JP2022112522A - Methods for cell-specifically controlling proteins that edit target genes - Google Patents

Abstract

To provide more specific and safe methods capable of cell-specific editing of genomes using miRNA activity as an indicator.SOLUTION: Disclosed is a method for cell-specifically controlling the editing of a target gene, the method comprising a step of introducing to a cell: (a) mRNA encoding a first protein that edits the target gene; (b) miRNA-responsive mRNA encoding a second protein that inactivates the first protein; and (c) sgRNA having a guide sequence specifically recognized by the target gene, where the miRNA-responsive mRNA includes: (i) a nucleic acid sequence specifically recognized by miRNA; and (ii) a nucleic acid sequence corresponding to a coding region of the second protein, and the miRNA is specifically highly active in the cell.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for cell-specifically controlling a protein that edits a target gene, and a kit that can be used for the method.

In recent years, a Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (CRISPR/Cas) System has been widely used in genome editing. CRISPR / Cas9 System is a nuclease Cas9 protein (Cas9) and a single guide RNA (sgRNA) that incorporates 20 bases complementary to the target sequence to cleave the target gene. However, in genome editing, cutting unintended sequences or editing the genome of non-target cells may lead to unexpected results. Therefore, it is important to control the tools that perform genome editing.

MicroRNAs (hereinafter referred to as miRNAs) are attracting attention as indicators for identifying cells (see, for example, Patent Document 1). Using this miRNA, a method of controlling nucleases such as Cas9 in a cell-specific manner has been devised (see, for example, Non-Patent Documents 1, 2, and Patent Document 1). In addition, the present inventors have also attempted to use RNA-binding proteins to edit genomes when target miRNAs have high activity (see, for example, Non-Patent Document 2 and Patent Document 2).

Recently, AcrllA4 was discovered that inhibits the activity of Cas9 of Streptococcus pyogenes. By controlling this AcrllA4 with miRNA, it was attempted to edit the genome when the activity of the target miRNA is high (see Non-Patent Document 3). This method is DNA delivery, and there is a risk that the transgene will be integrated into the genome. In addition, gene activation is evaluated using a reporter (luciferase) using plasmid DNA.

International publication WO2015/105172 International publication WO2018/003339

BiotechnolJ. 2014 Nov;9(11):1402-1412 Nucleic Acids Res (2017) 45:e118 Nucleic Acids Res (2019) 1, doi: 10.1093/nar/gkz271

The nuclease control methods disclosed in Non-Patent Documents 1, 2, and Patent Document 2 are systems in which the genome is not edited when the target miRNA activity is high. Such systems are also referred to herein as OFF systems. In order to edit the genome in a target cell-specific manner using the OFF system, it is necessary to search for miRNAs with low specific activity in specific target cells, which is complicated. On the other hand, in the method using the RNA-binding protein disclosed in Non-Patent Document 2 and Patent Document 2, Cas9 expression leak was observed when Cas9 expression is desired to be suppressed. In order to improve the specificity, it is necessary to suppress this leak.

Moreover, it is unclear whether the method disclosed in Non-Patent Document 3 can actually activate endogenous genes, that is, genes on the genome of target cells. Furthermore, this method requires the use of a viral vector, and there are concerns about the safety of cells in clinical use.

There is a need for a safe method that can edit target genes in a cell-specific and more precise manner without affecting unintended genes or cells using miRNA activity as an index.

As a result of intensive studies, the present inventors found that target cell-specific genome editing can be achieved by regulating the expression of a protein that inactivates a target gene-editing protein using miRNA activity as an index. I discovered it and came to complete the present invention.

That is, the present invention includes the following aspects.
[1] A method for cell-specifically controlling target gene editing, comprising:
(a) an mRNA encoding a first protein that edits a target gene;
(b) a miRNA-responsive mRNA encoding a second protein that inactivates the first protein;
(c) introducing into a cell an sgRNA comprising a guide sequence specifically recognized by said target gene, wherein said miRNA-responsive mRNA is:
(i) a nucleic acid sequence specifically recognized by a miRNA; and (ii) a nucleic acid sequence corresponding to the coding region of said second protein;
The method, wherein the miRNA is a miRNA that is specifically highly active in the cell.
[2] the first protein is a Cas9 protein or an analogue thereof,
The method of [1], wherein the second protein is AcrllA4 or an analogue thereof.
[3] the first protein is a dCas9-VPR protein or an analogue thereof;
The method of [1], wherein the second protein is AcrllA4 or an analogue thereof.
[4] The method of [1], wherein (a), (b) and/or (c) are introduced into the cell in the form of a synthetic RNA molecule without using a DNA vector or viral vector.
[5] The method according to any one of [1] to [4], wherein (i) a plurality of nucleic acid sequences specifically recognized by miRNA are contained in the untranslated region of the miRNA-responsive mRNA.
[6] The method of [2] or [3], wherein the second protein is AcrllA4 having a nuclear localization signal fused to its N-terminus.
[7] A kit for cell-specific control of target gene editing, comprising:
(a) an mRNA encoding a first protein that edits a target gene;
(b) a miRNA-responsive mRNA encoding a second protein that inactivates the first protein;
(c) an sgRNA comprising a guide sequence specifically recognized by said target gene, wherein said miRNA-responsive mRNA is:
(i) a nucleic acid sequence specifically recognized by a miRNA; and (ii) a nucleic acid sequence corresponding to the coding region of said second protein;
The kit, wherein the miRNA is a miRNA that is specifically highly active in the cell.
[8] the first protein is a Cas9 protein or an analogue thereof,
The kit of [7], wherein the second protein is AcrllA4 or an analogue thereof.
[9] the first protein is a dCas9-VPR protein or an analogue thereof;
The kit of [7], wherein the second protein is AcrllA4 or an analogue thereof.
[10] The kit according to any one of [7] to [9], wherein the 5′-UTR contains a plurality of (i) nucleic acid sequences specifically recognized by miRNA.
[11] The kit of [8] or [9], wherein the second protein is AcrllA4 having a nuclear localization signal fused to its N-terminal side.

According to the method of the present invention, miRNA-responsive mRNA switches, such as miR-AcrllA4, i.e., mRNA molecules that express specific proteins in response to miRNAs, are used to target expressed in specific cell types. We were able to detect miRNA and realize cell-specific genome editing. By combining this method with a gene editing system such as the CRISPR-Cas9 system, it can be applied to various research fields including cell engineering and clinical therapy.

INDUSTRIAL APPLICABILITY The present invention can realize a system in which the genome is edited when the activity of the target miRNA is high, that is, an ON system. In such a method, the expression of a protein that inactivates a gene-editing protein can be controlled by miRNA-responsive mRNAs such as the miR-AcrllA4 switch. can be a useful tool.
[1] Compared to OFF systems, ON systems can easily and selectively activate gene-editing proteins (such as nucleases) in target cells.
[2] In order to identify gene function in model organisms, miRNA-responsive mRNA switches can perform cell-specific genome editing or tissue-specific genome editing, that is, loss of function by genome knockout or gain of function by CRISPRa. to enable.
[3] miRNA-responsive mRNA switches, such as the miR-AcrllA4 switch, can be implemented by RNA delivery methods. This avoids genomic integration and off-target effects, which are concerns in DNA delivery methods.
[4] miRNA-responsive mRNA switches, such as the miR-AcrllA4 switch, can minimize genome editing in non-target cells. This is important in clinical applications, for example in cancer therapy, where genome editing in normal cells can be avoided and side effects can be reduced.
[5] miRNA-responsive mRNA switches, such as the miR-AcrllA4 switch, respond not only to endogenous miRNAs, but also to synthetic oligonucleotides, such as miR-mimic, and are therefore used for inducible or transient genome editing. be able to.

FIG. 1 is a conceptual diagram representing the scheme of the miRNA-responsive CRISPR-Cas9 ON switch. AcrllA4 is a key player that inhibits the activity of Cas9, and the expression of this AcrllA4 is controlled by endogenous miRNAs by inserting the target site of the miRNA into the 5'-UTR of AcrllA4 mRNA. That is, since the expression of AcrllA4 is suppressed when the activity of the target miRNA is high, gene editing is performed by Cas9 and gene activation is performed by dCas9-VPR. FIG. 2A is a photograph of representative cells when inhibition of Cas9 activity by AcrllA4 was assessed by EGFP gene knockout. FIG. 2B is a representative histogram when inhibition of Cas9 activity by AcrllA4 was assessed by knockout of the EGFP gene. FIG. 2C is a bar graph of the results of three experiments in which inhibition of Cas9 activity by AcrllA4 was assessed by knockout of the EGFP gene. The results are expressed as mean±SD. FIG. 3A is a conceptual diagram of the miRNA-responsive CRISPR-Cas9 ON system that senses miRNA and controls genome editing by the miR-AcrllA4 switch. Cas9 mRNA, sgRNA, miRNA-responsive AcrllA4 switch is introduced into cells. When the activity of the target miRNA is low, the AcrllA4 protein is expressed, binds to the Cas9-sgRNA complex, and inhibits Cas9 DNA binding, preventing genome editing (left). AcrllA4 protein is not expressed when the activity of the target miRNA is high. Therefore, Cas9 binds to DNA and edits the genome (right). FIG. 3B is a photograph of representative cells where Cas9 activity was assessed by EGFP gene knockout. FIG. 3C is a representative histogram of Cas9 activity assessed by knockout of the EGFP gene. FIG. 3D shows a bar graph of the results of triplicate experiments in which Cas9 activity was assessed by knockout of the EGFP gene. The results are expressed as mean±SD and ***P<0.001 (Student's test). FIG. 4A is a schematic diagram of the miRNA-responsive CRISPRa ON system in which the miR-AcrllA4 switch regulates CRISPRa through endogenous miRNAs. dCas9-VPR plasmid (a), sgRNA plasmid (c), miR-AcrllA4 plasmid (b) are introduced into HeLa cells in which TRE-P _miniCMV -hmAG1 is assembled into the genome. AcrllA4 inhibits DNA binding of the dCas9-VPR-sgRNA complex and suppresses hmAG1 expression. When target miRNA activity is high, hmAG1 is expressed as the dCas9-VPR-sgRNA complex binds to the target site. In the figure below, the target sequence is underlined, and the boxed sequence is the protospacer adjacent motif (PAM). FIG. 4B is a photograph of representative cells where the activity of dCas9-VPR was assessed by hmAG1 expression. FIG. 4C is a representative dot plot where the activity of dCas9-VPR was assessed by hmAG1 expression. FIG. 4D shows a bar graph of the results of three experiments in which the activity of dCas9-VPR was assessed by hmAG1 expression. The results are expressed as mean±SD and ***P<0.001 (Student's test). Figure 5 introduces Cas9 mRNA and 5 ng of miRNA-responsive AcrllA4 mRNA into iPS-EGFP cells, which are iPS cells that have integrated the EGFP gene on the genome, controls genome editing by sensing miRNA, Cas9 Results of experiments in which activity was assessed by knockout of the EGFP gene are shown. FIG. 5A is a photograph of cells obtained as a result of a control experiment without miRNA-responsive AcrllA4 mRNA (left panel without addition of AcrllA4, right panel without addition of sgRNA _EGFP recognized by the target gene). be. FIG. 5B is a photograph of cells obtained as a result of a control experiment without miRNA-responsive AcrllA4 mRNA (left panel: addition of AcrllA4, middle panel: addition of inactivated AcrllA4 mutant, right panel: untreated). is. FIG. 5C shows miRNA sensing using miR-302a-responsive AcrllA4 mRNA with one sequence specifically recognized by miR-302a at the target site, and Cas9 activity assessed by knockout of the EGFP gene. It is a photograph showing the results of the experiment. FIG. 5D shows miRNA sensing using 4×mi-302a-responsive AcrllA4 mRNA with four sequences specifically recognized by miR-302a at the target site, and Cas9 activity by knockout of the EGFP gene. It is a photograph showing the results of the evaluated experiment. FIG. 6 is a dot plot representation of the experimental results shown in FIG. FIG. 7 is a graph showing the ratio (%) of the EGFP-negative cell population in cells subjected to each operation as a result of the experiment shown in FIG. Figure 8A-D shows Cas9 mRNA and 10 ng of miRNA-responsive AcrllA4 mRNA are introduced into iPS-EGFP cells, which are iPS cells that have integrated the EGFP gene on the genome, to control genome editing by sensing miRNA, The results of experiments in which Cas9 activity was evaluated by EGFP gene knockout are shown. FIG. 8A is a photograph of cells obtained as a result of a control experiment without miRNA-responsive AcrllA4 mRNA (left panel without addition of AcrllA4, right panel without addition of sgRNA _EGFP recognized by the target gene). be. FIG. 8B is a photograph of cells obtained as a result of a control experiment without miRNA-responsive AcrllA4 mRNA (left panel: addition of AcrllA4, middle panel: addition of inactivated AcrllA4 mutant, right panel: untreated). is. FIG. 8C shows miRNA sensing using miR-302a-responsive AcrllA4 mRNA with one sequence specifically recognized by miR-302a at the target site, and Cas9 activity assessed by knockout of the EGFP gene. It is a photograph showing the results of the experiment. FIG. 8D shows miRNA sensing using 4× miR-302a-responsive AcrllA4 mRNA with four sequences specifically recognized by miR-302a at the target site, and Cas9 activity was detected by knockout of the EGFP gene. It is a photograph showing the results of the evaluated experiment. FIG. 9 is a dot plot representation of the experimental results shown in FIG. FIG. 10 is a graph showing the ratio (%) of the EGFP-negative cell population in cells subjected to each operation as a result of the experiment shown in FIG. Figure 11 shows a schematic of the construct. (A) shows a structural schematic diagram of miRNA-responsive mRNA that has a miRNA target site in the 5'-UTR and encodes Anti-CRISPR that functions as the second protein of the present invention. (B) is a conceptual diagram showing an example of the miRNA target sequence of miRNA-responsive mRNA, showing that one repeat or four repeats of the miRNA target site can be incorporated. (C) shows a conceptual diagram of the second protein of the miRNA-responsive mRNA. The upper diagram shows the sequence encoding the wild-type AcrllA4 protein, and the lower diagram fuses the nuclear localization signal M9 to the N-terminus. FIG. 4 conceptually shows the sequence encoding M9_AcrllA4.

Embodiments of the present invention are described below. However, the present invention is not limited by the embodiments described below.

According to one embodiment, the present invention provides a method for cell-specific control of target gene editing, comprising:
(a) an mRNA encoding a first protein that edits a target gene;
(b) a miRNA-responsive mRNA encoding a second protein that inactivates the first protein;
(c) introducing into a cell an sgRNA comprising a guide sequence specifically recognized by said target gene, wherein said miRNA-responsive mRNA is:
(i) a nucleic acid sequence specifically recognized by a miRNA; and (ii) a nucleic acid sequence corresponding to the coding region of said second protein;
The method relates to a method, wherein the miRNA is a miRNA that is specifically highly active in the cell.

In the present invention, "cells" are not particularly limited and may be arbitrary cells. For example, it may be a cell harvested from a multicellular organism, or a cell obtained by culturing an isolated cell. The cells are particularly cells collected from mammals (e.g., humans, mice, monkeys, pigs, rats, etc.), cells isolated from mammals, or cells obtained by culturing mammalian cell lines. be. Examples of somatic cells include keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., surface epithelial cells of the tongue), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., , adrenal medulla cells), metabolic and storage cells (e.g., hepatocytes), luminal epithelial cells that make up the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain ducts (e.g., blood vessels endothelial cells), ciliated cells with carrying capacity (e.g. airway epithelial cells), cells secreting extracellular matrix (e.g. fibroblasts), contractile cells (e.g. smooth muscle cells), blood and immune system cells (e.g. T lymphocytes), sensory cells (e.g. rod cells), autonomic nervous system neurons (e.g. cholinergic neurons), supporting cells of sensory and peripheral neurons (e.g. companion cells), central nervous system neurons and glial cells (eg, astrocyte), pigment cells (eg, retinal pigment epithelial cells), and their progenitor cells (tissue progenitor cells). There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected. It can be used as a cell in the invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

Cells in the present invention may be cells artificially manipulated after collection of prophase somatic cells. For example, a cell group comprising pluripotent stem cells prepared from the somatic cells, or a cell group obtained after differentiating pluripotent stem cells, which contains differentiated cells other than desired cells. It may be a group of cells obtained. The pluripotent stem cells that can be used in the present invention are stem cells that have pluripotency capable of differentiating into all cells existing in a living body and that also have proliferative potential. Specific examples of these pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (“GS cells”). ”), embryonic germ cells (“EG cells”), induced pluripotent stem (iPS) cells, and the like. Preferred pluripotent stem cells are ES cells, ntES cells and iPS cells. More preferred pluripotent stem cells are human pluripotent stem cells, particularly preferred are human ES cells and human iPS cells. Furthermore, the cells that can be used in the present invention are not only pluripotent stem cells, but also a group of cells induced by so-called "direct reprogramming", in which differentiation into desired cells is directly induced without going through pluripotent stem cells. There may be. Alternatively, the cells in the present invention may be a cell group that may contain cells for which gene editing is desired and cells for which gene editing is not desired, such as a cell group that may contain cancer cells and normal cells. .

Cells in the present invention are particularly preferably in a viable state. In the present invention, a cell in a viable state means a cell in a state of maintaining metabolic capacity. In the present invention, even after the RNAs of (a), (b), and (c) above are introduced into cells, they remain viable, particularly maintain their mitotic potential, without losing their innate characteristics. , cells that can be used for subsequent applications.

A "target gene" in the present invention is a gene located on the genome of a specific target cell. The type of gene is not particularly limited, and any desired gene can be used as the target gene by designing the sgRNA described below.

In the present invention, "editing" of a target gene refers to acting on the target gene to change the function of the gene, including, for example, reducing, losing, acquiring, or enhancing the function of the gene. be More specifically, "editing" includes cutting genes, activating and repressing gene expression, base editing, labeling (genome labeling), DNA methylation/demethylation, and histone acetylation. It shall include performing epigenome editing such as.

In the present invention, the term "regulate" means that the translation of the miRNA-responsive mRNA encoding the second protein is suppressed, thereby preventing the first protein that edits the target gene from functioning ( (OFF state) to a state in which it can function (ON state).

In addition, the term “cell-specific control” refers to control of editing of a target gene in a specific cell using miRNA that is specifically highly active in the cell as an indicator. Editing control using miRNA as an indicator can be performed using the miRNA-responsive mRNA of (b) above. The structure and function of each RNA are described below.

(a) mRNA encoding the first protein that edits the target gene
The mRNA of (a) encodes the first protein that edits the target gene, is translated in the cell, and expresses the first protein. The first protein that edits the target gene is generally a protein that has enzymatic activity capable of editing the target gene. The first protein includes, but is not limited to, a nuclease capable of cleaving the target gene and an inactivating nuclease capable of activating the target gene.

Nucleases include, but are not limited to, Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 9 (Cas9) transcription activator-like effector nucleases (TALENs), homing endonucleases, zinc finger nucleases. For any of the nucleases, analogues having the same function, that is, target gene-cleaving activity, or derivatives thereof having the same activity can be used. For example, analogs of Cas9 proteins include Cas9 family proteins, specific Cas9 family proteins include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Francisella novicida Cas9 (FnCas9), Campylobacter ( CjCas9), Neisseria meningitidis Cas9 (NmCas9, or NmeCas9), Geobacillus stearothermophilus Cas9 (GeoCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). Nucleases also include fusion proteins in which Cas9 proteins, analogs or derivatives are fused to other proteins.

The inactivating nuclease can include an inactivating nuclease fused to a transcriptional activator, and an inactivating Cas9 (dCas9) nuclease fused to a transcriptional activator VP64 dCas9-VP64, dCas9- VPR, dCas9-SunTag, dCas9-VP16, dCas9-VP160, dCas9-P300 can be used, but are not limited to these. In addition, with dCas9-VP64, MS2-P65-HSF1 fusion protein and SAM system using (c) sgRNA having MS2 binding sequence in the guide strand, and Tree system using VPR with dCas9-SunTag are also used. be able to. The proteins used in these systems can be encoded by mRNA separate from the mRNA encoding the first protein, or can be encoded on the mRNA encoding the first protein. In addition, analogues and derivatives having functions similar to those of these inactivated nucleases can also be used.

The mRNA of (a) preferably has a Cap structure (7-methylguanosine 5' phosphate) or a Cap such as Anti-Reverse Cap Analog (ARCA) manufactured by Ambion in the 5' to 3' direction from the 5' end. It has a 5'-UTR containing the analogue, an initiation codon, an open reading frame encoding the first protein, and a 3'-UTR containing the polyA tail. The design of the 5'-UTR and 3'-UTR may be arbitrary. The mRNA of (a) does not contain a nucleic acid sequence specifically recognized by miRNA in any of 5'-UTR, open reading frame and 3'-UTR. This allows the mRNA encoding the first protein of (a) to be introduced into cells, translated without being affected by miRNA activity, and express the first protein.

(b) miRNA-responsive mRNA encoding a second protein that inactivates the first protein
The (b) miRNA-responsive mRNA is an mRNA that encodes a second protein and can express the second protein under the translational control of the miRNA in cells. A second protein is a protein that can inactivate the first protein. The second protein can be determined in relation to the first protein described for the mRNA in (a). The second protein may be a protein fusion that inactivates the first protein.

For example, when the first protein is Cas9 nuclease, inactivated Cas9 nuclease, or analogues thereof, the second protein is an anti-CRISPR capable of inactivating these nucleases. be able to. In particular, anti-CRISPRs capable of inactivating Cas9 of Streptococcus pyogenes (spCas9) include, but are not limited to, AcrllA2, AcrllA4, AcrllA5, AcrllA7, AcrllA8, AcrllA9, AcrllA10. The second protein may be an analogue of anti-CRISPR that has a similar function as the exemplified anti-CRISPR, ie is capable of inactivating nucleases. The anti-CRISPR that inactivates NmCas9 of the Cas9 family protein exemplified above includes AcrllC1, AcrllC2, AcrllC3, AcrllC4, and AcrllA5, and the anti-CRISPR that inactivates CjCas9 includes AcrllC1. Is, anti-CRISPR that inactivates GeoCas9 includes AcrllC1, anti-CRISPR that inactivates St1Cas9 includes AcrllA5 and AcrllA6, and anti-CRISPR that inactivates SaCas9 includes AcrllA5. is mentioned. The second protein may be a protein fusion containing anti-CRISPR or an analogue thereof, for example, a fusion protein in which nuclear localization signal M9 is fused to the N-terminal side of anti-CRISPR such as AcrllA4. good too. A fusion protein in which the nuclear localization signal M9 is fused to AcrllA4 can be used to enhance cleavage or activation of the target gene edited by the first protein.

Preferred combinations of the first protein and the second protein include, but are not limited to, AsCas12a and AcrVA1, LbCas12a and AcrVA4 or AcrVA5 (AcrVA1), NmeCas9 and AcrllC1 or AcrllC3.

A miRNA-responsive mRNA is a messenger RNA whose translation of the encoded protein is controlled in response to the activity of the miRNA, and is also referred to herein as a miRNA switch. The general structure of miRNA-responsive mRNAs is detailed in WO2015/105172, which is incorporated herein by reference. The (b) miRNA-responsive mRNA used in the present invention comprises (i) a nucleic acid sequence specifically recognized by the miRNA, and (ii) a nucleic acid sequence corresponding to the coding region of the second protein, and (i) the miRNA and (ii) a nucleic acid sequence corresponding to the coding region of the second protein are operably linked.

For the miRNA of (i), a miRNA having a high specific activity in a certain cell can be appropriately selected for the purpose of editing a specific target gene with the first protein in the cell. In this specification, the miRNA of i) may be referred to as the "target sequence" of the miRNA, and such miRNA may be referred to as the target miRNA. A miRNA that is specifically highly active in a specific cell refers to a miRNA that is expressed in the cell in a higher amount than in other cells. “Expression” of miRNA refers to the presence of miRNA in a state where mature miRNA interacts with a plurality of predetermined proteins to form an RNA-induced silencing complex (RISC). "Mature miRNAs" are single-stranded RNAs (20-25 bases) that arise from pre-miRNAs by cleavage by Dicer outside the nucleus, and "pre-miRNAs" are partially cleaved by an intranuclear enzyme called Drosha to It arises from pri-mRNA, a single-stranded RNA transcribed from DNA. The miRNA in the present invention is appropriately selected from at least 10,000 or more miRNAs. Specifically, miRNA registered in database information (for example, http://www.mirbase.org/ or http://www.microrna.org/) and/or literature information described in the database are appropriately selected from the miRNAs described in . That is, in the present invention, the miRNA of (i) is not limited to miRNAs composed of specific sequences. Such miRNAs of (i) are not limited to miRNAs identified at the time of filing of the present application, and include all miRNAs whose existence and function will be identified in the future. miRNAs highly expressed in specific cells are 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more compared to the expression in other cells % or more, 90% or more, or more highly expressed miRNAs are exemplified. Such miRNAs that are highly expressed in specific cells can also be appropriately selected based on the information in the database.

In the present invention, the target sequence of miRNA is preferably, for example, a sequence completely complementary to the miRNA. Alternatively, it may have a mismatch (mismatch) with a completely complementary sequence as long as it can be recognized by the miRNA. The mismatch from the sequence that is completely complementary to the miRNA may be a mismatch that can be normally recognized by the miRNA in the desired cell, and the original intracellular function in vivo is a mismatch of about 40 to 50%. There may be Such mismatches include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases or 1%, 5, or 1% of the total recognition sequence. %, 10%, 20%, 30%, or 40% discrepancies are exemplified. Also, in particular, like the miRNA target sequence on the mRNA provided in the cell, in particular, the 5' in the target sequence corresponding to a portion other than the seed region, that is, about 16 bases on the 3' side of the miRNA The flanking regions may contain multiple mismatches, and portions of the seed region may contain no mismatches, 1-, 2-, or 3-base mismatches. Such a sequence may have a base length that includes the number of bases that RISC specifically binds to, and the length is not particularly limited. It is a sequence of more than 22 bases and less than 22 bases. In the present invention, a nucleic acid sequence specifically recognized by miRNA introduces a miRNA-responsive mRNA that has the target sequence into desired cells and other cells and encodes a marker protein such as a visible fluorescent protein. can be appropriately determined and used by confirming that the expression of the corresponding marker protein is suppressed only in the desired cells.

In the present invention, functionally linking the nucleic acid sequence (i) specifically recognized by miRNA and the nucleic acid sequence (ii) corresponding to the coding region of the second protein means an open reading sequence encoding the second protein. It means that at least one miRNA target sequence is provided within the 5′-UTR, 3′-UTR, and/or the open reading frame of the frame (including the initiation codon). The miRNA-responsive mRNA preferably, from the 5' end, in a 5' to 3' orientation, is open reading to encode a Cap structure (7-methylguanosine 5' phosphate) or a Cap analog such as ARCA, a second protein. It comprises a frame and a poly A tail and comprises at least one miRNA target sequence within the 5'-UTR, within the 3'-UTR, and/or within the open reading frame. Hereinafter, when referring to the Cap structure, the Cap analog is also included. The position of the miRNA target sequence in the mRNA may be in the untranslated region and may be in the 5'-UTR or 3'-UTR. Alternatively, the target sequence of the miRNA may be positioned within the open reading frame (3' to the start codon). All of the untranslated region and the open reading frame may be provided with miRNA target sequences. Thus, the number of miRNA target sequences may be one or more, such as two, three, four, five, six, seven, eight or more. It can be. For example, it is preferable to incorporate 4 or more miRNA target sequences in the 5'-UTR.

FIG. 11 is a diagram conceptually showing a design example of miRNA-responsive mRNA in (b). FIG. 11(A) conceptually shows an example of the overall structure of miRNA-responsive mRNA. In the figure, miRNA target site indicates the nucleic acid sequence of (i) above, and Anti-CRISPR indicates the nucleic acid sequence of (ii). The open circle on the left represents the Cap structure at the 5' end, and Poly(A) on the right represents the poly A tail at the 3' end. The binding mode of the nucleic acid sequence (i) and the nucleic acid sequence (ii) in FIG. 11 is an example, and does not limit the present invention. Figure 11 (B) shows the nucleic acid sequence of (i) above, the upper figure is the nucleic acid sequence (i) having one repeat of the miRNA target sequence, and the lower figure is the nucleic acid sequence (i) having 4 repeats of the miRNA target sequence. Illustrate. FIG. 11 (C) is a conceptual diagram showing an example of the nucleic acid sequence of (ii). In the upper diagram, the second protein is AcrllA4, and in the lower diagram, the second protein is a fusion protein of the nuclear localization signal M9 and AcrllA4. It is a conceptual diagram in the case of .

Preferably, the miRNA-responsive mRNA has the nucleic acid sequences of (i) and (ii) linked in that order in the 5' to 3' direction. At this time, the number of bases and the type of bases between the Cap structure and the miRNA target sequence may be arbitrary as long as they do not form a stem structure or three-dimensional structure. For example, the number of bases between the Cap structure and the miRNA target sequence can be designed to be 0-50 bases, preferably 10-30 bases. Moreover, it is preferable that the number and type of bases between the miRNA target sequence and the initiation codon do not affect the interaction between the miRNA and the miRNA-responsive mRNA, such as inhibiting their binding. As an example, the number of bases between the miRNA target sequence and the initiation codon can be designed to be 0 to 50 bases, preferably 10 to 30 bases.

In the present invention, it is preferred that the miRNA target sequence of (i) does not have an AUG as an initiation codon. For example, when the miRNA target sequence exists in the 5'-UTR and contains AUG in the target sequence, it is designed to be in-frame with respect to the marker gene linked to the 3' side. preferably. Alternatively, when AUG is contained in the target sequence, it is also possible to convert the AUG in the target sequence to GUG and use it. Also, in order to minimize the effect of AUG within the target sequence, the location of the target sequence within the 5'-UTR can be changed as appropriate. For example, the number of bases between the Cap structure and the AUG sequence in the target sequence is 0-60 bases, such as 0-15 bases, 10-20 bases, 20-30 bases, 30-40 bases, 40-50 bases. , 50-60 bases. The number of bases in the 5'-UTR and 3'-UTR of the miRNA-responsive mRNA in (b) is approximately the same as the number of bases in the 5'-UTR and 3'-UTR of the mRNA in (a), respectively. can be designed to be

The miRNA-responsive mRNA of (b) preferably contains modified bases such as pseudouridine and 5-methylcytidine instead of normal uridine and cytidine. This is to reduce cytotoxicity. In both uridine and cytidine, the positions of the modified bases can be all or part of them independently, and when they are part, they can be random positions at any ratio.

The miRNA-responsive mRNA of (b) is introduced into cells, and the translation of the second protein is regulated in response to the miRNA. When the activity of the specific miRNA that specifically binds to the target sequence is high in the transfected cells, the translation of the second protein is suppressed. On the other hand, when the activity of the specific miRNA that specifically binds to the target sequence is low in the transfected cell, the second protein is translated and expressed in the cell.

(c) an sgRNA comprising a guide sequence specifically recognized by the target gene
The sgRNA of (c) is RNA comprising a guide sequence that is specifically recognized by the target gene edited by the first protein. sgRNA(c) can be designed to incorporate a sequence of about 20, eg, about 18-22 bases near the 5' end that specifically recognizes the target gene. A nucleotide sequence that specifically recognizes a target gene is preferably a sequence completely complementary to the target gene. Alternatively, it may have a mismatch (mismatch) with a completely complementary sequence as long as it can be recognized by the target gene. The mismatch from the sequence completely complementary to the target gene may be the same as defined for miRNA and its target sequence in the design of miRNA-responsive mRNA in (b). In addition, on the 5' side of the nucleotide sequence that specifically recognizes the gene to be edited by the first protein, there may be a sequence of about 1 to 5 bases, or a sequence of about 80 bases. good. By providing such a sequence, it may be possible to bind to the target sequence and control genome editing. Sequence Furthermore, the sequence after the 3' side of the base sequence that specifically recognizes the target gene is an sgRNA sequence known to function as an sgRNA for a gene editing system such as the CRISPR / Cas9 System, Examples of Tetra loop, stem loop 2, modified 3′ end, and chemically modified RNA constituting sgRNA are known. Depending on the CRISPR/Cas9 System used, the tetraloop and/or stem loop of the sgRNA may be added with a binding sequence for a related protein. However, the sequence is not limited to a specific sequence as long as it forms a complex with the first protein and enables editing, such as cleavage or activation, at the target site of the target gene.

When the first protein is present in an active state, the sgRNA of (c) forms a complex with the first protein to edit the target gene, for example, cleave the target gene at the desired site, or can be activated at any desired site.

The mRNA of (a), the miRNA-responsive mRNA of (b), and the sgRNA of (c) are designed and sequenced according to the above, and can be synthesized by a person skilled in the art by any method known in genetic engineering. can be done. In particular, it can be obtained by an in vitro transcription synthesis method using a template DNA containing a promoter sequence as a template, but the synthesis method is not particularly limited.

In one embodiment of the present invention, the method for introducing the mRNA of (a), the miRNA-responsive mRNA of (b), and the sgRNA of (c) into cells includes introduction into cells in the form of synthetic RNA molecules. be done. For example, it may be introduced into somatic cells by techniques such as lipofection and microinjection. Alternatively, the RNAs of (a), (b), and (c) can be introduced in the form of DNA such as viral vectors, plasmid vectors, episomal vectors, etc., and the same introduction method as above is used. be able to. The three RNAs (a), (b), and (c) are preferably introduced by the same method.

In the method of the present invention, the miRNA-responsive mRNA of (b) introduced into the cells may be one type, two types, three types, four types, five types, six types, seven types, or eight types. There may be more than one type. When introducing two or more miRNA-responsive mRNAs, for example, mRNAs that have different miRNA target site sequences in (i) and respond to different miRNAs can be used. By using a plurality of miRNA-responsive mRNAs with different sequences in (i), for example, the activity of the second protein is controlled in multiple types of cells that may be included in the cell group into which the miRNA-responsive mRNAs are introduced. becomes possible.

When introducing the RNAs of (a), (b) and (c) into cells, it is preferable to co-introduce the mRNAs of (a) and (b) into the cell group. This is because the intracellular ratio of the two or more types of co-introduced mRNAs is maintained in individual cells, and the activity ratio of proteins expressed from these mRNAs is constant within the cell population. The amount of introduction at this time varies depending on the cell group to be introduced, the mRNA to be introduced, the method of introduction and the type of introduction reagent. These can be selected. The sgRNA of (c) is preferably co-introduced with the mRNAs of (a) and (b), but may be introduced independently at an appropriate timing without necessarily being co-introduced.

As a more specific example, the method according to the first aspect of the present invention when using Cas9 protein as the first protein and AcrllA4 as the second protein will be described. The present invention, according to one embodiment, the mRNA encoding the Cas9 protein of (a), miRNA-responsive mRNA encoding AcrllA4 of (b), and the step of introducing sgRNA of (c) into cells . In the following description, the mRNA encoding the Cas9 protein of (a) is also referred to as Cas9 mRNA, miRNA-responsive mRNA encoding AcrllA4 of (b) is also referred to as miRNA-responsive AcrllA4 mRNA.

The left side of FIG. 1 schematically shows the action in the cell when the Cas9 mRNA of (a), the miRNA-responsive AcrllA4 mRNA of (b), and the sgRNA of (c) are introduced into the cell. is. When the activity of miRNA recognized by the target sequence of miRNA-responsive AcrllA4 mRNA is high, translation of AcrllA4 protein from miRNA-responsive AcrllA4 mRNA is suppressed, or degradation of miRNA-responsive AcrllA4 mRNA molecule occurs, resulting in AcrllA4 protein Not expressed. On the other hand, Cas9 mRNA expresses Cas9 protein regardless of miRNA activity. Since the Cas9 protein is not inactivated by the AcrllA4 protein, the sgRNA and the Cas9 protein form a complex to perform genome editing, specifically cleavage of the target gene.

Next, when the activity of the miRNA recognized by the target sequence of miRNA-responsive AcrllA4 mRNA is low, the miRNA-responsive AcrllA4 mRNA is translated into AcrllA4 protein, and the AcrllA4 protein is expressed. AcrllA4 protein inactivates Cas9 protein expressed from Cas9 mRNA.

As another specific example, the method according to the first aspect of the present invention when using dCas9-VPR protein as the first protein and AcrllA4 as the second protein will be described. According to one embodiment of the present invention, (a) the mRNA encoding the dCas9-VPR protein, (b) the miRNA-responsive mRNA encoding AcrllA4, and (c) the step of introducing the sgRNA into the cell including. In the following description, mRNA (a) encoding dCas9-VPR protein is also referred to as dCas9-VPR mRNA (a).

The right side of Figure 1, dCas9-VPR mRNA, miRNA-responsive AcrllA4 mRNA, and sgRNA when introduced into the cell, is a diagram schematically showing the action in the cell. When the activity of miRNA recognized by the target sequence of miRNA-responsive AcrllA4 mRNA is high, translation of AcrllA4 protein from miRNA-responsive AcrllA4 mRNA is suppressed, or degradation of miRNA-responsive AcrllA4 mRNA molecule occurs, resulting in AcrllA4 protein Not expressed. On the other hand, dCas9-VPR mRNA expresses dCas9-VPR protein regardless of miRNA activity. Since the dCas9-VPR protein is not inactivated by the AcrllA4 protein, sgRNA and dCas9-VPR protein form a complex, genome editing, specifically activation of the target gene.

When the activity of miRNA recognized by the target sequence of miRNA-responsive AcrllA4 mRNA is low, AcrllA4 protein is translated from miRNA-responsive AcrllA4 mRNA, and AcrllA4 protein is expressed. AcrllA4 protein inactivates Cas9 protein expressed from dCas9-VPR mRNA.

According to the method of the present invention, the activity of a protein that enables target gene editing can be controlled in a cell-specific manner using the activity of miRNA as an index. Therefore, it has become possible to construct an ON system that executes target gene editing in response to highly active miRNAs in specific cells.

The invention is explained in more detail below using examples. The following examples do not limit the invention.

(1) Inhibition of Cas9 activity by AcrllA4 First, it was verified whether AcrllA4 protein produced from mRNA encoding AcrllA4 (AcrllA4 mRNA) can suppress Cas9 activity. The present inventors have reported the results of evaluating Cas9 activity using HeLa cells (HeLa-EGFP) in which the EGFP gene was integrated into the genome (Hirosawa M, et al. (2017), Non-Patent Document 2 ). In this experiment, AcrllA4 mRNA, mRNA encoding Cas9 (Cas9 mRNA), and the sgRNA _EGFP targeting the EGFP gene were transfected into HeLa-EGFP. Cas9 activity was assessed by quantifying the EGFP-negative cell population by flow cytometry. Details are given in the Methods section.

Cas9 mRNA activity was assessed with and without AcrllA4 mRNA transfection. The results are shown in Figures 2A, 2B, and 2C. In the absence of AcrllA4 mRNA, approximately 70% of the cells were EGFP-negative, indicating that Cas9 mRNA and sgRNA _EGFP efficiently edited the EGFP gene. In contrast, the addition of AcrllA4 mRNA decreased the number of EGFP-negative cells. This result indicates that AcrllA4 mRNA (10-50 ng) efficiently suppressed Cas9 activity to the same extent as untargeted and untreated cells. A defective AcrllA4 mutant (E70R) without Cas9 binding activity (Dong D, et al. (2017) Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature 546: 436-439.) to negative cells, EGFP population had no effect. This suggests that efficient suppression of Cas9 activity is due to the interaction of Cas9 and AcrllA4. Thus, AcrllA4 reliably controls genome editing. That is, the off-target in the OFF state is minimized. In the experiments below (Figure 3), 10 ng of AcrllA4 mRNA was used.

(2) miRNA sensing and genome editing control by miR-AcrllA4 switch In order to induce Cas9 activity in response to any miRNA, miRNA-responsive AcrllA4 mRNA (also referred to as miR-AcrllA4 switch, Figure 3A) 5' - Inserted the miRNA target site (antisense sequence of miRNA) into the UTR. When specific miRNA activity is low, AcrllA4 is expressed from AcrllA4 mRNA and binds to the Cas9-sgRNA complex, suppressing Cas9 binding to double-stranded DNA (dsDNA) and disrupting genome editing ( FIG. 3A, left). On the other hand, when the specific miRNA activity is high, AcrllA4 mRNA does not express AcrllA4, forming a Cas9-sgRNA-dsDNA complex and promoting genome editing (Fig. 3A, right). Thus, a Cas9-ON system driven by miRNAs was obtained.

We investigated whether the miR-AcrllA4 switch can sense miRNA activity and activate Cas9. Due to high miR-21 activity in HeLa cells, we constructed a miR-21-responsive AcrllA4 mRNA (T21_AcrllA4) and transfected T21_AcrllA4 mRNA, Cas9 mRNA, and sgRNA _EGFP into HeLa-EGFP. Three days after transfection, the EGFP-negative cell population increased based on Cas9 activity. The results are shown in Figures 3B, 3C and 3D. This indicates that the expression of AcrllA4 protein from T21_AcrllA4 mRNA was suppressed by miR-21.

We determined whether endogenous miR-21 can cause genome editing. The miR-21 inhibitor reduced the size of the EGFP-negative cell population to a similar extent to cells not treated with Cas9 (Cas9-OFF). A negative control that does not interfere with miRNA (inhibitor NC) had no effect on the EGFP-negative cell population (Cas9-ON). These results indicate that the T21_AcrllA4 switch regulates Cas9 activity through endogenous miR-21 and revealed background levels of Cas9 activity in the OFF state in the presence of miR-21 inhibitor. That is, in the OFF state, Cas9 activity was efficiently suppressed, and Cas9 was activated by detecting target miRNA. In addition, tandem insertion of multiple miRNA target sequences into the 5′-UTR of AcrllA4 mRNA improved the ON state without affecting the OFF state (Fig. 3D, 4xT21_AcrllA4 vs. T21_AcrllA4). inhibitor N. C. , the EGFP-negative cell population when transfected with 4xT21_AcrllA4 mRNA was higher compared to the EGFP-negative cell population when transfected with T21_AcrllA4 mRNA, 41.1% for the former and 31.7% for the latter. %Met. However, using the miR-21 inhibitor, the EGFP-negative cell population was 15.6% when transfected with 4xT21_AcrllA4 mRNA, and the EGFP-negative cell population was 15.9% when transfected with T21_AcrllA4 mRNA. was a number. This indicates improved sensitivity to target miRNAs while avoiding off-target effects on non-targeted cells.

To confirm the generality of this strategy, we targeted a different miRNA, miR-302a-5p (miR-302a). miR-302a is known as a marker for human pluripotent stem cells (hPSCs). Due to the low activity of miR-302a in HeLa cells, miR-302a mimics were used to trigger Cas9 activation. Cas9 mRNA, sgRNA _EGFP and T302a-AcrllA4 mRNA were co-transfected with miR-302a mimic or miR-302a negative control (mimic NC). As expected, the EGFP-negative cell population increased in the presence of miR-302a mimic, but miR-302a mimic N. It did not increase in the presence of C, and the background level of Cas9 activity remained in the OFF state (Figures 3B, 3C, 3D). This indicates that the miR-AcrllA4 switch is modular. Thus, by controlling AcrllA4, we have successfully engineered a Cas9-ON system that is delivered by synthetic RNA and responds to miRNA.

(3) MiR-AcrllA4 switch miRNA sensing and CRISPR control searched for For this purpose, as reporter cells, HeLa cells (HeLa-TRE _hmAG1 ) were constructed in which the humanized monomeric Azami-Green1 (hmAG1) gene regulatable by the tetracycline responsive element (TRE) was integrated into the genome (Fig. 4A). For activation of hmAG1, the dCas9-VPR system was used (Chavez A, et al. (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12: 326-328.). First, we observed efficient expression of hmAG1 without AcrllA (Fig. 4D, Without AcrllA4). To investigate the regulation of the CRISPRa system using the miR-AcrllA4 switch, plasmids encoding each of dCas9-VPR, sgRNA _TRE and T21-AcrllA4 mRNA were transformed into miR-21 inhibitor or miR-21 inhibitor N. C. was co-transfected with HeLa-TRE _hmAG1 . As a result, the addition of the miR-21 inhibitor selectively and significantly reduced the hmAG1-positive cell population, whereas the miR-21 inhibitor N. C. did not reduce the hmAG1-positive cell population, indicating that hmAG1 expression depends on the activity of miR-21 (FIGS. 4B, 4C, 4D, T21_AcrllA4). This result indicates that the miR-AcrllA4 switch can activate genome-encoded genes in response to miRNA activity. In addition, by fusing the N-terminus of AcrllA4 to M9, a nuclear localization signal (NSL), hmAG1 expression levels could be improved without sacrificing Cas9 suppression efficiency. (FIGS. 4B, 4C, 4D, T21-M9_AcrllA4 (SEQ ID NO: 55). Furthermore, by varying the level of AcrllA4, we were able to optimize the expression level of the hmAG1 gene triggered by miR-21 ( Furthermore, we experimentally confirmed that similar results were obtained by changing the switch to one targeting miR-302a, confirming the generality of this strategy (details not shown). These results suggest that changing the expression level of AcrllA4 can achieve the optimal ON/OFF state of the CRISPR-Cas9 ON system using miRNA as an input signal.

(4) miRNA sensing and genome editing control by miR-AcrllA4 switch (5 ng) The same experiment as in (1) above was performed in iPS-EGFP cells, which are iPS cells in which the EGFP gene was integrated into the genome, and the EGFP gene Cas9 activity was assessed by knockout of iPS-EGFP cells were seeded at 5×10 ⁴ cells/well in 24 well plates 17-24 hours before transfection. AcrllA4-mRNA (5 ng), mRNA encoding Cas9 (Cas9 mRNA), and sgRNA _EGFP targeting the EGFP gene were transfected into iPS-EGFP cells using StemFect RNA Transfection Reagent according to the protocol. Three days after transfection, cells were captured by Cytell Cell Imaging System (GE Healthcare Life Sciences) prior to flow cytometric analysis. Cells were washed with PBS and treated with 100 μL Accumax for 10 min at 37° C., 5% CO ₂ . 200 μL of PBS was then added to suspend the detached cells. Cells were measured by flow cytometer. Flow cytometer measurement conditions were described in Experimental methods, EGFP knockout assay. FIG. 5A is a photograph of cells obtained as a result of a control experiment using no miRNA-responsive AcrllA4 mRNA. The left panel is the result without addition of AcrllA4, and the right panel is the result without addition of sgRNA _EGFP recognized by the target gene. indicates FIG. 5B is also a photograph of the cells obtained as a result of the control experiment, the left panel showing the addition of AcrllA4, the middle panel showing the addition of the inactivated AcrllA4 mutant, and the right panel showing the results of untreated). FIG. 6 shows the dot plot results, and FIG. 7 shows the percentage of EGFP-negative cell population in cells subjected to each manipulation. The results of FIGS. 5A, 5B, 6, and 7 showed almost the same tendency as the results obtained from FIG.

An experiment similar to (2) was then performed in iPS-EGFP cells. T302a-AcrllA4 switch or 4xT302a-AcrllA4 switch (both 5 ng) having 1 repeat or 4 repeats of the miR-302a complementary sequence as the target sequence was used as the switch. Cell seeding and transfection were performed in the same manner as the AcrllA4 mRNA transfection into iPS-EGFP cells. In both T302a-AcrllA4 switch and 4xT302a-AcrllA4 switch transfections, in addition to the switch, H ₂ O, miR-302 inhibitor, miR-302 inhibitor N.C. C. was added. Cas9 activity was assessed by EGFP gene knockout. FIG. 5C is a photograph of cells resulting from transfection with the T302a-AcrllA4 switch, and FIG. 5D is a photograph of cells resulting from transfection with the 4×T302a-AcrllA4 switch. FIG. 6 shows the dot plot results, and FIG. 7 shows the percentage of EGFP-negative cell population in cells subjected to each manipulation. The results of FIGS. 5C, 5D, 6 and 7 showed a trend similar to that obtained from FIG.

(5) miRNA sensing and genome editing control by miR-AcrllA4 switch (10 ng) Transfection into iPS-EGFP cells was performed in the same manner as in (4), except that 10 ng of AcrllA4 mRNA or miR-AcrllA4 switch was added. performed an injection. The results are shown in FIGS. 8A-D, 9 and 10. FIG. Even when the amount of miR-AcrllA4 switch to be introduced was changed, the same results as in the experiment (4) were shown.

In this study, we constructed a miR-AcrllA4 switch as a Cas9 regulator to cleave or activate target genes in response to highly active miRNAs. Because AcrllA4 delivered as mRNA efficiently suppressed Cas9 activity (Fig. 2A-C), regulation of AcrllA4 by miRNAs could be an attractive tool for genome editing and activation depending on cell state. In the absence of the input miRNA, the miR-AcrllA4 switch expressed AcrllA4 to effectively repress the activity of Cas9 or dCas9-VPR, resulting in an OFF state. On the other hand, the presence of the target miRNA formed an ON state that repressed the expression of AcrllA4 and upregulated the activity of Cas9 or dCas9-VPR. Hirosawa M, et al. The conventional Cas9-ON switch system disclosed in (Non-Patent Document 2) used two types of mRNA, Cas9 mRNA and L7Ae mRNA, which suppresses the translation of Cas9. Compared to this conventional system, the miR-AcrllA4 switch of the present invention reduced Cas9 leak expression in the OFF state. It is believed that the interaction of AcrllA4 and Cas9 effectively blocked Cas9 activity. On the other hand, in the conventional control system using L7Ae, since two types of mRNA, Cas9 mRNA and L7Ae mRNA, were co-transfected, it is considered that the translation of Cas9 could not be completely shut down. Furthermore, control of AcrllA4 by varying the amount and stability of input AcrllA4 could improve the ON state performance.

[experimental method]
[Construction of plasmid]
All PCR reactions were performed using KOD-Plus-Neo (TOYOBO) and all PCR products were purified using Min Elute PCR purification kit (QIAGEN). For ligation, Ligation high ver. 2 (TOYOBO) was used. All plasmid DNA was E. E. coli strain DH5α and purified using the Jetstar plasmid Midiprep Kit (Genomed) or the QIAGEN plasmid Midi Kit (QIAGEN) according to the manufacturer's instructions. Primer and oligonucleotide numbers are shown in Table 1 below.

[pUC19-AcrllA4]
Synthesis of AcrllA4 was contracted to gBlocks® Gene Fragments (IDT). This fragment was directly ligated into the pUC19 vector by digestion with the restriction enzyme SmaI.

[pHL-EF1α-AcrllA4-iP-A]
A DNA fragment containing AcrllA4 was obtained from pUC19-AcrllA4 with forward primer No. 1 and reverse primer no. 2 was used to amplify by PCR. The fragment was inserted between the EcoRI and SalI sites of pHL-EF1α-SphcCas9-iP-A (provided by Dr. Akitsu Hotta) to generate pHL-EFα-AcrllA4-iP-A.

[pHL-EF1α-AcrllA4_E70R-iP-A]
pHL-EF1α-AcrllA4_E70R-iP-A was constructed from pHL-EF1α-AcrllA4-iP-A using the KOD-Plus-Mutagenesis Kit according to the manufacturer's (TOYOBO) protocol. To introduce mutation, iPCR forward primer No. 3 and iPCR reverse primer No. 4 was used.

[pHL-EF1α-miR_AcrllA4-iP-A: miR=T21, T302a, 4xT21]
Each DNA fragment containing the miRNA target site was forward primer No. 5, reverse primer No. 6, and oligo DNA template Nos. 7, 8, 9 were amplified by PCR. oligo DNA template No. 7 is T21, oligo DNA template No. 8 is T302a, oligo DNA template No. 9 corresponds to 4xT21.
pHL-EF1α-AcrllA4-iP-A is a forward primer No. 10, and reverse primer no. 11 was used to linearize by inverse PCR. The resulting insert and linearized vector were fused using the In-Fusion® HD Cloning Kit according to the manufacturer's (Clontech) protocol.

[pHL-EF1α-M9_AcrllA4-iP-A and pHL-EF1α-T21_M9_AcrllA4-iP-A]
A DNA fragment containing M9 was obtained from pAptamerCassette-tagBFP-M9 (unpublished) with forward primer No. 12 and reverse primer no. 13 was used to amplify by PCR. pHL-EF1α-AcrllA4-iP-A and pHL-EF1α-T21_AcrllA4-iP-A are forward primer Nos. 14, and reverse primer No. 15 was used to linearize by inverse PCR. The M9 fragment and the linearized vector were fused using the In-Fusion® HD Cloning Kit according to the manufacturer's (Clontech) protocol.

[pHL-gRNA[EGFP]-mEF1α-mRFP-RiH]
A DNA fragment containing the target gene EGFP was forward primer No. 16 and reverse primer no. 18 was used to amplify by PCR. The DNA fragment was inserted between the BamHI site and the EcoRI site of pHL-gRNA[DMD]-mEF1α-mRFP-RiH (provided by Dr. Akitsu Hotta) to obtain pHL-gRNA[EGFP]-mEF1α-mRFP-RiH. generated.

[pHL-gRNA[TRE]-mEF1α-mRFP-RiH]
A DNA fragment containing the target gene TRE was forward primer No. 17 and reverse primer no. 18 was used to amplify by PCR. The DNA fragment was inserted between the BamHI and EcoRI sites of pHL-gRNA[EGFP]-mEF1α-mRFP-RiH to generate pHL-gRNA[TRE]-mEF1α-mRFP-RiH.

[pHL-gRNA [Target]-mEF1α-iRFP670-RiH: Target = TRE and EGFP]
ｉＲＦＰ６７０を含むＤＮＡフラグメントは、ｐｉＲＦＰ６７０－Ｎ１（Ａｄｄｇｅｎｅ， ｐｌａｓｍｉｄ ＃４５４５７，Ｓｈｃｈｅｒｂａｋｏｖａ ＤＭ，Ｖｅｒｋｈｕｓｈａ ＶＶ．（２０１３）Ｎｅａｒ－ｉｎｆｒａｒｅｄ ｆｌｕｏｒｅｓｃｅｎｔ ｐｒｏｔｅｉｎｓ ｆｏｒ ｍｕｌｔｉｃｏｌｏｒ ｉｎ ｖｉｖｏ ｉｍａｇｉｎｇ． Ｎａｔ Ｍｅｔｈｏｄｓ １０：７５１－７５４）から、ｆｏｒｗａｒｄ ｐｒｉｍｅｒ Ｎｏ . 19 and reverse primer no. 20 was used to amplify by PCR. The DNA fragment was inserted between the EcoRV and AvrII sites of pHL-gRNA[TRE]-mEF1α-mRFP-RiH and pHL-gRNA[EGFP]-mEF1α-mRFP-RiH to replace iRFP670 with mRFP.

[pTRE-Tight-hmAG1]
A DNA fragment containing hmAG1 was obtained from p5'RTM-hmAG1(deltapA)-ABHD12Bexon13 (unpublished) using forward primer No. 21 and reverse primer no. 22 and amplified by PCR. The DNA fragment was inserted between the EcoRI and EcoRV sites of pTRE-Tight (Clontech) to generate pTRE-Tight-hmAG1.

[PB-TRE_hmAG1]
A DNA fragment containing TRE-AG1 was obtained from pTRE-Tight-hmAG1 with forward primer No. 23 and reverse primer no. 24 and amplified by PCR. ＰＢ－ＴＲＥ－ＩＲＥＳ－ＡＧ（Ｎａｋａｎｉｓｈｉ Ｈ， ｅｔ ａｌ．（２０１７） Ｍｏｎｉｔｏｒｉｎｇ ａｎｄ ｖｉｓｕａｌｉｚｉｎｇ ｍｉｃｒｏＲＮＡ ｄｙｎａｍｉｃｓ ｄｕｒｉｎｇ ｌｉｖｅ ｃｅｌｌ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ｕｓｉｎｇ ｍｉｃｒｏＲＮＡ－ｒｅｓｐｏｎｓｉｖｅ ｎｏｎ－ｖｉｒａｌ ｒｅｐｏｒｔｅｒ ｖｅｃｔｏｒｓ． Ｂｉｏｍａｔｅｒｉａｌｓ １２８：１２１－１３５．）は、ｆｏｒｗａｒｄ ｐｒｉｍｅｒ Ｎｏ． 25 and reverse primer no. 26 was used to linearize by inverse PCR. The resulting DNA fragment and linearized vector were fused using the In-Fusion® HD Cloning Kit according to the manufacturer's (Clontech) protocol.

[DNA template for in vitro transfection (IVT)]
5'-UTRs without miRNA target sites (control 5'-UTR) and 3'-UTRs were extended by hybridizing synthetic oligo DNA. Cas9 protein and AcrllA4 protein coding regions were amplified by PCR from pHL-EF1α-SphcCas9-iP-A (Addgene, plasmid #60599) and pHL-EF1α-AcrllA4-iP-A, respectively, using appropriate primers. The AcrllA4_E70R protein coding region was amplified from pHL-EF1α-AcrllA4_E70R-iP-A. Plasmid DNA was removed by DpnI. The 5′-UTR, protein-coding region, and 3′-UTR were fused to construct Cas9 and AcrllA4 mRNA templates for IVT by further PCR reactions. sgRNA templates are described in Hirosawa M, et al. It was constructed by the method described in (Non-Patent Document 2). All PCR reactions were performed using KOD-Plus-Neo (TOYOBO) and all PCR products were purified using Min Elute PCR purification kit (QIAGEN). Primer and template combinations for PCR reactions are shown in Tables 2 and 3 below.

[Synthesis and purification of Cas9-mRNA, AcrllA4-mRNA and sgRNA]
All mRNAs were generated using the MEGAscript™ T7 Transcription Kit (Ambion). pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink Bio Technologies) instead of native uridine triphosphate and cytidine triphosphate, respectively, to reduce the interferon response induced by long RNAs was used. Guanosine 5′-triphosphate was diluted 5-fold with Anti-Reverse Cap Analog (TriLink Bio Technologies) prior to the IVT reaction. sgRNAs were constructed using the MEGAshortscript™ T7 Transcription Kit (Ambion). Natural bases such as rNTPs were used to prepare the sgRNA. To remove the DNA template, samples were treated with TURBO DNase (Ambion) for 30 minutes at 37°C. FavorPrep Blood/Cultured Cells total RNA extraction columns (Favorgen Biotech) were then generated, incubated with Antarctic Phosphatase (New England Biolabs) for 30 minutes at 37°C, and purified again using RNeasy MinElute ELISA. . The sgRNA was electrophoresed for further purification, extracted from a 10% polyacrylamide gel containing 8.3 M urea, and ethanol precipitated.

[Sequences of mRNA and sgRNA]
The sequences of the mRNA and sgRNA sequences used are shown in Table 4 below. In Table 4, open reading frames are shown in bold and start and stop codons are boxed. The miRNA target sequences on mRNA as well as the sequences recognized by target genes on sgRNA are underlined.

Generation of HeLa-TRE _hmAG1 cell line.
HeLa CCL2 cells (ATCC) were seeded in 24-well plates (2.5×10 ⁴ cells/well). 250 ng of PB-TRE_hmAG1 (SEQ ID NO: 56) and 250 ng of pCAG-PBase were co-transfected into cells using Lipofectamine™ 3000 transfection reagent according to the manufacturer's (ThermoFisher) protocol. Cells were passaged in 6-well plates, cultured and selected with puromycin (Invivogen).

[Cell culture]
HeLa-EGFP cells were cultured in DMEM High glucose (nakalai tesque) supplemented with 10% FBS (JBS). HeLa-TREhmAG1 cells were added with 10% FBS (JBS), 0.1 mM MEM-non-essential amino acids (Life technologies), 2 mM L-glutamine (Life technologies), and 1 mM sodium pyruvate in DMEM High glucose ( nakalai tesque). iPS_EGFP (AAVS1-CAG :: GFP iPS cells, Woltjen Lab (CiRA, Kyoto University, Japan) was cultured on a laminin-coated {laminin-511 E8 (iMatrix-511 silk, nippi)} plate using StemFit (Ajinomoto) medium. All cell lines were cultured at 37° C., 5% CO ₂ .

[Cas9 activity assay (EGFP knockout assay)]
HeLa-EGFP cells were seeded in 24-well plates (5×10 ⁴ cells/well). Cas9 mRNA, sgRNA, AcrllA4 mRNA, human miRNA-21-5p inhibitor/negative control (ThermoFisher, optional), and human miRNA-302a-5p mimic/negative control (ThermoFisher, optional) were obtained with Lipofectamine™ MessengerMAX (trademark) Cells were transfected using transfection reagent according to the manufacturer's (ThermoFisher) protocol. Cells were captured by an IX 81 microscope (Olympus) equipped with a CCD camera before flow cytometry analysis. Seventy-two hours after transfection, cells were washed with PBS and treated with 100 μL of 0.25% trypsin-EDTA for 5 minutes at 37° C., 5% CO ₂ . Then, 100 μL of medium was added to suspend the detached cells. Samples were analyzed on an Accuri™ C6 (BD Bioscience) flow cytometer using FL-1 (530/30 nm) filters. EGFP-negative and positive cell populations were gated from histograms (HeLa-EGFP) or dot plots (iPS-EGFP). The EGFP-negative cell population corresponds to successful EGF gene knockout by Cas9, and the EGFP-positive cell population corresponds to unsuccessful EGF gene knockout by Cas9.

[dCas9-VPR activation assay (hmAG1 activation assay)]
HeLa-TREhmAG1 cells were seeded in 24-well plates (5×10 ⁴ cells/well). dCas9-VPR plasmid, sgRNA plasmid, AcrllA4 plasmid, human miR-21-5p inhibitor / negative control (optional), and human miR-302a-5p mimic / negative control (optional) using Lipofectamine (trademark) 3000 transfection reagent , cells were transfected according to the manufacturer's instructions (ThermoFisher). Cells were captured by Cytell Cell Imaging System (GE Healthcare Life Sciences) prior to flow cytometric analysis. Forty-eight hours after transfection, cells were washed with PBS and treated with 100 μL of 0.25% trypsin-EDTA for 5 minutes at 37° C., 5% CO ₂ . Then, 100 μL of medium was added to suspend the detached cells. Samples were analyzed on an Accuri™ C6 flow cytometer using FL-1 (530/30 nm) and FL-4 (675/25 nm) filters at 99% attenuation. The iRFP670 positive cell population was gated to determine transfected cells. The hmAG1 positive and negative cell populations were then gated. The hmAG1 positive cell population corresponds to the successful hmAG1 gene activation by dCas9-VPR and the hmAG1 negative cell population corresponds to the failed hmAG1 gene activation by dCas9-VPR.

[Statistical analysis]
Student's t-test and Welch's t-test were used for statistical analysis. Levels of significance were indicated as follows. *P<0.05, **P<0.01, ***P<0.001. Student's t-test and Welch's t-test were performed using Excel (Microsoft).

Claims (11)

A method for cell-specific control of target gene editing, comprising:
(a) an mRNA encoding a first protein that edits a target gene;
(b) a miRNA-responsive mRNA encoding a second protein that inactivates the first protein;
(c) introducing into a cell an sgRNA comprising a guide sequence specifically recognized by said target gene, wherein said miRNA-responsive mRNA is:
(i) a nucleic acid sequence specifically recognized by a miRNA; and (ii) a nucleic acid sequence corresponding to the coding region of said second protein;
The method, wherein the miRNA is a miRNA that is specifically highly active in the cell. the first protein is a Cas9 protein or an analogue thereof;
2. The method of claim 1, wherein said second protein is AcrllA4 or an analogue thereof. the first protein is a dCas9-VPR protein or an analogue thereof;
2. The method of claim 1, wherein said second protein is AcrllA4 or an analogue thereof. 2. The method of claim 1, wherein (a), (b) and/or (c) are introduced into the cell in the form of synthetic RNA molecules without the use of DNA vectors or viral vectors. 5. The method according to any one of claims 1 to 4, wherein (i) a plurality of nucleic acid sequences specifically recognized by miRNA are contained in the untranslated region of the miRNA-responsive mRNA. 4. The method according to claim 2 or 3, wherein said second protein is AcrllA4 having a nuclear localization signal fused to its N-terminal side. A kit for cell-specific control of target gene editing, comprising:
(a) an mRNA encoding a first protein that edits a target gene;
(b) a miRNA-responsive mRNA encoding a second protein that inactivates the first protein;
(c) an sgRNA comprising a guide sequence specifically recognized by said target gene, wherein said miRNA-responsive mRNA is:
(i) a nucleic acid sequence specifically recognized by a miRNA; and (ii) a nucleic acid sequence corresponding to the coding region of said second protein;
The kit, wherein the miRNA is a miRNA that is specifically highly active in the cell. the first protein is a Cas9 protein or an analogue thereof;
8. The kit of claim 7, wherein said second protein is AcrllA4 or an analogue thereof. the first protein is a dCas9-VPR protein or an analogue thereof;
8. The kit of claim 7, wherein said second protein is AcrllA4 or an analogue thereof. The kit according to any one of claims 7 to 9, wherein the (i) nucleic acid sequence specifically recognized by miRNA is contained in the 5'-UTR. 10. The kit according to claim 8 or 9, wherein said second protein is AcrllA4 having a nuclear localization signal fused to its N-terminus.

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