items described in “Composition for Accumulating Effector Protein in Target Gene]” will not be repeated.

[3-1. Method for producing cells in which effector protein is accumulated in target gene]
The cell which concerns on one Embodiment of this invention is not specifically limited, For example, cells, such as bacteria, yeast, an insect, an animal, a plant, are mentioned. The cell according to one embodiment of the present invention is preferably an animal cell, more preferably a mammalian cell, and particularly preferably a human cell.

  The method for introducing a composition, a polynucleotide or an expression vector containing the polynucleotide according to one embodiment of the present invention into a cell is not particularly limited. For example, electroporation method, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, transduction, infection using virus vector, and the like. Such methods are described in many standard laboratory manuals such as [Leonard G. Davis et al., “Basic methods in molecular biology”, New York: Elsevier, 1986]. Moreover, the introduction method can be appropriately selected depending on the type of cells to be introduced.

  In one embodiment of the present invention, introduction of the polynucleotide or an expression vector containing the polynucleotide into a cell is a step performed in vitro. In this case, among the introduction methods described above, it is preferable to employ electroporation, cationic lipid-mediated transfection, and infection using a viral vector.

  A fusion of a guide RNA according to an embodiment of the present invention, a Cas protein, a protein that binds to the second region of the guide RNA, and a peptide that serves as a tag for binding a plurality of first effector proteins, and a plurality The method for introducing the first effector protein is not particularly limited. Examples include electroporation and cationic lipid mediated transfection.

[3-2. Method for producing mammals (except humans) in which effector proteins are accumulated in the target gene]
A composition according to an embodiment of the present invention, a polynucleotide or an expression vector containing the polynucleotide, or a guide RNA, a Cas protein, a protein that binds to the second region of the guide RNA, and a plurality of first effector proteins A method of introducing a fusion with a peptide serving as a tag for binding and a plurality of the first effector proteins into mammals (excluding humans) is as described in [3-1].

[4. Disease Treatment Method Using Third Generation System]
In another embodiment of the present invention, there is provided a method for treating a disease in a subject, comprising administering the composition, polynucleotide or polynucleotide according to one embodiment of the present invention to the subject. By administering the composition, the polynucleotide or the polynucleotide to a subject, the effector protein is accumulated in the target gene in the subject, and as a result, the disease of the subject can be treated.

  In one embodiment of the present invention, “treatment” means an action that produces a treatment effect on a subject in need of treatment. The treatment effect may be a concept including a prophylactic effect and a therapeutic effect.

  In one embodiment of the present invention, the disease to be treated is not particularly limited as long as the disease produces a treatment effect as a result of enhanced gene expression by the third generation system. Examples of the disease to be treated include cancer, neurological disease, diabetes and the like. For example, when the disease to be treated is cancer, E-Cadherin (encoded by CDH1 gene) involved in cell adhesion is targeted and its expression is enhanced to prevent migration and invasion of cancerous cells. be able to.

  In one embodiment of the present invention, any method used in the field of gene therapy or the like can be used to administer the composition, polynucleotide or the polynucleotide to a subject.

  In one embodiment of the present invention, the disease to be treated is not particularly limited, and is, for example, a mammal, preferably a human.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Hereinafter, as an example of the third generation system of the present invention, an embodiment of a third generation artificial gene expression enhancing system using a transcriptional activator as an effector protein will be described, but the present invention is not limited thereto. That is, as described above, since the effector protein can be selected according to the purpose, if it is a third generation system having a function of accumulating the effector protein in the target gene, the effect other than the gene expression enhancing effect can be obtained. The third generation system shown is also within the scope of the present invention.

[1 Basic operation for cell culture]
[1.1 Cell culture]
In this example, MIA-PaCa2 cells, HEK293T cells and HCT116 cells were used. The cultured cells were cultured under conditions of 37 ° C. and 5% CO ₂ . The e-Myco ™ Mycoplasma PCR Detection Kit (from iNtRON Biotechnology) was used to test that cells in culture were not contaminated with mycoplasma. Furthermore, the cell line was authenticated by STR analysis (contract analysis by TaKaRa).

[1.1.1 Preparation of complete medium]
[1.1.1.1 Preparation of complete medium for MIA-PaCa2 cells]
D-MEM (Dulbecco's Modified Eagle Medium) (High Glucose) (Wako) containing L-glutamine and phenol red is 86.5% by volume, FBS (fetal bovine serum, Thermo Fisher) with respect to the total volume of the medium. (Scientific) was mixed with 10% by volume, Horse Serum (horse serum, manufactured by Thermo Fisher Scientific) was 2.5% by volume, and ST-PN (penicillin-streptomycin, manufactured by Wako) was 1% by volume. -Complete medium for PaCa2 cells was made.

[1.1.1.2 Preparation of complete medium for HEK293T cells and HCT116 cells]
D-MEM (High Glucose) is 88% by volume, FBS is 10% by volume, NEAA (non-essential amino acid, manufactured by Thermo Fisher Scientific) is 1% by volume, and ST-PN is 1% by volume with respect to the total volume of the medium. To prepare a complete medium for HEK293T cells and HCT116 cells.

[1.1.2 Cell passage]
[1.1.2.1 Passage of MIA-PaCa2 cells and HCT116 cells]
The medium during culture was sucked with an aspirator, and 3 ml of EDTA-PBS (−) containing 0.25% trypsin was added. It was placed in a 37 ° C., 5% CO ₂ incubator and allowed to react for 3-4 minutes. 9 ml of complete medium was added, the solution was sucked with an electric pipettor and sprayed onto the bottom of the dish repeatedly to peel off the cells. The detached cell suspension was sucked with an electric pipetter, and the tip of the pipette was lightly applied to the bottom of the dish and blown out repeatedly to make the cell clumps fine. The suspension was placed in a 50 ml tube and centrifuged at 1,000 rpm for 3 minutes at 20 ° C. The supernatant was aspirated with an aspirator, and 10 ml of complete medium was added and suspended. A portion of the cell suspension was transferred to a new dish containing a total of 12 ml of complete medium in advance. The amount transferred was 3 ml when the next passage was performed the next day, 1.5 ml when the second passage was performed, and 700 μl when the passage was performed three days later, and was adjusted according to the degree of cell proliferation. The cells were shaken to be uniform and cultured in a 37 ° C., 5% CO ₂ incubator.

[1.1.2.2 Passage of HEK293T cells]
The medium during culture was sucked with an electric pipettor and sprayed on the bottom of the dish to peel off the cells. The detached cell suspension was sucked with an electric pipetter, and the tip of the pipette was lightly applied to the bottom of the dish and blown out repeatedly to make the cell clumps fine. A portion of the cell suspension was transferred to a new dish containing a total of 12 ml of complete medium in advance. The amount transferred was 3.6 ml when the next passage was performed the next day, 1.2 ml when the next passage was performed 2 days later, and 500 μl when the passage was performed 3 days later, and was adjusted according to the degree of cell proliferation. The cells were shaken to be uniform and cultured in a 37 ° C., 5% CO ₂ incubator. In addition, when it was necessary to count the number of cells, it was performed by the same operation as the passage of MIA-PaCa2, but the treatment time of trypsin was 1 minute.

[1.2 Introduction of plasmid into cultured cells by lipofection]
25 μl of D-MEM to which nothing was added (hereinafter referred to as “serum-free D-MEM”) was placed in a well used in a 96-well plate. The plasmid mixture to be introduced was adjusted in advance to a total volume of 6 μl and added to serum-free D-MEM. 25 μl of a mixture of 25 μl of serum-free D-MEM and 0.7 μl of Lipofectamine ™ LTX (Thermo Fisher Scientific) was added per well and allowed to stand at room temperature for 30 minutes. During this time, a cell suspension was prepared by the technique described in the subculturing procedure, 10 μl was applied to the cell measurement slide, and the cell concentration was measured with a cell counter (manufactured by LUNA). The suspension and complete medium were mixed so that the cells had the desired concentration. After completion of the standing, 100 μl of the suspension of which the concentration was adjusted was added to each well and cultured in a 37 ° C., 5% CO ₂ incubator.

[2 Basic operations for plasmid construction]
[2.1 PCR amplification]
[2.1.1 PCR using PrimeSTAR (registered trademark) Max (manufactured by TaKaRa)]
0.2 μl of template solution, 2 × PrimeSTAR (registered trademark) Max Premix (manufactured by TaKaRa) 5.0 μl, 10 μM forward and reverse primers 0.5 μl each, autoclaved Milli-Q water (hereinafter referred to as “sterile water”) 3 8 μl was mixed. Using a thermal cycler, after preheating at 94 ° C. for 2 minutes, heat denaturation at 98 ° C. for 10 seconds, annealing at an appropriate temperature for 5 seconds, and adding 72 seconds at 72 ° C. for 5 seconds per 1 kb After performing a time extension reaction for 25 to 35 cycles, it was post-heated at 72 ° C. for 2 minutes and stored at 4 ° C.

[2.1.2 PCR using PrimeSTAR (registered trademark) GXL (TaKaRa)]
4. Template solution 1.0 μl, PrimeSTAR (registered trademark) GXL DNA Polymerase (TaKaRa) 0.2 μl, 2.5 mM each dNTP Mix (TaKaRa) 0.8 μl, 10 μM forward and reverse primers 0.3 μl, sterile water 5. 4 μl was mixed. Using a thermal cycler, after preheating at 94 ° C. for 2 minutes, heat denaturation at 98 ° C. for 10 seconds, annealing at an appropriate temperature for 30 seconds, and 68 seconds at 20 ° C. per 1 kb for 30 seconds. After performing a time extension reaction for 25 to 35 cycles, it was post-heated at 72 ° C. for 2 minutes and stored at 4 ° C.

[2.1.3 PCR using KOD FX Neo (Toyobo)]
Template solution 1.0 μl, KOD FX Neo (Toyobo) 0.2 μl, 2 × PCR Buffer (Toyobo) 5.0 μl for KOD FX Neo, 2 mM each dNTP Mix 2.0 μl, 10 μM forward and reverse primer 0. 3 μl and 1.2 μl of sterile water were mixed. Using a thermal cycler, after preheating at 94 ° C. for 2 minutes, heat denaturation at 98 ° C. for 10 seconds, annealing at an appropriate temperature for 30 seconds, and 30 seconds per 68 kb at 68 ° C. as a preliminary time was added. The time extension reaction was performed for 25 to 35 cycles and then stored at 4 ° C.

[2.2 Restriction enzyme treatment]
Mix DNA solution (400 ng for plasmid), restriction enzyme stock 0.3 μl, 10 × buffer 1.0 μl, if necessary 10 × BSA 1.0 μl, and sterilized water to make a total volume of 10 μl, and in a 37 ° C. incubator (When using BssHII as a restriction enzyme, it was incubated at 50 ° C. using a thermal cycler or a hybrid oven) for 1 hour to overnight. If necessary, 0.5 μl of rAPid Alkaline Phosphatase (Roche Life Science) was added to the solution after the reaction, and the mixture was further incubated at 37 ° C. for 1 hour.

[2.3 Agarose gel electrophoresis]
While heating in a microwave oven, 1 to 3% (w / v) Agarose S (manufactured by Nippon Gene) was dissolved in 1 × TAE buffer, poured into a gel maker, and a comb was inserted. The gel was solidified by standing at room temperature for about 30 minutes, and the solidified gel and 1 × TAE buffer were placed in the electrophoresis tank. If it is necessary to add the loading buffer to the sample solution and DNA size marker so that the final concentration is 1 × or more and apply to the well, and then DNA needs to be extracted from the gel, 50 V for about 1 hour, otherwise 100 V for 30 Electrophoresis was performed for about a minute. 2 μl of ethidium bromide was added with deionized water so that the gel soaked in the tapper, and the gel after electrophoresis was added and shaken for about 20 minutes. The gel was photographed with a UV transilluminator to confirm the band. When DNA extraction was necessary, the target band was cut out with a razor.

[2.4 Extraction of DNA from gel pieces]
[2.4.1 Method Using NucleoSpin (registered trademark) Gel and PCR Clean-up (TaKaRa)]
The mass of the cut-out agarose gel was measured, and 200 μl of Buffer NTI (manufactured by TaKaRa) was added per 100 mg of the gel piece. When there were a plurality of cut out samples, the amount of Buffer NTI (manufactured by TaKaRa) added was adjusted to the heaviest gel piece. While vortexing every 2-3 minutes, the gel pieces were dissolved by incubating at 50 ° C. for 10 minutes using a heat block. A column (NucleoSpin (registered trademark) Gel and PCR Clean-up Column, manufactured by TaKaRa) was set in a 2 ml collection tube, and the gel piece solution was applied. Centrifugation was performed at 11,000 × g for 30 seconds at 20 ° C., and the filtrate was discarded. 700 μl of Wash Buffer NT3 (TaKaRa) was applied to the column, centrifuged at 11,000 × g for 30 seconds at 20 ° C., and the filtrate was discarded. Without applying anything to the column, the membrane was dried by centrifugation at 11,000 × g for 30 seconds at 20 ° C., and the filtrate was discarded. The column was transferred to a new 1.5 ml tube for collection and 15 μl of sterile water was applied. The mixture was incubated at room temperature for 1 minute and centrifuged at 11,000 × g for 30 seconds at 20 ° C. The filtrate was returned onto the column and centrifuged at 11,000 × g for 30 seconds at 20 ° C. The filtrate was stored at -20 ° C.

[2.4.2 Method Using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega)]
The mass of the cut-out agarose gel was measured, and 100 μl of Membrane Binding Solution (manufactured by Promega) was added per 100 mg of the gel piece. When there were a plurality of cut out samples, the volume of the added membrane binding solution was adjusted to the heaviest gel piece. The gel pieces were dissolved by incubating at 55 ° C. for 10 minutes using a heat block while vortexing every 2 to 3 minutes. A column (SV Column, manufactured by Promega) was set in a collection tube, and a solution of gel pieces was applied. The mixture was incubated at room temperature for 5 minutes, centrifuged at 13,000 rpm at 4 ° C. for 1 minute, and the filtrate was discarded. 700 μl of Membrane Wash Solution (in EtOH) was applied to the column, centrifuged at 13,000 rpm at 4 ° C. for 1 minute, and the filtrate was discarded. 500 μl of Membrane Wash Solution (in EtOH) was applied to the column, centrifuged at 13,000 rpm for 1 minute at 4 ° C., and the filtrate was discarded. The column was transferred to a new 1.5 ml tube for collection, 15 μl of sterile water was applied to the column and incubated at room temperature for 1 minute. Centrifugation was performed at 4 ° C. and 13,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 13,000 rpm for 1 minute at 4 ° C. The filtrate was stored at -20 ° C.

[2.5 Ligation of linear DNA by In-Fusion (registered trademark) reaction]
The DNA solution on the vector side and the DNA solution on the insert side extracted from the gel were subjected to In-Fusion (registered trademark) reaction. The composition of the reaction solution was basically 5 × In-Fusion (registered trademark) Mix (manufactured by TaKaRa) 0.4 μl, vector-side DNA solution 0.8 μl, and insert-side DNA solution 0.8 μl. When it was performed again less, the scale was increased or the proportion of the DNA solution on the insert side was increased. Incubation was performed at 50 ° C. for 15 minutes using a thermal cycler or a thermostatic bath.

[2.6 Ligation of linear DNA by ligation reaction]
A vector-side DNA (end-dephosphorylated) solution 0.4 μl extracted from an agarose gel, an insert-side DNA solution 0.6 μl, 2 × Ligation Mix (manufactured by NIPPON GENE) 1.0 μl are mixed, and a 16 ° C. incubator is mixed. And incubated for 1 hour.

[2.7 Transformation into E. coli]
In a 1.5 ml tube, the sample solution was mixed with 10 times or more competent cells (XL10-Gold (Agilent Technologies) or Stable Competent E. coli (New England Biolabs)). The mixture was allowed to stand on ice for 10 minutes or more, incubated at 42 ° C. for 30 seconds using a heat block or a thermostatic bath, and ice-cooled for 2 minutes or more. When performing SOC recovery culture, 400 μl to 1 ml of SOC medium was added to the bacterial solution, and each 1.5 ml tube was placed in a 50 ml tube and cultured at 37 ° C. for 1 hour or more. The culture solution was centrifuged at 3,000 rpm for 5 minutes at 20 ° C., and a part of the supernatant was discarded and the rest was suspended. When the sample has an ampicillin resistance gene, the LB plate containing ampicillin (hereinafter referred to as “LB + ampicillin plate”), and when the sample has a spectinomycin resistance gene, the LB plate containing spectinomycin (hereinafter referred to as “LB + spectinoline”). The bacterial solution was spread on a “mycin plate” and cultured in a 37 ° C. incubator for 14 to 16 hours.

[2.8 Colony PCR]
[2.8.1 Colony PCR using KOD FX Neo]
A reaction solution was prepared by mixing 0.16 μl of KOD FX Neo, 4.0 μl of 2 × PCR Buffer for KOD FX Neo, 1.6 μl of 2 mM each dNTP Mix, 0.24 μl each of 10 μM forward and reverse primers, and 1.76 μl of sterile water. . The colony of Escherichia coli was picked up with the chip, and after touching the replica plate (LB + ampicillin plate), the chip was immersed in the reaction solution for a while. When performing multiple colony PCR from one colony, the chip with the colony was once immersed in 5 μl of sterilized water for a while, touched with two chips, and immersed in each PCR reaction solution for a while. Using a thermal cycler, after preheating at 94 ° C. for 2 minutes, heat denaturation at 98 ° C. for 10 seconds, annealing at an appropriate temperature for 30 seconds, and 30 seconds per 68 kb at 68 ° C. as a preliminary time was added. After 27 cycles of time extension reaction, the mixture was post-heated at 68 ° C. for 2 minutes and stored at 4 ° C. The whole or part of the product was electrophoresed on an agarose gel, and it was confirmed that a band of the desired length appeared.

[2.8.2 Colony PCR using SapphireAmp (registered trademark) Fast PCR Master Mix (TaKaRa)]
2 × SapphireAmp (registered trademark) Fast PCR Master Mix (manufactured by TaKaRa) 4.0 μl, 10 μM forward and reverse primers 0.16 μl each, and sterile water 3.68 μl were mixed. In the same manner as in the case of using KOD FX Neo, colonies of E. coli were added and clones were grown on replica plates. Using a thermal cycler, after preheating at 94 ° C for 2 minutes, heat denaturation at 98 ° C for 10 seconds, annealing at 60-66 ° C (depending on the primer), 5 seconds, preparatory for 10 seconds per kb at 72 ° C After 27 cycles of an extension reaction for 30 seconds as a time, the sample was stored at 4 ° C. after 72 minutes of post-heating at 72 ° C. The whole or part of the product was electrophoresed on an agarose gel, and it was confirmed that a band of the desired length appeared.

[2.9 Small-scale culture of E. coli]
If the sample has an ampicillin resistance gene in 3 ml of LB liquid medium in a test tube, 25 mg / ml ampicillin 12-30 μl or 10 mg / ml carbenicillin 30 μl, and if the sample has a spectinomycin resistance gene, 20 μl 10 mg / ml spectinomycin Was added. The replica plate colony of the clone from which the target band was obtained by colony PCR was picked with a chip, and the whole chip was dropped on the medium in the test tube. When re-amplifying an existing plasmid, a plurality of colonies were collected with a chip and dropped together with the chip into a medium in a test tube. The test tube was cultured with shaking at 37 ° C. for 14 to 16 hours.

[2.10 Small-scale preparation of plasmid from E. coli]
[2.10.1 Preparation of E. coli pellet from small-scale culture]
A small amount of culture medium (3 ml) was put into a 1.5 ml tube and centrifuged at 13,000 rpm for 1 minute at 4 ° C. The supernatant was discarded, and the rest of the culture was placed in a tube and centrifuged at 13,000 rpm for 1 minute at 4 ° C. The supernatant was discarded and proceeded to the next treatment or stored temporarily at -20 ° C.

[2.10.2 Small-scale preparation of plasmid using GenElute ™ Plasmid Miniprep Kit (Sigma-Aldrich)]
200 μl of a Resuspension Solution containing RNase A (Sigma-Aldrich) cooled at 4 ° C. was added, and the precipitate was suspended by vortexing. 200 μl of Lysis Solution (manufactured by Sigma-Aldrich) was added and mixed by inversion, and allowed to stand at room temperature for 3 minutes. 350 μl of Neutralization / Binding Buffer (manufactured by Sigma-Aldrich) was added and mixed by inversion, followed by centrifugation at 13,000 rpm for 10 minutes at 4 ° C. To prepare the column during centrifugation of the lysate, the column was set in a collection tube, and 500 μl of Column Preparation Solution (Sigma-Aldrich) was applied to the column and centrifuged at 13,000 rpm at 4 ° C. for 1 minute. The filtrate was discarded, and the centrifuged lysate supernatant was applied to the column and centrifuged at 13,000 rpm for 1 minute at 4 ° C., and the filtrate was discarded. 500 μl of Wash Solution 1 (manufactured by Sigma-Aldrich) was applied to the column, centrifuged at 13,000 rpm for 1 minute at 4 ° C., and the filtrate was discarded. 750 μl of Wash Solution 2 (manufactured by Sigma-Aldrich) containing EtOH was applied to the column and centrifuged at 13,000 rpm for 1 minute at 4 ° C., and the filtrate was discarded. Without applying anything to the column, the membrane was dried at 13,000 rpm for 1 minute at 4 ° C. to dry the membrane. The column was transferred to a 1.5 ml tube for collection, and 40 μl of Elution Buffer (manufactured by Sigma-Aldrich) was applied and centrifuged at 13,000 rpm for 1 minute at 4 ° C. The filtrate was returned to the column and centrifuged at 13,000 rpm for 1 minute at 4 ° C. The filtrate was saved.

[2.10.3 Measurement and adjustment of plasmid concentration and volume]
A small amount of the prepared plasmid solution was applied to NanoDrop (manufactured by NanoDrop Technologies), and the DNA concentration was measured. The entire volume of the remaining solution was sucked with a 200 μl pipetman, and the volume was measured by turning the dial to remove air. Based on these results, the amount of sterilized water necessary for the DNA concentration to be 200 ng / μl (or 100 ng / μl when the measurement result is 200 ng / μl or less) was calculated and added, and diluted.

[2.11 Sequence analysis]
[2.11.1 Analysis by ABI sequence]
[2.11.1.1.1 Cycle sequence reaction]
Plasmid 400 ng, BigDye (registered trademark) Terminator Ready Reaction Mix (Applied Biosystems) 1.0 μl, 5 × Sequence Buffer (Applied Biosystems) 1.5 μl, 3.2 μM primer 1.0 μl, sterilized so that the total volume is 10 μl Water was mixed. Using a thermal cycler, after preheating at 96 ° C. for 1 minute, heat denaturation at 96 ° C. for 10 seconds, annealing at 50 ° C. for 5 seconds, and extension reaction at 60 ° C. for 4 minutes were carried out for 25 cycles at 4 ° C. saved.

[2.1.1.1.2 Purification of cycle sequence reaction product]
This operation was performed while shielding light as much as possible. Immediately before the operation, 3 mM sodium acetate and 500 mM EDTA were mixed to prepare 1.5 mM sodium acetate / 250 mM EDTA. The cycle sequence reaction (10 μl) was mixed with 1.0 μl of 1.5 mM sodium acetate / 250 mM EDTA. 40 μl of 100% ethanol was added and suspended by vortexing. It left still at room temperature for 15 minutes, and centrifuged at 20 degreeC and 14,500 rpm for 15 minutes. The supernatant was discarded by decantation, and 100 μl of 70% ethanol was added and mixed by inversion, followed by centrifugation at 20 ° C. and 14,500 rpm for 5 minutes. The supernatant was discarded by decantation, and 100 μl of 70% ethanol was added again and mixed by inversion, followed by centrifugation at 20 ° C. and 14,500 rpm for 5 minutes. The supernatant was carefully removed and vacuum dried for about 10 minutes.

[2.1.1.1.3 Formamide treatment and ABI sequence analysis]
To the purified and vacuum dried cycle sequence reaction, 20 μl of Hi-Diformamide was added. 10 μl of the solution was dispensed into a 96-well plate, and the plate was incubated at 95 ° C. for 2 minutes using a thermal cycler and ice-cooled. In some cases, the heat treatment is first performed using a heat block and then dispensed into a plate. The plate was transported to a genetic experiment facility while being ice-cooled and shielded from light, and subjected to a genetic analyzer 3130 × 1 (Applied Biosystems) for sequence analysis.

[2.11.2 Sequence analysis by subcontracting]
400 ng of plasmid, 2.0 μl of 3.2 μM primer, and 10 μl of sterilized water were mixed in a 96-well plate, and sequence analysis was requested by FASMAC.

[3. Preparation of sgRNA2.0 and dCas9, and sgRNA2.0 and dCas9-VP64 all-in-one expression vector]
An all-in-one expression vector in which a plurality of sgRNA expression cassettes having an MS2 stem loop in the scaffold region and an expression cassette of a fusion of -dCas9 or dCas9 and a transcriptional activator was integrated was prepared ((a) of FIG. 3) .

[3.1 Preparation of STEP 1 for pX330A-MS2-1 × n-dCas9-VP64 and pX330S-MS2-2-7]
[3.1.1 Preparation of pX330A-MS2-1 × n]
Cas9 expression cassette and BsaI cleavage sequences 1 and 2 amplified by inverse PCR from a plasmid with normal sgRNA and Cas9 expression cassette and BsaI cleavage sequences 1 and 2 (pX330A-1 × 2, Addgene # 58766) SgRNA having an MS2 stem loop in Scaffold, amplified by PCR from a vector-side template having sgRNA and a plasmid (sgRNA (MS2) cloning backbone (Addgene # 61424)) having an sgRNA expression cassette having MS2 stem loop in Scaffold The template on the insert side with the expression cassette of 1 was ligated by In-Fusion (registered trademark), and the MS2 stem loop was connected to Scaffold. To sgRNA and Cas9 expression cassettes and BsaI plasmids with cleavage sequences 1 and 2 of the (pX330A-MS2-1 × 2) was prepared. An sgRNA expression cassette having an MS2 stem loop in Scaffold and a vector-side template having a BsaI cleavage sequence 1 excised from pX330A-MS2-1 × 2 prepared above with XhoI and BglII, and a normal sgRNA and Cas9 Of Cas9, which was excised with XhoI and BglII from the expression cassette and the plasmid with the BsaI cleavage sequences 1 and 3, 4, 5, 6, or 7 (pX330A-1 × 3-7, Addgene # 58767-58771) The cassette and the template on the insert side having the BsaI cleavage sequence 3, 4, 5, 6, or 7 were ligated together by ligation, and the expression cassette of sgRNA and Cas9 having the MS2 stem loop in Scaffold. Plasmids with Tsu cleavage sequence bets and BsaI 1 and 3, 4, 5, 6 or 7, (pX330A-MS2-1 × 3~7) were prepared.

[3.1.2 Preparation of pX330A-MS2-1 × n-dCas9-VP64]
Vector-side template opened downstream of dCas9 by inverse PCR of normal sgRNA and dCas9 expression cassettes and plasmids (pX330A-dCas9-1 × 5, Addgene # 63599) having BsaI cleavage sequences 1 and 5 VP64 sequences were ligated together with In-Fusion® to produce a plasmid (pX330A-1 × 5-dCas9-VP64) having the normal sgRNA and dCas9-VP64 expression cassette and BsaI cleavage sequences 1 and 5 did. Using the above-prepared pX330A-1 × 5-dCas9-VP64 and a primer having BsaI cleavage sequence 2, 3, 4, 6, or 7 added to the 5 ′ side, BsaI cleavage sequence 5 Plasmids that are opened by inverse PCR, self-closed by In-Fusion®, and have normal sgRNA and dCas9-VP64 expression cassettes and BsaI cleavage sequences 1 and 2, 3, 4, 6, or 7 (PX330A-1 × 2-4, 6, 7-dCas9-VP64) was prepared. The vector-side template having the sgRNA expression cassette having the MS2 stem loop in Scaffold and the BsaI cleavage sequence 1 excised from the pX330A-MS2-1x2 prepared above with NotI and XhoI, and the pX330A-1 prepared above A template on the insert side having the expression cassette of dCas9-VP64 and the cleavage sequence 2, 3, 4, 5, 6, or 7 of dCas9-VP64 excised from Not. 2-7-dCas9-VP64 with NotI and XhoI was ligated by ligation. The ligated and plasmid containing the expression cassette of sgRNA with MS2 stem loop in Scaffold and dCas9-VP64 and the cleavage sequence 1 and 2, 3, 4, 5, 6, or 7 of BsaI (pX330A- S2-1 was produced × 2~7-dCas9-VP64).

[3.1.3 Preparation of pX330S-MS2-2-7]
A vector-side template having a Cas9 expression cassette and a BsaI cleavage sequence, PCR-amplified from a plasmid (pX330S-2, Addgene # 58778) having a normal sgRNA and Cas9 expression cassette and a BsaI cleavage sequence 2, and a sgRNA ( MS2) A template on the insert side having an sgRNA expression cassette having MS2 stem loop in Scaffold, which was PCR-amplified from cloning backbone (Addgene # 61424), was ligated by In-Fusion (registered trademark), and MS2 stem loop was connected to Scaffold. A plasmid (pX330S-MS2-2) having an sgRNA and Cas9 expression cassette and a BsaI cleavage sequence 2 was prepared. Inverse such that pX330S-MS2-2 prepared above is removed from BsaI cleavage sequence 2 using a primer having a sequence having BsaI cleavage sequence 3, 4, 5, 6, or 7 added to the 5 ′ side. Plasmids with sgRNA having MS2 stem loop in Scaffold and dCas9-VP64 expression cassette and BsaI cleavage sequence 3, 4, 5, 6, or 7 opened by PCR, self-closed by In-Fusion® pX330S-MS2-3-7) was prepared.

[3.2 Design of sgRNA using Web tools]
A nucleotide sequence in a range where sgRNA design is desired was input to CRISPR-Direct (https://crispr.dbcls.jp/), and candidate sgRNA was output. The extent to which they act on which sequence on the genome was examined by COSMID (https://crispr.bme.gatech.edu/). In order to avoid the off-target effect, a parameter indicating that the smaller the value is, the more likely it is to act, is 0.5 or more in a sequence other than the target.

[3.3 STEP1: Preparation of each sgRNA expression cassette]
Each sgRNA expression cassette to be integrated into an all-in-one expression vector was first made on a separate vector. When n sgRNA expression cassettes are ligated, the first template is the pX330A-MS2-1 × n-dCas9, pX330A-MS2-1 × n-dCas9-VPR prepared in the above item, and the inventors' The 2nd to n-th templates were inserted into the existing pX330A-MS2-1 × n-dCas9-VP64 in the laboratory and the existing pX330S-MS2-2 to n in the inventor's laboratory.

[3.3.1 Annealing of template DNA for target sequence]
0.5 μl each of sense strand and antisense strand oligo DNA (100 μM) ordered for the template of the target sequence of sgRNA, 1.0 μl of 10 × annealing buffer, and 8.0 μl of sterile water were mixed. After incubating at 95 ° C. for 5 minutes using a thermal cycler, the temperature was lowered to 25 ° C. over 90 minutes by the slope function, and the two oligo DNAs were annealed. The oligo DNA sequences used are shown in Table 1.

[3.3.2 Insertion of template into sgRNA expression cassette]
50 ng / μl vector 0.3 μl, annealed oligo DNA 0.5 μl, BpiI 0.1 μl, Quick Ligase 0.1 μl, 10 × T4 Ligase Buffer 0.2 μl, and sterilized water 0.8 μl were mixed. After a restriction enzyme reaction at 37 ° C. for 5 minutes, a ligation reaction at 16 ° C. for 10 minutes was taken as 1 cycle, and 3 cycles were performed using a thermal cycler and stored at 4 ° C. To the reaction solution (2.0 μl), 0.1 μl of BpiI and 0.2 μl of 10 × Buffer G were added and incubated at 37 ° C. for 60 minutes using a thermal cycler to cleave the vector in which ligation did not occur. Thereafter, the enzyme was inactivated by incubation at 80 ° C. for 5 minutes, and stored at 4 ° C. The reaction solution was transformed into Escherichia coli, and colonies were formed on the LB + ampicillin plate for the first template and on the LB + spectinomycin plate for the second to nth templates. Colonies were transferred to LB medium and plasmids were extracted.

[3.3.3 Confirming template insertion by BpiI processing]
The plasmid into which the template was inserted or the plasmid before insertion was treated with BpiI for 1 hour, and the reaction solution was electrophoresed on a 1% agarose gel. BpiI differs from the DNA recognition sequence in the cleavage site, and is designed so that the BpiI recognition sequence is lost after template insertion. Therefore, it was confirmed that the template was inserted because no cleavage by BpiI occurred. Moreover, it was confirmed that the restriction enzyme treatment was correctly performed by cutting the plasmid before inserting the template with BpiI.

[3.4 Ligation of sgRNA expression cassette by Golden-Gate Assembly]
The second and subsequent sgRNA expression cassettes were inserted and integrated into a vector having the first sgRNA expression cassette and the dCas9 expression cassette, and a vector having the first sgRNA expression cassette and the dCas9-Effector expression cassette.

[3.4.1 Golden-Gate Assembly and Transformation]
5 ng / μl of template inserted pX330A-MS2-1 × n-dCas9 (or pX330A-MS2-1 × n-dCas9-VP64, pX330A-MS2-1 × n-dCas9VPR) 0.3 μl, template inserted 100 ng / Μl of pX330S-MS2-2 to n 0.3 μl each, BsaI-HF 0.2 μl, Quick Ligase 0.2 μl, 10 × T4 Ligase Buffer 0.4 μl, and sterilized water were mixed so that the total amount was 4.0 μl. After a restriction enzyme reaction at 37 ° C. for 5 minutes and a ligation reaction at 16 ° C. for 10 minutes as one cycle, using a thermal cycler, two sgRNA expression cassettes are ligated six cycles, and four ligation reactions are 25 ˜35 cycles were performed and stored at 4 ° C. The reaction solution was transformed into Escherichia coli, spread on an LB + ampicillin plate previously spread with 75 μl of SOC medium, 5.0 μl of IPTG, and 20 μl of x-gal, and incubated at 37 ° C. for 16 hours.

[3.4.2 Confirmation of sgRNA expression cassette ligation by colony PCR]
pX330A-MS2-1 × n-dCas9 (dCas9-VP64-VPR) expresses the LacZ gene in E. coli, and blue colonies are formed from the medium containing X-gal. On the other hand, when an sgRNA expression cassette is inserted into this, the LacZ gene is disrupted and does not function, and a white colony is formed. A white colony was subjected to colony PCR at an annealing temperature of 66 ° C. using a primer pair sandwiching a region where sgRNA expression cassettes are lined up, and electrophoresed on a 1-1.8% agarose gel. From the length of the band, it was confirmed that the desired number of sgRNA expression cassettes were contained. A plasmid was extracted from the confirmed clone.

[4. Preparation of MS2-22sTag expression vector]
[4.1 Preparation of MS2-4 × and 8 × 22sTag expression vectors]
First, a vector (MS2-10 × SunTag) expressing a conventional SunTag linked to MS2 was prepared. The MS2-10 × SunTag vector is a vector having a CMV promoter and a polyA addition sequence (ptCMV-136 / 63-VR-NG, Addgene # 50700), and the MS2 portion as a coding sequence is MS2-P65-HSF1_GFP (Addgene # 61423). Thus, 10 × SunTag portions were respectively amplified from pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP (Addgene # 60903) and inserted by In-Fusion (registered trademark). Thereafter, the 10 × SunTag sequence was replaced with the 4 × and 8 × 22 sTag sequences, respectively (FIG. 3B).

[4.1.1 Replacement of 10 × SunTag array to 4 × 22 sTag and 8 × 22 sTag by In-Fusion®]
Primers were designed so as to remove the 10 × SunTag region from MS2-10 × SunTag, and inverse PCR was performed at an annealing temperature of 67 ° C. and 35 cycles to amplify the sequence on the vector side. In addition, by combining different primers, three types of insert sequences for MS2-4 × 22 sTag, MS2-8 × 22 sTag for 1st to 4th and MS2-8 × 22 sTag for 5th to 8th are synthesized artificially (IDT , GBlock (registered trademark) 4 × 22 sTag, PCR amplification was performed at an annealing temperature of 63 ° C. and 25 cycles. For MS2-4 × 22sTag, 5 μl In-Fusion (registered trademark) Mix 1.0 μl, vector side DNA solution 1.6 μl, and insert side DNA solution 1.6 μl were mixed. Each reaction solution was electrophoresed to cut out a band and extract DNA. For MS2-8 × 22sTag, 5 × In-Fusion (registered trademark) Mix 1.0 μl, vector 0.5 μl, first to fourth Tag inserts 3.0 μl (increase due to weak band signal), 5-5 Mix 0.5 μl of 8th Tag insert. Each was subjected to In-Fusion (registered trademark) reaction and transformed into E. coli. The primers used are shown in Table 2.

[4.1.2 Screening by colony PCR]
The 4 × and 8 × 22 sTag inserts have similar sequence repeats, and the number of tags may change due to recombination. Therefore, colony PCR was performed at an annealing temperature of 60 ° C. using a primer pair designed on the vector side so as to sandwich the insert region. The product was electrophoresed on a 2% agarose gel, and a plasmid was extracted from a clone from which a band considered to be the desired length was obtained.

[4.1.3 Screening by restriction enzyme treatment]
The extracted plasmid was treated with SacI (cutting both the vector side and the insert side) overnight, and electrophoresed on a 1.6% agarose gel to confirm that the desired band appeared. The results are shown in FIG.

[4.1.4 Confirmation of In-Fusion (Registered Trademark) Region and Insert Sequence by Sequence Analysis]
Primers in the direction toward the In-Fusion (Registered Trademark) region were designed in a region on the vector side that was approximately 200 bp away from the two In-Fusion (Registered Trademark) regions, and sequence analysis was performed. As a result of the analysis, in all samples, a 51 bp insertion was included in the linker connecting the last Tag and NLS sequences. Later, it was discovered that the In-Fusion (registered trademark) primer design was incorrect, and this was considered to be the cause. However, since there was no stop codon within 51 bp and no frame shift occurred, it was judged that there was no effect on functionality and was used as it was. When actually used, the functionality was confirmed.

[4.2 Preparation of MS2-16 × and 24 × 22 sTag expression vectors]
One or two 8 × 22 sTag sequences were inserted into the MS2-8 × 22 sTag of the above items to prepare vectors expressing MS2-16 × and 24 × 22 sTag (FIG. 3B).

[4.2.1 Insertion of sequence by In-Fusion (registered trademark)]
For amplification on the vector side, a primer was designed so as to open the downstream of MS2-8 × 22sTag. For amplification on the insert side, an additional sequence for In-Fusion (registered trademark) is attached, and a primer for amplifying 8 × 22 sTag from MS2-8 × 22 sTag is used as a primer for MS2-16 × 22 sTag, MS2- Designed for 9th to 16th of 24 × 22 sTag and 17th to 24th of MS2-24 × 22 sTag, respectively. These were used for PCR amplification at an annealing temperature of 67 ° C. and 25 cycles. Each reaction solution was electrophoresed to cut out a band and extract DNA. For MS2-16 × 22sTag, the vector side solution and the insert side solution were mixed at a volume ratio of 1: 1. For MS2-24 × 22sTag, the vector side solution and the two types of insert side solutions were mixed at a ratio of 2: 1: 1. In-Fusion (registered trademark) reaction was performed to transform E. coli.

[4.2.2 Screening by colony PCR]
Colony PCR was performed in the same manner as MS2-4x and 8x22sTag. The product was electrophoresed on a 1% agarose gel. Since the band was unclear, the plasmid was extracted from a clone that might have a band of the desired length.

[4.2.3 Screening by restriction enzyme treatment]
The extracted plasmid was treated overnight with NotI (cleaved on both sides of n × 22sTag) and electrophoresed on a 1% agarose gel to confirm that a band with a length corresponding to the number of tags of interest was produced. (FIG. 4).

[4.2.4 Confirmation of In-Fusion (Registered Trademark) Region Sequence by Sequence Analysis]
The 3′-most In-Fusion (registered trademark) sequence was confirmed by sequence analysis using a primer on the vector side. The remaining one or two In-Fusion (registered trademark) regions exist in the 22sTag repetitive sequence. Thus, overnight treatment was performed with BssHII cut at two sites per 8 × 22 sTag and electrophoresis was performed, and a band having a length including the In-Fusion (registered trademark) region was cut out to extract DNA. Although it contains fragments at a plurality of locations in the vector, they all have the same sequence, and therefore, a sequence analysis was conducted collectively to confirm that there was no mutation in the In-Fusion (registered trademark) sequence. Moreover, the arrangement | sequence of the other area | region was also confirmed in the range which can be read.

[5. Preparation of MS-Effector and scFv-Effector expression vectors]
MS2-p65-HSF1 (Addgene # 61423) was cloned into a CMV expression vector to produce an expression vector for MS2-p65-HSF1. The MS2-VPR expression vector was prepared by replacing p65-HSF1 of MS2-p65-HSF1 cloned into the CMV expression vector with VPR (VP64-p65-Rta) (Addgene # 63798). scFv-sfGFP-VP64-GB1 (Addgene # 60904) was cloned into a CMV expression vector to prepare an expression vector for scFv-sfGFP-VP64-GB1. The scFv-sfGFP-VP64-GB1 VP64 cloned into the CMV expression vector was replaced with p65-HSF1 and VPR, respectively, and scFv-sfGFP-p65-HSF1-GB1 and scFv-sfGFP-VPR-GB1 expression vectors were prepared. ((C) of FIG. 3).

[6 Preparation of luciferase reporter vector]
Luc2 cDNA (manufactured by Promega) was ligated downstream of the promoter sequence and 5′UTR sequence of the target gene (FIG. 5B and FIG. 18B).

[6.1 Extraction of genome from cultured cells]
[6.1.1 Cell recovery]
The same operation as the above-mentioned passage of MIA-PaCa2 cells was performed until suspension after centrifugation. 10 μl of the suspension was applied to a cell measurement slide, and the cell concentration was measured with a Cell Counter. The suspension was placed in a 1.5 ml tube so that it would be 5 × 10 ⁶ cells or less, which is the limit of treatment in the kit described later, and centrifuged at 20 ° C. and 1,000 rpm for 3 minutes, and the supernatant was discarded. 1 ml of PBS (-) was added to suspend, and centrifuged at 20 ° C and 1,000 rpm for 3 minutes to discard the supernatant. The precipitate was suspended in 200 μl of PBS (−).

[6.1.2 Extraction of genome from cells using DNeasy Blood & Tissue kit (from Quiagen)]
To the cell suspension, 20 μl of Proteinase K (manufactured by Quiagen) and 200 μl of Buffer AL (manufactured by Quiagen) were added and suspended by vortexing. Incubation was carried out at 56 ° C. for 10 minutes using a thermostatic bath. 200 μl of 100% EtOH was added, suspended by vortexing, and applied to a column (DNeasy Mini Spin Column, manufactured by Quiagen) set in a 2 ml collection tube. The mixture was centrifuged at 20 ° C. and 8,000 rpm for 1 minute, and the eluate was discarded. 500 μl of Buffer AW1 (manufactured by Quiagen) containing EtOH was applied to the column, centrifuged at 8,000 rpm at 20 ° C. for 1 minute, and the filtrate was discarded. 500 μl of Buffer AW2 (manufactured by Quiagen) containing EtOH was applied to the column, centrifuged at 14,000 rpm for 3 minutes at 20 ° C., and the filtrate was discarded. Without applying anything to the column, it was centrifuged at 14,000 rpm for 3 minutes at 20 ° C. to dry the membrane. The column was transferred to a 1.5 ml tube for recovery, 50 μl of Buffer AE (manufactured by Quiagen) was applied, and centrifuged at 20 ° C. and 8,000 rpm for 1 minute. The filtrate was returned to the column and centrifuged at 20 ° C. and 8,000 rpm for 1 minute. The DNA concentration and volume were measured in the same manner as in the above plasmid concentration adjustment, and diluted with sterilized water to a concentration of 100 ng / μl.

[6.2 Replacement of sequence by In-Fusion (registered trademark)]
Using a primer that removes the pminCMV sequence from pminCMV-Luc2 with an additional sequence for In-Fusion (registered trademark), inverse PCR is performed at an annealing temperature of 62 ° C. and 35 cycles to amplify the vector sequence. did. In addition, a primer that amplifies the promoter sequence of the target gene (from the most upstream target sequence to immediately upstream of the start codon) is designed, PCR is performed from the genome extracted from HEK293T cells at an annealing temperature of 68 ° C. at 35 cycles, and then inserted. The side sequence was amplified. Each reaction solution was electrophoresed to cut out a band and extract DNA. The DNA solution on the vector side and the insert side were mixed and subjected to In-Fusion (registered trademark) reaction, and transformed into E. coli.

[6.3 Screening by colony PCR]
Colony PCR was performed using a primer pair designed for each of the vector side and the insert side. The product was electrophoresed and it was confirmed that a band of the desired length appeared. A plasmid was extracted from the confirmed clone.

[6.4 Screening by restriction enzyme treatment]
Treat pCDH1-Luc2 with EcoT14I and pRANKL-Luc2 with SacI (both cut one or more of the vector region and insert region, respectively), and confirm that a band of the desired length appears by electrophoresis of the reaction solution. did.

[6.5 Confirmation of sequence by sequence analysis]
The sequence was read from the primer designed on the vector side so as to sandwich the insert region, and it was confirmed that the sequence of the In-Fusion (registered trademark) region and the insert was correct. Several sequences differing from the database were observed in units of one base, but they were not used as targets, so they were used as they were.

[7 Luciferase Reporter Assay]
Whether the system constructed above was working was examined by luciferase reporter assay.

[7.1 Transfection into cells]
100 ng of the luciferase reporter vector prepared in 6, 20 ng of the R-Luc expression vector for correction, and 100 ng of the total amount of the expression vector of the constructed system were introduced into 6.0 × 10 ⁴ cells by the method of 1.2.

[7.2 Measurement of activity with Dual-Glo (registered trademark) Luciferase Assay System (Promega)]
75 μl of 150 μl of medium in each well of the culture plate was removed, and 75 μl of Luciferase substrate (in Dual-Glo® Luciferase buffer (manufactured by Promega)) was added. The mixture was allowed to stand for 10 minutes or more while being shielded from light, and the activity of luciferase was measured using TriStar LB941 Multimode Microplattenser (Berthold Technologies). Subsequently, 75 μl of R-Luc substrate (Dual-Glo (registered trademark) Stop% Glo (registered trademark) buffer (manufactured by Promega)) was added, and the mixture was allowed to stand for 10 minutes or more while being shielded from light. Thereafter, R-Luc activity was measured with TriStar LB941 to correct Luciferase activity.

[8 Evaluation of mRNA expression induction for endogenous genes]
The transcription-inducing activity at the mRNA level for the endogenous locus of the system constructed above was evaluated by real-time PCR.

[8.1 Transfection into cells]
A total amount of 200 ng of the expression vector of the system constructed above was introduced into 3.0 × 10 ⁴ cells by the method of 1.2.

[8.2 Synthesis of cDNA from cells by SuperPrep (registered trademark) Cell Lysis Kit for qPCR (Toyobo Co., Ltd.)]
[8.2.1 Preparation of cell lysate]
The culture medium of cells in culture was removed from a 96-well plate, and 100 μl of PBS was added. PBS was aspirated, 49.7 μl of Lysis Solution (Toyobo Co., Ltd.) and 0.3 μl of gDNA Remover (Toyobo Co., Ltd.) were added and incubated at room temperature for 5 minutes to disrupt the cells and decompose the genomic DNA. A mixed solution of 9.5 μl of Stop Solution (Toyobo) and 0.5 μl of RNase inhibitor (Toyobo) was added to stop the lysis reaction, incubated at room temperature for 2 minutes or more, and placed on ice.

[8.2.2 RT-PCR]
5 × RT Master Mix (Toyobo Co., Ltd.) 8.0 μl, Nuclease-free Water 24 μl, and lysate 8.0 μl were mixed. When taking no-RT Control, 5 × no-RT 8.0 μl, Nuclease-free Water 24 μl, and lysate 8.0 μl were mixed. Using a thermal cycler, the mixture was reacted at 37 ° C. for 15 minutes, 50 ° C. for 5 minutes, and 98 ° C. for 5 minutes, and stored at 4 ° C. Then, it moved to -20 degreeC.

[8.3 Real-time PCR]
[8.3.1 Preparation of reaction solution]
Templates are divided into three types: Standard Sample (calibration curve), Unknown Sample, and Negative Control. For the Standard Sample, the cDNA of a cell in which the gene is expressed to some extent was used, and in the absence, the sample of the cDNA expected to have the highest increase in expression was used. A calibration curve was prepared by diluting in steps of 2 to 1/10 and plotting 6 points. For the Unknown Sample, the cDNA of the sample to be measured was appropriately diluted with RNase-free water so that the CT value was within the plot of the calibration curve. RNase-free water and no-RT Control were used for Negative Control, and no-RT Control was diluted at the same magnification as Unknown Sample. 2 × KOD SYBR® qPCR Mix (Toyobo) 10 μl, 50 × Rox Reference Dye (Toyobo) 0.4 μl, 10 μM forward and reverse primers 0.2 μl each, template 1.0 μl, RNase-free water 8.2 μl Were mixed. The primers used are shown in Table 3.

[8.3.2 Real-time PCR by StepOnePlus ™ Real-time PCR System (Applied Biosystems)]
After preheating at 98 ° C. for 2 minutes, 40 cycles of thermal denaturation at 98 ° C. for 10 seconds, annealing at the studied temperature for 10 seconds, and extension reaction at 68 ° C. for 30 seconds were performed. In each cycle, the fluorescence intensity of the intercalator was measured during the extension reaction. After 40 cycles, melting curve analysis was performed at 95 ° C. for 15 seconds, 60 ° C. for 1 minute, and 95 ° C. for 15 seconds. By measuring the fluorescence intensity stepwise in the process of raising the temperature from 60 ° C. to 95 ° C., it was confirmed that the sequence other than the target was not amplified. After completion of the measurement, the expression level of the target gene was divided by the expression level of RPL8 of the internal control and corrected.

[9 Immunostaining]
Regarding the CDH1 gene in MIA-PaCa2 cells, whether or not a protein (E-Cadherin, manufactured by Abcam (ab40772)) was expressed by the system constructed above was evaluated by immunostaining.

[9.1 Transfection into cells]
A total amount of 200 ng of the expression vector of the system constructed above was introduced into 6.0 × 10 ⁴ cells by the method of 1.2.

[9.2 Transfer from 96-well plate to 24-well plate]
24 hours after the transfection, the medium was removed from the well, 50 μl of EDTA-PBS (−) containing 0.25% trypsin was added, and the mixture was reacted at 37 ° C. in a 5% CO ₂ incubator for 3 minutes. The reaction was stopped by adding 150 μl of complete medium, and the whole amount was added to a 24-well plate in which 300 μl of complete medium had been placed in advance. The cells were cultured in a 37 ° C. incubator. The medium was changed after 24 hours.

[9.3 Immunostaining]
After 72 hours from the transfection, the medium was removed from the wells, and the cells were washed twice with 1 ml of 1 × PBS (−). The cells were fixed by adding 250 μl of 4% paraformaldehyde and allowing to stand for 15 minutes. The cells were washed 3 times with 1 ml of 1 × PBS (−), and 250 μl of PBS (−) containing 1% Triton X-100 was added and allowed to stand for 20 minutes to permeabilize the cells. The plate was washed 5 times with 1% PBS (−), and 250 μl of PBS containing 1% BSA was added and left to stand for 1 hour for blocking. 200 μl of a primary antibody reaction solution (1: 100 in PBS containing 1% BSA) was added and reacted at 4 ° C. overnight. Washing with 1 × PBS (−) for 5 minutes × 3 times, reaction solution of secondary antibody conjugated with fluorescent substance (Alexa647-conjugated anti-rabbit secondary antibody (manufactured by Thermo Fisher Scientific), in PBS containing BSA, 1: 1,000) 200 μl was added and reacted at room temperature for 1 hour. After washing 3 times with 1 × PBS (−), 200 μl of DAPI (1: 100 in 1 × PBS (−)) was added and allowed to react at room temperature for 15 minutes. Washed twice with 1 × PBS (−). The sample was observed with an OLYMPUS FV-1000 confocal laser microscope in a state where PBS was contained.

[10 Western blotting]
Whether the target gene was expressed at the protein level by the system constructed above was evaluated by Western blotting.

[10.1 Transfection into cells]
A 4-well portion of a 96-well plate was used as a unit for one sample. A total amount of 200 ng of the expression vector of the constructed system per well was introduced into 6.0 × 10 ⁴ cells by the method of 1.2.

[10.2 Transfer from 96-well plate to 6-well plate]
24 hours after the transfection, the cells were detached from the wells in the same manner as in 8.2. 1.2 ml of complete medium was placed in a 6-well plate, and a cell suspension for 4 wells of a 96-well plate was added. The cells were cultured in a 37 ° C. incubator. The medium was changed after 24 hours.

[10.3 Preparation of protein sample]
[10.3.1 Extraction of protein from cells]
72 hours after the transfection, the medium was removed from the wells and washed twice with 1 ml of 1 × PBS (−). Add 60 μl of Lysis Buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 × Protease Inhibitor Cocktail (Roche)), peel the cells with a cell scraper, and remove 1.5 ml tube. Recovered. While cooling the tube with ice, the ultrasonic wave was flushed 10 times and centrifuged at 12,000 rpm at 4 ° C. for 1 minute. The supernatant was collected in a new 1.5 ml tube and stored at -70 ° C.

[10.3.2 Measurement of protein concentration]
The Protein assay solution and 5 volumes of Milli-Q water were mixed and centrifuged at 4 ° C. and 3,000 rpm for 5 minutes. 500 μl of the supernatant and 5 μl of the protein solution were mixed and allowed to stand at room temperature for 5 minutes, and the absorbance at 595 nm was measured with a spectrophotometer. At this time, a calibration curve was prepared by measuring the absorbance of PBS containing various concentrations of BSA up to 0.5 μg / ml. The protein sample was appropriately diluted with 1 × PBS (−) and measured so as to be within the range of the calibration curve plot.

[10.4 Western blotting]
[10.4.1 SDS-PAGE]
Separation gel buffer (10% Acrylamide, 380 mM Tris-HCl (pH 8.8), 0.1% SDS, 0.1% APS) was prepared, and 6.0 μl of TEMED was added and poured into a glass plate. From the top, 1 ml of sterilized water was poured to prevent polymerization inhibition due to contact with air, left at room temperature for 1 hour or at 37 ° C. for 15 minutes, and stored at 4 ° C. overnight. A stacking gel buffer (4.9% Acrylamide, 125 mM Tris-HCl (pH 6.8), 0.1% SDS, 0.1% APS) was prepared by removing sterilized water, and 8.0 μl of TEMED was added to the stacking gel. Shed on top. The comb was inserted and allowed to stand for 1 hour at room temperature or 30 minutes at 37 ° C. Meanwhile, 1/3 volume of 4 × sample buffer (250 mM Tris-HCl (pH 6.8), 8% SDS, 40% Glycerol, 0.02% BPB (bromophenol blue), 40 mM DTT (dithiothreitol) was added and heat-treated at 98 ° C. for 5 minutes using a heat block. The completed gel was placed in an electrophoresis apparatus containing an appropriate amount of 1 × running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) in the lower layer, and 1 × running buffer was also placed in the upper layer. The comb was removed, the wells were cleaned, and 2.5 μl of protein sample or protein size marker in an appropriate amount was applied. In addition, 2.5 μl of 4 × sample buffer was applied to empty lanes. The electrophoresis apparatus was connected to a power supply apparatus, and electrophoresis was performed at a constant current of 10 mA / gel for 180 minutes.

[10.4.2 Blotting on PVDF membrane]
The PVDF membrane was immersed in 100% methanol for 5 minutes, and then activated by immersing in 1 × blotting buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 27% methanol) for 10 minutes. Place three filter papers moistened with 1 × blotting buffer, activated PVDF membrane, separation gel after migration, 3 filter papers moistened with 1 × blotting buffer, on top of the negative electrode. I put it on. The electrode was connected to a power supply device and energized for 90 minutes at a constant current of 150 mA / gel.

[10.4.3 Antibody Reaction]
The membrane after blotting was soaked in PBS containing 5% skim milk and shaken for 1 hour to block. The membrane was rinsed twice with 1 × PBST (1 × PBS, 0.05% Tween-20), and further washed with shaking for 15 minutes × 1, 5 minutes × 2 times. The membrane was sandwiched between the hybrid bags and encapsulated with 3 ml of a primary antibody reaction solution (1: 1000 in Can Get Signal (registered trademark) Solution 1 (manufactured by Toyobo)). The reaction was allowed to proceed overnight at 4 ° C. The next day, the membrane was washed with 1 × PBST by shaking for 5 minutes × 3 times, and sandwiched with 3 ml of secondary antibody reaction solution (Can Get Signal (registered trademark) Solution 2 (Toyobo Co., Ltd., 1: 250)) sandwiched between hybrid bags. did. The reaction was allowed to proceed for 1 hour at room temperature.

[10.4.4 Band Detection]
The membrane after the secondary antibody reaction was shaken and washed with 1 × PBST for 5 minutes × 3 times, placed on parafilm, and a SuperSignal ™ West Pico PLUS Chemiluminescent Substrate (manufactured by Thermo Scientific) reaction mixture (Luminol / Enhancer Solution: Peroxide Solution = 1: 1) 2 ml was placed and allowed to stand for 5 minutes. The membrane was sandwiched between hybrid bags, exposed to an X-ray film (manufactured by Fuji Film) in a dark room, and immersed in a developer. When the band was visible, it was transferred to the fixing solution, and when the film turned blue, it was transferred to the stop solution. While changing the exposure time, it was repeated several times until it became a band of comparable intensity. The developed film was read with a scanner, and the intensity of the band was measured with Image J (http://imagej.nih.gov/ij/).

[11 Functional verification of 3rd generation artificial gene expression enhancement system targeting CDH1 gene]
In the verification of this item, unless otherwise specified, the method described in the items 1 to 9 was used.

[11.1 Verification-1]
In order to verify the functionality of the third generation system in one embodiment of the present invention, an assay is performed in the presence / absence of three vectors, dCas9-VP64, MS2-4 × 22sTag, and scFv-p65-HSF1. It was. Further, targeting the promoter region of the CDH1 gene, five types of sgRNAs were designed as shown in FIG. 5 (a) and FIG. Using the designed five types of sgRNA, the functionality was compared with the first-generation and second-generation artificial gene expression enhancement systems by a plasmid-based reporter assay. As the cells, MIA-PaCa2 cells were used. The results are shown in FIG. 6 and FIG.

  The third generation artificial gene expression enhancement system (FIG. 1 (c)), which includes all of dCas9-VP64, MS2-4 × 22sTag, and scFv-p65-HSF1, converts MS2-4 × 22sTag and scFv-p65-HSF1. 1st generation artificial gene expression enhancing system (FIG. 1 (a)) and 2nd generation artificial gene expression enhancing system containing MS2-p65-HSF1 instead of MS2-4 × 22sTag and scFv-p65-HSF1 ( It became clear that the activity of luciferase becomes higher compared to (b) of FIG. That is, it is clear that the effect of enhancing gene expression is higher than that of the first-generation and second-generation artificial gene expression enhancement systems, whether it is five sgRNAs or only a single sgRNA. It became.

[11.2 Verification-2]
Subsequently, dCas9-VP64 and MS2-n × 22sTag (n = 4, 8, 16, 24) in which the number of 22sTag is 4, 8, 16, 24 ((a) to (d) in FIG. 2) , ScFv-p65-HSF1 or scFv-VPR, and a reporter assay was performed in the same manner as in 10.1. The total amount of expression vector in the constructed system was 50 ng and 100 ng. The results are shown in FIG. 8 and FIG.

  Among the four types of MS2-n × 22sTag, it was revealed that MS2-8 × 22sTag had the highest luciferase activity, so that the effect of enhancing gene expression was enhanced. It was also found that the effect of enhancing gene expression was increased in a dose-dependent manner with respect to the amount of expression vector.

[11.3 Verification-3]
CDH1 gene expression was confirmed at the mRNA level by quantitative RT-PCR. dCas9-VP64, MS2-n × 22sTag (n = 4, 8) or MS2-p65-HSF1, and scFv-p65-HSF1 or scFv-VPR were used. All five types of sgRNA were used. The results are shown in FIG.

  In the first generation artificial gene expression enhancing system containing only dCas9-VP64, mRNA was hardly expressed. When p65-HSF1 is used as the first transcriptional activator, the second generation artificial gene expression enhancing system is about 1.6 times as compared with the positive control in which the constructed expression vector is not transfected. mRNA was expressed. In the third-generation artificial gene expression enhancing system, about 2.7 to 3.7 times as much mRNA was expressed as compared to the positive control. When VPR was used as the first transcriptional activator, the second generation artificial gene expression enhancing system expressed about 0.5 times as much mRNA as the positive control. In the third generation artificial gene expression enhancing system, about 1.7 to 2.2 times as much mRNA was expressed as compared to the positive control. Therefore, it is clear from the mRNA level that the third-generation artificial gene expression enhancing system is more effective in enhancing gene expression than the first-generation and second-generation artificial gene expression enhancing systems. became.

[11.4 Verification-4]
It was evaluated whether the target gene was expressed at the protein level by the constructed system (FIGS. 10, 11, 16, and 17).

  In the same sample as in 10.3, the expression state of E-cadherin encoded by the CDH1 gene was confirmed by immunostaining. As a result, the expression of E-cadherin was confirmed in the third generation artificial gene expression enhancing system.

  In addition, the expression state of E-cadherin was confirmed by Western blotting in samples similar to 10.3 (other than samples containing VPR) (FIGS. 12 and 15). As a result, the expression of E-cadherin was confirmed in the third generation artificial gene expression enhancing system.

[12 Functional verification of 3rd generation artificial gene expression enhancement system targeting RANKL gene]
In the verification of this item, unless otherwise specified, the method described in the items 1 to 9 was used.

[12.1 Verification-5]
Whether the superiority of the third generation artificial gene expression enhancement system in one embodiment of the present invention is also seen in different target genes, and the expression of endogenous genes can be efficiently activated with a smaller number of sgRNAs I verified. For this purpose, two types of sgRNAs were designed as shown in FIG. 18 (a) and FIG. 21, targeting the promoter region of the RANKL gene. The two designed sgRNAs were used to compare their functionality with the 1st generation and 2nd generation artificial gene expression enhancement systems by a plasmid-based reporter assay. The cells used were HEK293T cells. A reporter assay was performed in the same manner as in 10.2, except that the targeted gene, sgRNA, and cells were changed. The results are shown in FIG.

  In addition, the expression level of mRNA was confirmed by quantitative RT-PCR in the same manner as in 10.3 except that the targeted gene, sgRNA, and cells were changed. The total amount of expression vector in the constructed system was 50 ng and 100 ng. The results are shown in FIG. 20 and FIG.

  Even when the RANKL gene is targeted, the third generation artificial gene expression enhancing system is compared with the first and second generation artificial gene expression enhancing systems, as in the case of targeting the CDH1 gene. Thus, it was revealed that the effect of enhancing gene expression is enhanced.

  Also, even when two types of sgRNA are used, the third generation artificial gene expression enhancing system enhances gene expression compared to the first and second generation artificial gene expression enhancing systems. It became clear that the effect became high.

  Furthermore, it was found that the gene expression enhancing effect was increased in a dose-dependent manner with respect to the amount of the expression vector.

[12.2 Verification-6]
Comparison was made with another artificial gene expression enhancement system of the second generation in which 22sTag was fused to dCas9. dCas9-VP64 or dCas9-n × 22sTag (n = 4, 8), MS2-VP64, MS2-n × 22sTag (n = 4, 8), MS2-p65-HSF1 or MS2-VPR, scFv-VP64, A reporter assay was performed in the same manner as in 11.1 using scFv-p65-HSF1 or scFv-VPR. The results are shown in FIG.

  As a result of examination using three types of VP64 and p65-HSF1-VPR, in any case, the MS2 coat protein was used more than when n × 22sTag (n = 4, 8) was directly fused to dCas9. Higher activity was obtained when nx22 sTag (n = 4, 8) was fused. Therefore, it is clear that the third generation artificial gene expression enhancing system is more effective in enhancing gene expression than the second generation other artificial gene expression enhancing system in which 22sTag is fused to dCas9. became.

  Since the third generation system of the present invention can accumulate effector proteins in a target gene, it can be widely used in fields such as medicine, agriculture, forestry and fisheries, life science, biotechnology, and gene therapy.

Claims (19)

(A) a polynucleotide comprising a base sequence encoding a guide RNA that specifically binds to a target gene in the first region, or an expression vector comprising the polynucleotide,
(B) a polynucleotide comprising a base sequence encoding Cas protein or an expression vector comprising the polynucleotide,
(C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide A composition for accumulating an effector protein in a target gene, comprising: an expression vector comprising: (D) a polynucleotide comprising a base sequence encoding the first effector protein or an expression vector comprising the polynucleotide. (A ′) a guide RNA that specifically binds to the target gene in the first region,
(B ′) Cas protein,
(C ′) a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, and (D ′) a plurality of the first effectors protein,
A composition for accumulating an effector protein in a target gene.   The composition for accumulating an effector protein in the target gene according to claim 1 or 2, wherein the Cas protein is fused with a second effector protein.   The composition for accumulating an effector protein in the target gene of any one of Claims 1-3 whose said Cas protein is dCas9. The composition for enhancing expression of a target gene according to claim 4, wherein the dCas9 is encoded by a polynucleotide having a base sequence represented by any of the following (a1) to (a3):
(A1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1,
(A2) A polynucleotide comprising a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 1, and the protein expressed from the polynucleotide is the guide RNA of (A) above, (C) a fusion, and a polynucleotide having an activity of accumulating an effector protein in a target gene in cooperation with the first effector protein of (D), and (a3) a base sequence represented by SEQ ID NO: 1 A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary base sequence, and the protein expressed from the polynucleotide is a guide RNA of (A) above, a fusion of (C) above, And the effector in the target gene in cooperation with the first effector protein of (D) above A polynucleotide having an activity of accumulating proteins.   The composition for accumulating an effector protein in the target gene according to any one of claims 1 to 5, wherein the second region of the guide RNA has a stem-loop structure. The second region of the guide RNA is encoded by a polynucleotide having a base sequence represented by any of the following (b1) to (b3), to accumulate the effector protein in the target gene according to claim 6. Composition of:
(B1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2,
(B2) A polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2, and RNA transcribed from the polynucleotide binds to the fusion of (C) above. (B3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2, and from the polynucleotide A polynucleotide in which transcribed RNA has an activity of binding to the fusion of (C) above.   The composition for accumulating an effector protein in the target gene of any one of Claims 1-7 whose protein couple | bonded with the 2nd area | region of the said guide RNA is MS2 coat protein. The composition for accumulating an effector protein in a target gene according to claim 8, wherein the MS2 coat protein is encoded by a polynucleotide having a base sequence represented by any of the following (c1) to (c3):
(C1) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3,
(C2) a polynucleotide comprising a nucleotide sequence having a nucleotide sequence of 90% or more with the nucleotide sequence represented by SEQ ID NO: 3, and a protein expressed from the polynucleotide is the number of the guide RNA of the above (A) A polynucleotide having an activity of binding to the region 2, and (c3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3, and The polynucleotide in which the protein expressed from the polynucleotide has an activity of binding to the second region of the guide RNA of (A). The first effector protein comprises an antigen or an antibody;
The composition for accumulating effector protein in the target gene of any one of Claims 1-9 provided with the antibody or antigen for the peptide used as the said tag to couple | bond with the said antigen or antibody.   The composition for accumulating an effector protein in the target gene according to claim 10, wherein the peptide serving as the tag comprises 4 to 24 antigens. The first effector protein comprises an antibody;
The composition for accumulating an effector protein in the target gene according to claim 10, wherein the antibody is scFv.   The composition for integrating | stacking an effector protein on the target gene of Claim 10 which has a linker between the antigens or antibodies with which the peptide used as said tag is provided.   The composition for accumulating an effector protein in the target gene according to claim 13, wherein the linker is 5 to 60 amino acid residues.   The composition for accumulating an effector protein in the target gene according to claim 3, wherein the second effector protein is VP64.   The composition for accumulating an effector protein in the target gene of any one of Claims 1-15 whose said 1st effector protein is a transcriptional activator. (A) a polynucleotide comprising a base sequence encoding a guide RNA that specifically binds to a target gene in the first region, or an expression vector comprising the polynucleotide,
(B) a polynucleotide comprising a base sequence encoding Cas protein or an expression vector comprising the polynucleotide,
(C) a polynucleotide comprising a base sequence encoding a fusion of a protein that binds to the second region of the guide RNA and a peptide that serves as a tag for binding a plurality of first effector proteins, or the polynucleotide A kit for accumulating an effector protein in a target gene, comprising: an expression vector comprising: (D) a polynucleotide comprising a base sequence encoding the first effector protein or an expression vector comprising the polynucleotide.   The step of introducing the composition according to any one of claims 1 to 16, the polynucleotide provided in the kit according to claim 17, or the expression vector provided in the kit according to claim 17 into a cell in vitro. A method for producing a cell in which an effector protein is accumulated in a target gene.   The composition according to any one of claims 1 to 16, the polynucleotide provided in the kit according to claim 17, or the expression vector provided in the kit according to claim 17 is a mammal (excluding humans). A method for producing a mammal (but excluding humans) in which an effector protein is accumulated in a target gene.