mRNA switch and method for controlling protein expression using the same

Abstract

The object of the present invention is to develop a mRNA switch which enables the construction of a precise translation regulation circuit and can be used as a transformation regulation module having versatility and orthogonality.　A mRNA switch comprising an artificial mRNA molecule which comprises (i) a nucleic acid sequence capable of being recognized specifically by an input protein comprising Cas protein or a variant thereof and (ii) a nucleic acid sequence encoding an output protein, wherein the nucleic acid sequence (i) is located on the 5'- or 3'-side of the nucleic acid sequence (ii) and the nucleic acid sequences (i) and (ii) are linked to each other operably.

Description

[Technical field]

[0001] The present invention relates to an mRNA switch and a method for controlling protein expression using the same. [Background technology]

[0002] In recent years, research has been conducted in gene therapy and cell therapy to improve therapeutic effects and reduce side effects by "programming" cell behavior with DNA or RNA molecules introduced from outside the cell. Programmed cells are expected to detect signals in the environment and autonomously select appropriate therapeutic molecules and output them, as well as the timing, duration, and strength of their expression. However, to realize such a precise control mechanism, it is necessary to create multiple modules that control transcription and translation, and to construct a logic operation circuit (translation control circuit) that performs complex information processing like a computer by combining these multiple modules in a complex manner. In this respect, translation control circuits have the advantage of being safer than circuits based on transcription control, since there is virtually no risk of insertion into the genome. In addition, since there is no need to go through the transcription step, they respond to inputs more quickly than circuits based on transcription control, and they have attracted attention because it is possible to construct a mechanism that can respond more quickly to environmental signals.

[0003] As a translation control module, an mRNA switch is known. An mRNA switch is an artificial mRNA that has an RNA sequence (aptamer) that binds to a specific molecule (input molecule), and the translation state of the protein it codes for (output protein) changes when the input molecule binds to the aptamer. There are OFF switches, in which the translation of the output protein is suppressed when the input molecule binds, and ON switches, in which the translation of the output protein is induced. This switching is mainly caused by (1) steric hindrance caused by the input molecule-aptamer complex, or (2) cleavage of the mRNA by the input molecule.

[0004] An example of (1) is an OFF switch (Patent Document 1) developed by the present inventors, which has a kink-turn sequence in the 5'UTR that is specifically recognized by L7Ae (a protein that constitutes the large subunit of the archaeal ribosome), and in which the expression of L7Ae forms an L7Ae-kink-turn complex on the 5'UTR, thereby suppressing translation. An example of (2) is an OFF / ON switch that has a target sequence of miRNA, and is degraded in the presence of the miRNA to suppress or de-suppress expression (Patent Document 2). Furthermore, an OFF / ON switch that has a pre-crRNA sequence of the Csy4 (Cas6f), Cpf1 (Cas12a), Cas6, or CasE (ygcH, Cas6e, Cse3) protein of the CRISPR-Cas system, and is cleaved by the expression of the Cas protein to suppress or de-suppress expression has also been reported (Non-Patent Documents 1, 2, Patent Document 3, etc.).

[0005] The inventors have shown that by introducing three types of mRNA switches into a cell and having the output protein expressed from the first mRNA switch function as the input protein of the second mRNA switch, and having the output protein of the second mRNA switch function as the input protein of the third mRNA, it is possible to construct a highly precise translation control mechanism in which expression of the third output protein is doubly controlled (Patent Document 4, Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 040395 [Patent Document 2] International Publication No. 2015 / 105172 [Patent Document 3] US Patent Application Publication No. 2019 / 0032054 [Patent Document 4] International Publication No. 2016 / 040395 [Non-patent literature]

[0007] [Non-Patent Document 1] Borchardt, EK et al. RNA 21, 1921-1930 (2015) [Non-Patent Document 2] Zhong, G., Wang, H., Li, Y., Tran, MH & Farzan, M. Nat. Chem. Biol. 13, 839-841 (2017) [Non-Patent Document 3] Matsuura, S., et al. Nat. Commun. 9, 4847-4854 (2018) Summary of the Invention [Problem to be solved by the invention]

[0008] However, to realize a more precise translational control mechanism, the translation of one or more final output proteins needs to be controlled by many modules consisting of RNA-protein interactions. To achieve this, it is necessary to prepare the number of highly versatile and orthogonal translational control modules required for the desired gene circuit, which do not interfere with each other. However, there are too few modules (only a few) that satisfy this requirement, which has been a problem.

[0009] (1) In an mRNA switch based on the steric hindrance of an input molecule-aptamer complex, the stability of the complex is very important as well as the specificity of sequence recognition, so it was not easy to find a combination of an input molecule and an aptamer that satisfies these requirements. In addition, (2) in an mRNA switch that uses miRNA as an input molecule, the input molecule is preferably an endogenous miRNA, and therefore it is necessary to select an input molecule for each target cell, which causes a problem in terms of versatility. Furthermore, the mRNA switch that uses the Cas protein as an input molecule utilizes the reaction in which the Cas protein converts (processes) its own pre-crRNA into crRNA, so the Cas proteins that can be used as input molecules are very limited. This is because the Cas proteins that have RNA cleavage activity in heterologous cells and are responsible for the processing reaction are very limited.

[0010] Thus, it is extremely difficult to develop a translational control module that is both versatile and orthogonal, which has hindered the construction of precise translational control circuits. [Means for solving the problem]

[0011] As a result of intensive research, the present inventors have found that by using the crRNA or sgRNA of the Cas protein as an aptamer, the Cas protein can be used as an input molecule regardless of the presence or absence of RNA cleavage activity. This is a method in which the Cas protein is used simply as an RNA-binding protein, rather than as an enzyme that cleaves RNA in a sequence-dependent manner, and it has become clear that this makes it possible to form a highly stable complex containing the Cas protein and the aptamer on the mRNA. Based on these findings, the present inventors have developed a novel mRNA switch, and further found that by combining these mRNA switches in a complex manner, a precise translation control circuit can be constructed in human cells, thereby completing the present invention.

[0012] That is, the present invention includes the following aspects. [1] (i) a nucleic acid sequence that is specifically recognized by an input protein, the input protein comprising a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein; Including, An mRNA switch comprising an artificial mRNA molecule in which (i) is present on the 5' or 3' side of (ii), and (i) and (ii) are operably linked. [2] The mRNA switch described in [1], wherein the nucleic acid sequence (i) is a crRNA or sgRNA sequence of the input protein, or a modified version thereof. [3] The mRNA switch according to [1] or [2], wherein the Cas protein is one selected from the group consisting of SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, and Cas14a1. [4] The mRNA switch described in any one of [1] to [3], wherein the artificial mRNA molecule further comprises an RNA inverter sequence between the nucleic acid sequences of (i) and (ii), and the RNA inverter sequence consists of a nucleic acid sequence including a bait open reading frame, an intron, and an internal ribosome entry site. [5] A vector comprising a nucleic acid sequence encoding the mRNA switch described in any one of [1] to [4]. [6] The vector described in [5], further comprising a transcription control sequence located upstream of the nucleic acid sequence encoding the mRNA switch, the transcription control sequence being a sequence that is specifically recognized by a transcription control protein. [7] The vector described in [6], wherein the transcriptional control protein includes a Cas protein or a modified version thereof, and further includes a nucleic acid sequence encoding a small RNA used for transcriptional control or translational control, which is located downstream of the nucleic acid sequence encoding the mRNA switch. [8] A cell comprising an mRNA switch described in any one of [1] to [4] or a vector described in any one of [5] to [7]. [9] (a) the mRNA switch according to any one of [1] to [4] or a vector encoding the mRNA switch; and (b) A kit for controlling protein expression, comprising the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA.

[10] (a) The mRNA switch according to any one of [1] to [4], or a vector encoding the mRNA switch; (b) a first trigger mRNA comprising a nucleic acid sequence encoding a first fusion protein comprising a first fragment of the input protein and a first heterodimerization domain, or a vector encoding the first trigger mRNA; and (c) A protein expression control kit comprising a second trigger mRNA comprising a nucleic acid sequence encoding a second fusion protein comprising a second fragment of the input protein of (b) and a second heterodimerization domain, or a vector encoding the second trigger mRNA.

[11] The expression control kit described in

[10] , further comprising an agent that promotes heterodimerization by the first and second heterodimerization domains.

[12] A kit for controlling expression of a protein described in any one of [9] to

[11] , further comprising an input inhibitor that inhibits recognition of the nucleic acid sequence (i) by the input protein.

[13] An mRNA switch set comprising n types of mRNA switches consisting of a first mRNA switch to an nth mRNA switch or a vector encoding the mRNA switches, wherein n is selected from an integer of 2 to 25; (A) The kth mRNA switch (i) a nucleic acid sequence specifically recognized by an input protein consisting of a kth protein, including a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein consisting of a (k+1)th protein; The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, the kth protein and the (k+1)th protein are different proteins; k is an integer from 1 to (n-1); (B) The nth mRNA switch is (i) a nucleic acid sequence that is specifically recognized by an nth protein that is an output protein of an (n-1)th mRNA switch; (ii) a nucleic acid sequence encoding an output protein that is the (n+1)th protein; Including, The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, A set of mRNA switches, wherein the (n+1)th protein is any protein.

[14] The mRNA switch set according to

[13] , wherein the first protein to the nth protein are all different proteins.

[15] The switch set according to

[13] , wherein the (n+1) protein is a first input protein.

[16] The mRNA switch set according to any one of

[13] to

[15] , wherein the Cas protein or a variant thereof contained in the k and (k+1) proteins is one Cas protein or a variant thereof selected from the group consisting of SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, and Cas14a1.

[17] (a) the mRNA switch according to any one of [1] to [4] or a vector encoding the mRNA switch; and (b) A method for controlling protein expression, comprising the step of introducing into a cell the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA.

[18] (a) The mRNA switch according to any one of [1] to [4], or a vector encoding the mRNA switch; (b) a first trigger mRNA comprising a nucleic acid sequence encoding a first fusion protein comprising a first fragment of the input protein and a first heterodimerization domain, or a vector encoding the first trigger mRNA; and (c) A method for controlling protein expression, comprising the step of introducing into a cell a second trigger mRNA comprising a nucleic acid sequence encoding a second fusion protein comprising a second fragment of the input protein of (b) and a second heterodimerization domain, or a vector encoding the second trigger mRNA.

[19] A method for controlling expression of a protein described in

[18] , further comprising, after the introducing step, a step of contacting the cells with a drug that promotes heterodimerization by the first and second heterodimerization domains.

[20] A method for controlling expression of a protein described in any one of

[17] to

[19] , further comprising the step of introducing into a cell an input-inhibiting protein, an input-inhibiting mRNA encoding the input-inhibiting protein, or a vector encoding the input-inhibiting mRNA, which specifically inhibits the recognition of the nucleic acid sequence (i) by the input protein.

[21] A method for controlling protein expression, comprising the step of introducing an mRNA switch set described in any one of

[13] to

[16] into a cell.

[22] (a) The vector according to [6], wherein the transcriptional control protein comprises a Cas protein or a variant thereof, and further comprises a nucleic acid sequence encoding a crRNA or sgRNA that specifically recognizes the transcriptional control sequence, which is provided downstream of a nucleic acid sequence encoding the mRNA switch; and (b) a transcriptional control protein comprising a Cas protein or a variant thereof corresponding to a nucleic acid sequence encoding a crRNA or sgRNA that specifically recognizes the transcriptional control sequence of (a), a transcriptional activity control mRNA encoding the transcriptional control protein, or a vector encoding the transcriptional activity control mRNA. A kit for controlling protein expression comprising the steps of: Effect of the Invention

[0013] According to the present invention, a switch nucleic acid can be obtained that is orthogonal and has a translation control module that does not interfere with other modules. Furthermore, by combining a plurality of the translation control modules and by combining one or more transcription control modules, it becomes possible to construct a precise translation control circuit and a gene expression control circuit in a eukaryotic cell. The mRNA switch according to the present invention enables safer and more efficient cell programming and has various applications. [Brief description of the drawings]

[0014] [Figure 1]1 is a diagram showing an mRNA switch according to one embodiment of the present invention and translation control by an input protein, in which the upper left diagram shows a combination of an mRNA switch whose aptamer sequence is sgRNA and an input protein that specifically recognizes the sgRNA, and the lower left diagram shows a state in which the translation of an output protein is suppressed by the input protein. The upper right diagram shows a combination of an mRNA switch whose aptamer sequence is crRNA and an input protein that specifically recognizes the crRNA, and the lower left diagram shows a state in which the translation of an output protein is suppressed by the input protein. [Figure 2A] Figure 2A is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by SpCas9 and codes for an output protein, with a control mRNA (No aptamer), which does not have an aptamer and codes for an output protein, in the presence of the input protein SpCas9. [Figure 2B] Figure 2B is a graph comparing the translation efficiency in the presence of the input protein SaCas9 between an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by SaCas9 and encodes an output protein, and a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein. [Figure 2C] Figure 2C is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by CjCas9 and codes for an output protein, with a control mRNA (No aptamer), which does not have an aptamer and codes for an output protein, in the presence of the input protein CjCas9. [Figure 2D] Figure 2D is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by NmCas9 and encodes an output protein, with a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein, in the presence of the input protein NmCas9. [Figure 2E]Figure 2E is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by St1Cas9 and encodes an output protein, with a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein, in the presence of the input protein St1Cas9. [Figure 2F] Figure 2F is a graph comparing the translation efficiency of an OFF switch mRNA (Switch) that has an aptamer sequence specifically recognized by AsCas12a and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein, in the presence of the input protein AsCas12a. [Figure 2G] Figure 2G is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by PspCas13b and encodes an output protein, with a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein, in the presence of the input protein PspCas13b. [Figure 2H] Figure 2H is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by PguCas13b and encodes an output protein, with a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein, in the presence of the input protein PguCas13b. [Figure 2I] Figure 2I is a graph comparing the translation efficiency of an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by RanCas13b and encodes an output protein, with a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein, in the presence of the input protein RanCas13b. [Figure 2J]Figure 2J is a graph comparing the translation efficiency in the presence of the input protein CasRx between an OFF switch mRNA (Switch), which has an aptamer sequence specifically recognized by CasRx and encodes an output protein, and a control mRNA (No aptamer), which does not have an aptamer and encodes an output protein. [Diagram 3] Figure 3 is a graph comparing the translation efficiency of six types of mRNA switches (Nm_gRNA, Nm_gRNA_v1, Nm_gRNA_v2, Nm_gRNA_v3, Nm_gRNA_v4, Nm_gRNA_v5) with different aptamer sequences that have been created for mRNA switches that have an aptamer sequence specifically recognized by NmCas9 and encode an output protein, together with a control mRNA (No aptamer), in the presence of NmCas9. [Figure 4A] To confirm whether the suppression of expression of the mRNA switch is truly caused by translational suppression, wild-type SpCas9 [SpCas9(WT)], a mutant with DNA cleavage activity (nickase) [SpCas9(D10A)], a mutant with DNA cleavage activity (DNA cleavage null) [SpCas9(D10A_H840A)], and SpCas9 without NLS [SpCas9(ΔNLS)] were used as input proteins, and the translation efficiency of the mRNA switch specifically recognized by these and the control mRNA (No aptamer) was confirmed in the presence of each input protein. Figure 4A shows the results. [Figure 4B] Figure 4B shows the results of examining the translation efficiency of an mRNA switch (Switch) that specifically recognizes SpCas9 and a control mRNA (No aptamer) in the presence and absence of AcrIIA4 to confirm whether the anti-CRISPR protein (AcrIIA4), which inhibits the DNA binding ability of SpCas9, inhibits the suppression of expression of the mRNA switch. [Diagram 5]Figure 5 shows the results of confirming the translation efficiency of an mRNA switch (As_crRNA) specifically recognized by wild-type AsCas12a (AsCpf1) [Cpf1 WT] and the AsCas12a(H800A) mutant [Cpf1(H800A)], as well as a control mRNA (No aptamer), in the presence of each input protein to confirm whether mRNA cleavage is essential for translational repression. [Figure 6A] Figure 6A is a graph comparing the translation efficiency in the presence of the input protein SpCas9 between an ON switch mRNA (SpCas9-responsive) that has an aptamer sequence specifically recognized by SpCas9 and encodes an output protein, and a control mRNA (Control), which is a nucleic acid that does not have an aptamer sequence and has an inverter sequence. [Figure 6B] Figure 6B is a graph comparing the translation efficiency of an ON switch mRNA (PspCas13b-responsive) that has an aptamer sequence specifically recognized by PspCas13b and encodes an output protein, and a control mRNA (Control) in the presence of the input protein PspCas13b. [Figure 6C] Figure 6C is a graph comparing the translation efficiency of an ON switch mRNA (SaCas9-responsive) that has an aptamer sequence specifically recognized by SaCas9 and encodes an output protein, with a control mRNA (Control) in the presence of the input protein SaCas9. [Figure 6D] Figure 6D is a graph comparing the translation efficiency of an ON switch mRNA (CjCas9-responsive) that has an aptamer sequence specifically recognized by CjCas9 and encodes an output protein, and a control mRNA (Control) in the presence of the input protein CjCas9. [Figure 6E]Figure 6E is a graph comparing the translation efficiency of an ON switch mRNA (St1Cas9-responsive) that has an aptamer sequence specifically recognized by St1Cas9 and encodes an output protein, with a control mRNA (Control) in the presence of the input protein St1Cas9. [Figure 6F] Figure 6F is a graph comparing the translation efficiency of ON switch mRNAs (AsCas12a-responsive and AsCas12a(H800A)-responsive) that have aptamer sequences specifically recognized by AsCas12a and AsCas12a(H800A), respectively, and encode output proteins, with a control mRNA (Control) in the presence of input proteins AsCas12a and AsCas12a(H800A). [Figure 7A] Figure 7A is a diagram plotted by flow cytometry of the change in fluorescence of the switch depending on whether or not a trigger mRNA expressing an input protein was introduced. The upper figure is a dot plot for each case in which the trigger mRNA was introduced and not, and the lower figure is a histogram showing the distribution of the numerical values ​​of the fluorescence intensity ratio EGFP / iRFP670 for each case in which the trigger mRNA was introduced and not. [Figure 7B] Figure 7B is a graph showing the translation efficiency of the control mRNA (No aptamer) and the mRNA switch (Sp_gRNA) at each amount of trigger mRNA introduced. [Figure 8A] Figure 8A is a graph comparing the translation efficiency in the presence of the input protein FnCas9 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by FnCas9 and encode an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8B]Figure 8B is a graph comparing the translation efficiency in the presence of the input protein FnCpf1 between an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by FnCpf1 and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8C] Figure 8C is a graph comparing the translation efficiency in the presence of input protein LbCpf1 between an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by LbCpf1 and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8D] Figure 8D is a graph comparing the translation efficiency in the presence of the input protein MbCpf1 between an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by MbCpf1 and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8E] Figure 8E is a graph comparing the translation efficiency of two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by AkCas12b and code for an output protein, and a control mRNA (No aptamer) that does not have an aptamer and codes for an output protein, in the presence of the input protein AkCas12b. [Figure 8F] Figure 8F is a graph comparing the translation efficiency of an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by BvCas12b and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein, in the presence of the input protein BvCas12b. [Figure 8G]Figure 8G is a graph comparing the translation efficiency of an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by BsCas12b and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein, in the presence of the input protein BsCas12b. [Figure 8H] Figure 8H is a graph comparing the translation efficiency in the presence of the input protein PlmCasX between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by PlmCasX and code for an output protein, and a control mRNA (No aptamer) that does not have an aptamer and codes for an output protein. [Figure 8I] Figure 8I is a graph comparing the translation efficiency in the presence of the input protein CdCas9 between two types of OFF switch mRNA (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by CdCas9 and encode an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8J] Figure 8J is a graph comparing the translation efficiency of an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by ClCas9 and encodes an output protein, with a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein, in the presence of the input protein ClCas9. [Figure 8K] Figure 8K is a graph comparing the translation efficiency in the presence of the input protein NcCas9 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by NcCas9 and encode an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8L]Figure 8L is a graph comparing the translation efficiency in the presence of the input protein PlCas9 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by PlCas9 and code for an output protein, and a control mRNA (No aptamer) that does not have an aptamer and codes for an output protein. [Figure 8M] Figure 8M is a graph comparing the translation efficiency in the presence of the input protein SpaCas9 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by SpaCas9 and code for an output protein, and a control mRNA (No aptamer) that does not have an aptamer and codes for an output protein. [Figure 8N] Figure 8N is a graph comparing the translation efficiency in the presence of the input protein St3Cas9 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by St3Cas9 and encode an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. [Figure 8O] Figure 8O is a graph comparing the translation efficiency in the presence of the input protein Cas14a1 between two types of OFF switch mRNAs (Switch 1 and Switch 2) that have an aptamer sequence specifically recognized by Cas14a1 and code for an output protein, and a control mRNA (No aptamer) that does not have an aptamer and codes for an output protein. [Figure 9A] Figure 9A is a graph comparing the translation efficiency of an ON switch mRNA (CasRx-responsive) that has an aptamer sequence specifically recognized by CasRx and encodes an output protein, with a control mRNA (Control) in the presence of the input protein CasRx. [Figure 9B]Figure 9B is a graph comparing the translation efficiency of an ON switch mRNA (PguCas13b-responsive) that has an aptamer sequence specifically recognized by PguCas13b and encodes an output protein, with a control mRNA (Control) in the presence of the input protein PguCas13b. [Figure 9C] Figure 9C is a graph comparing the translation efficiency of an ON switch mRNA (AkCas12b-responsive) that has an aptamer sequence specifically recognized by AkCas12b and encodes an output protein, and a control mRNA (Control) in the presence of the input protein AkCas12b. [Figure 9D] Figure 9D is a graph comparing the translation efficiency of an ON switch mRNA (BvCas12b-responsive) that has an aptamer sequence specifically recognized by BvCas12b and encodes an output protein, and a control mRNA (Control) in the presence of the input protein BvCas12b. [Figure 9E] Figure 9E is a graph comparing the translation efficiency of an ON switch mRNA (PlmCasX-responsive) that has an aptamer sequence specifically recognized by PlmCasX and encodes an output protein, with a control mRNA (Control) in the presence of the input protein PlmCasX. [Figure 9F] Figure 9F is a graph comparing the translation efficiency of an ON switch mRNA (LbCas12a-responsive) that has an aptamer sequence specifically recognized by LbCas12a and encodes an output protein, and a control mRNA (Control) in the presence of the input protein LbCas12a. [Figure 9G] Figure 9G is a graph comparing the translation efficiency of an ON switch mRNA (FnCas12a-responsive) that has an aptamer sequence specifically recognized by FnCas12a and encodes an output protein, and a control mRNA (Control) in the presence of the input protein FnCas12a. [Figure 10A] Figure 10A is a schematic diagram showing that when both Cas9 fragments, SpCas9(N-term) and SpCas9(C-term), are present in a cell, full-length SpCas9 is generated and translational repression of the mRNA switch is triggered. [Figure 10B] Figure 10B is a table showing the experimental results, where the presence of SpCas9(N-term) and SpCas9(C-term) is represented as input (1) and no input (0), respectively, and the absence of SpCas9(N-term) is represented as output (1) when translational inhibition is not induced and the output protein is expressed, and the absence of output (0) when translational inhibition is induced and the output protein is not expressed. [Figure 10C] FIG. 10C is a graph showing the translation efficiency for each input in FIG. 10B and when unsegregated wild-type Cas is used (WT). [Figure 11A] Figure 11A is a conceptual diagram showing that split Cas9 was prepared by fusing the DmrA- or DmrC-binding domain to the C- or N-terminus of each fragment of SpCas9(N-term) and SpCas9(C-term), respectively, and that full-length SpCas9 is generated only when a drug is added, causing translational repression of the mRNA switch. [Figure 11B] Split 1-3 in Figure 11B shows the translation efficiency when a drug is added (Drug+) and when it is not added (Drug-) for each of Split 1-3, in the presence of both fragments, for an OFF switch mRNA (Switch) that has an aptamer sequence specifically recognized by SpCas9 and encodes an output protein, and for a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein. WT is a graph showing the translation efficiency when a drug is added (Drug+) and when it is not added (Drug-) for the same OFF switch mRNA (Switch) and control mRNA (No aptamer) in the presence of wild-type SpCas9. [Figure 12]Figure 12 shows the translation efficiency when no plasmid vector encoding AcrIIC2 was added (0 ng) or when 200 ng, 400 ng, 1000 ng, or 2000 ng of a plasmid vector encoding AcrIIC2 was added to an OFF switch mRNA (Switch) that has an aptamer sequence specifically recognized by SaCas9 and encodes an output protein, and a control mRNA (No aptamer) that does not have an aptamer and encodes an output protein, demonstrating that AcrIIC2 relieves translational inhibition by SaCas9. [Figure 13A] Figure 13A is a fluorescent photograph showing the results of confirming the orthogonality of translational repression between nine types of Cas proteins (SpCas9, SaCas9, CjCas9, St1Cas9, AsCas12a, PspCas13b, PguCas13b, RanCas13b, and CasRx) and nine types of mRNA switches that have aptamer sequences that are specifically recognized by each of them. For the combinations corresponding to the areas enclosed in white squares, unintended translational repression occurred, but orthogonality was confirmed for the other combinations. [Figure 13B] FIG. 13B shows the results of FIG. 13A, with shading based on translation efficiency. [Figure 13C] Figure 13C is a fluorescent photograph showing the results of confirming the orthogonality of translational repression between 14 types of Cas proteins and 14 types of mRNA switches each having an aptamer sequence specifically recognized by each of them, as well as the L7Ae protein, which is known as a protein-RNA binding motif (RNP motif), and the BoxCD sequence recognized by L7Ae. [Figure 14A] FIG. 14A is a diagram conceptually showing the configuration of a multilayer circuit in which switch sets each composed of six types of OFF switch mRNA are hierarchically combined. [Figure 14B] FIG. 14B shows fluorescence micrographs at each layer, histograms obtained from flow cytometry analysis, and calculated output levels. [Figure 15A]Panel (a) of Figure 15A is a fluorescent photograph showing the results of orthogonality testing for 29 types of OFF switch mRNAs, and panel (b) shows the results of 13 types of Cas-responsive OFF switch mRNA switches that showed clear orthogonality among the tested switches. [Figure 15B] FIG. 15B is a heat map showing the image quantification results for the 13 Cas-responsive OFF switch mRNAs in FIG. 15A, panel (b). [Figure 16] Figure 16 shows the results of a test to determine whether SpCas9 can be used to simultaneously control translation (OFF switch mRNA) and transcriptional activation of a DNA construct. Panel (a) shows an overview of the mechanism, and panel (b) shows a cell photograph. [Figure 17] Figure 17 shows a scheme of an AND gate circuit that can be constructed by combining three types of Cas-responsive OFF switch mRNAs. [Figure 18A] Figure 18A shows a scheme of a half-subtractor constructed using only two modules that simultaneously utilizes transcriptional and translational control by Cas proteins. [Figure 18B] FIG. 18B shows the truth table for the half subtractor. [Figure 18C] FIG. 18C shows a fluorescent micrograph of the cells corresponding to the traces. [Figure 19] Figure 19 is a graph comparing the translation efficiency of Nm_gRNA_v6 and Nm_gRNA_v7, which have an aptamer sequence specifically recognized by NmCas9 and encode an output protein, in addition to those shown in Figure 3, together with a control mRNA (No aptamer), in the presence of NmCas9. [Figure 20] FIG. 20 is a fluorescent photograph showing the results of orthogonality tests for 26 types of ON switch mRNAs. [Figure 21] Figure 21 shows the results of a test to simultaneously drive SaCas9-responsive translation OFF switch mRNA (red fluorescence) and translation ON switch mRNA (green fluorescence), where panel (a) shows a cell photograph and panel (b) shows the quantification results by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described. However, the present invention is not limited to the embodiment described below.

[0016] [1. mRNA switch] According to a first embodiment, the present invention relates to an mRNA switch, comprising: (i) a nucleic acid sequence that is specifically recognized by an input protein comprising a Cas protein or a variant thereof; and (ii) a nucleic acid sequence encoding an output protein, wherein (i) is present on the 5' or 3' side of (ii), and (i) and (ii) are operably linked.

[0017] The mRNA switch in this embodiment refers to an artificial mRNA molecule that codes for a specific output protein and controls the translation of the output protein in response to a specific input protein. Controlling translation in response to a specific input protein refers to the reversal of the translation state of the output protein (a state in which translation is occurring or a state in which translation is not occurring) between the presence and absence of a specific input protein. In this embodiment, the state in which translation is not occurring is caused by translation suppression, and may be called a translation suppression state. In addition, changing the degree of translation or translation suppression (translation efficiency) depending on the amount of a specific input protein is also included in "control". In this specification, an mRNA switch in which translation is performed in the presence of a specific input protein and translation is suppressed in its absence is referred to as an ON switch mRNA. Conversely, an mRNA switch in which translation is suppressed in the presence of a specific input protein and translation is performed in its absence is referred to as an OFF switch mRNA.

[0018] The input protein is a protein that specifically recognizes the mRNA switch, and includes at least a Cas protein or a variant thereof. The Cas protein may be any Cas protein, such as, for example, SpCas9 (Cas9 from Streptococcus pyogenes, aka SpyCas9, SEQ ID NO: 1), SaCas9 (Cas9 from Staphylococcus aureus, aka SauCas9, SEQ ID NO: 2), CjCas9 (Cas9 from Campylobacter jejuni, aka CjeCas9, SEQ ID NO: 3), NmCas9 (Cas9 from Neisseria meningitidis, aka NmeCas9, SEQ ID NO: 4), St1Cas9 (Cas9 from Streptococcus thermophilus, aka Sth1Cas9, SEQ ID NO: 5), FnCas9 (Cas9 from Francisella novicida, aka FnoCas9, SEQ ID NO: 6), CdCas9 (Cas9 from Corynebacterium diphtheriae, aka CdiCas9, SEQ ID NO: 7), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 8), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 9), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 10), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 11), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 12), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 13), ClCas9 (Cas9 from Campylobacter lari, aka CdiCas9, SEQ ID NO: 14), ClCas9 (Cas9 from Campylob CF89-12, SEQ ID NO: 8), PlCas9 (Cas9 from Parvibaculum lavamentivorans, SEQ ID NO: 9), NcCas9 (Cas9 from Neisseria cinerea, SEQ ID NO: 10), SpaCas9 (Cas9 from Streptococcus pasteurianus, SEQ ID NO: 11), St3Cas9 (Cas9 from Streptococcus thermophilus, also known as Sth3Cas9, SEQ ID NO: 12), AsCas12a (Cas9 from Acidaminococcus sp.Cas12a derived from BV3L6, alias AsCpf1, SEQ ID NO: 13), FnCas12a (Cas12a derived from Francisella novicida U112, alias FnCpf1, SEQ ID NO: 14), LbCas12a (Cas12a derived from Lachnospiraceae bacterium ND2006, alias LbCpf1, SEQ ID NO: 15), MbCas12a (Cas12a derived from Moraxella bovoculi 237, alias MbCpf1, SEQ ID NO: 16), AkCas12b (Cas12b derived from Alicyclobacillus kakegawensis, alias AkC2c1, SEQ ID NO: 17), BvCas12b (Cas12b derived from Bacillus sp. V3-13, alias BvC2c1, SEQ ID NO: 18), BsCas12b (Cas12b derived from Bacillus sp. Cas12 derived from NSP2.1, alias BsC2c1, SEQ ID NO: 19), PspCas13b (Cas13b derived from Prevotella sp., SEQ ID NO: 20), PguCas13b (Cas13b derived from Porphyromonas gulae, SEQ ID NO: 21), RanCas13b (Cas13b derived from Riemerella anatipestifer, SEQ ID NO: 22), CasRx (Cas protein derived from Ruminococcus flavefaciens XPD3002, alias RfxCas13d, SEQ ID NO: 23), PlmCasX (CasX derived from Planctomycetes, alias PlmCas12e, SEQ ID NO: 24), Cas14a1 (Cas14a derived from uncultured archaeon, alias Cas14a.1, SEQ ID NO: 25), AaCas12b. In addition, Cpf1, which is shown as part of the alias, is an abbreviation for CRISPR-associated endonuclease in Prevotella and Francisella 1. The Cas protein can be appropriately selected depending on the purpose and use of the mRNA switch. In the present invention, the Cas protein that is the input protein may or may not have DNA cleavage activity.

[0019] The input protein may be the Cas protein itself, or a variant thereof. The variant may be, for example, a fusion product in which an additional protein is fused to the Cas protein. The additional protein may be any protein that does not inhibit the recognition ability of the mRNA switch by the Cas protein, and may be a functional protein such as the transcription activator VP16, VP64, or VP64-p65-Rta (VPR), the transcription repressor Kruppel associated box domain (KRAB), the DNA methyltransferase DNMT3A, the DNA demethylase TET1, the histone acetyltransferase LSD1 or p300, the RNA degrading enzyme CNOT7 or DDX6, the translation factor eIF family, the virus-derived protein VPg, the RNA modifying enzyme ADAR, DNMT, METTL, WTAP, FTO or ALKBH5, or a fluorescent protein.

[0020] The modified Cas protein functioning as an input protein may be an assembly of Cas proteins divided into multiple fragments, or may be a molecule to which a molecule such as a protein used in the assembly is further bound. The assembly may be an assembly that has the ability to recognize the mRNA switch. The modified Cas protein may also be a Cas protein fragment obtained by removing a portion of the Cas protein that is not involved in the recognition of the RNA sequence. The Cas protein fragment may also be referred to as a miniaturized Cas protein.

[0021] The mRNA switch is typically an RNA molecule. In some embodiments, the mRNA switch may be a synthetic mRNA molecule. The synthetic mRNA molecule is not particularly limited, and may be, for example, an mRNA molecule synthesized in a test tube. The synthetic mRNA can be directly introduced into a cell in the form of an mRNA molecule and used for translation control. In this specification, an mRNA switch consisting of an mRNA molecule may be referred to as a switch mRNA, a protein-responsive mRNA, or a switch nucleic acid. For example, when the input protein is SpCas9, it may be referred to as a SpCas9-responsive mRNA.

[0022] In another embodiment, the mRNA switch may be an mRNA produced by transcription from a DNA construct in a cell, and the DNA construct may be a vector, etc. Hereinafter, the structure and sequence determination of the RNA molecule constituting the mRNA switch will be described.

[0023] (i) a nucleic acid sequence that is specifically recognized by an input protein, including a Cas protein or a variant thereof; The mRNA switch comprises a nucleic acid sequence that is specifically recognized by an input protein, including a Cas protein or a variant thereof. In this specification, the nucleic acid sequence of (i) may also be referred to as an aptamer sequence.

[0024] The nucleic acid sequence of (i) is not particularly limited as long as it is specifically recognized by an input protein including a Cas protein or a variant thereof and enables translational control, but is typically a crRNA or sgRNA sequence corresponding to the Cas protein. The nucleic acid sequence of (i) may be a variant of a crRNA or sgRNA sequence, and the variant refers to a variant that retains the specific binding ability to a Cas protein. The combination of a certain Cas protein and a corresponding crRNA or sgRNA sequence is widely known. A person skilled in the art can obtain the information from a protein database or literature and design the nucleic acid sequence of (i). In addition, when the pre-crRNA corresponding to the Cas protein is used as the nucleic acid sequence of (i), it may be undesirable because the mRNA switch may be cut in the pre-crRNA region, which may result in the loss of reversible translation control depending on the presence or absence of the input protein.

[0025] Specific examples of combinations of Cas protein (Target protein) and crRNA or sgRNA sequence include, but are not limited to, those shown in Table 1 below. Switch name represents the name of the mRNA switch. [Table 1A] [Table 1B] [Table 1C]

[0026] (ii) a nucleic acid sequence encoding an output protein The mRNA switch includes a nucleic acid sequence that codes for an output protein. The nucleic acid sequence that codes for the output protein can be appropriately determined by a person skilled in the art according to a desired output protein. The output protein is not particularly limited and may be any protein.

[0027] In some embodiments, the output protein may be a Cas protein or a variant thereof. The Cas protein or a variant thereof may be the same as that described in the definition of the input protein, and may be selected from the same options. Note that in one mRNA switch molecule, the output protein may be the same as or different from the input protein.

[0028] In another embodiment, the output protein may be an anti-CRISPR that inactivates a Cas protein. Anti-CRISPRs that can inactivate spCas9 include, but are not limited to, AcrIIA2, AcrIIA4, AcrIIA5, AcrIIA7, AcrIIA8, AcrIIA9, and AcrIIA10. Anti-CRISPRs that inactivate NmCas9 include AcrIIC1, AcrIIC2, AcrIIC3, AcrIIC4, and AcrIIA5; anti-CRISPRs that inactivate CjCas9 include AcrIIC1; anti-CRISPRs that inactivate St1Cas9 include AcrIIA5 and AcrIIA6; and anti-CRISPRs that inactivate SaCas9 include AcrIIC2 and AcrIIA5.

[0029] In another embodiment, the output protein may be a marker protein. The marker protein is a protein that is expressed from the mRNA switch and functions as a marker in a cell to identify the cell. The marker protein may be, for example, a protein that can be visualized and quantified by fluorescence, luminescence, color, or by assisting fluorescence, luminescence, or color. Examples of the fluorescent protein include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, and HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; and KusabiraOrange. (e.g., hmKO2), mOrange, and other orange fluorescent proteins; TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, and other red fluorescent proteins; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, mPlum, and other near-infrared fluorescent proteins, but are not limited to these.

[0030] An example of a luminescent protein is aequorin, but is not limited thereto. In addition, an example of a protein that assists fluorescence, luminescence, or color development is an enzyme that decomposes a fluorescent, luminescent, or color precursor, such as luciferase, phosphatase, peroxidase, or β-lactamase, but is not limited thereto. Here, in the present invention, when a protein that assists fluorescence, luminescence, or color development is used as an output protein, the fluorescence, luminescence, or color development can be observed by contacting the corresponding precursor with a cell into which an mRNA switch is introduced and introducing the corresponding precursor into the cell.

[0031] Other examples of marker proteins include proteins that directly affect the function of cells. Examples include, but are not limited to, cell proliferation proteins, cell death proteins, cell signaling factors, drug resistance genes, transcriptional regulators, translational regulators, differentiation regulators, reprogramming inducers, RNA-binding protein factors, chromatin regulators, and membrane proteins. For example, cell proliferation proteins function as markers by proliferating only cells that express them and identifying the proliferated cells. Cell death proteins cause cell death in the cells that express them, killing the cells themselves and functioning as markers indicating the life or death of the cells. Cell signaling factors function as markers by identifying the signals that cells that express them emit specific biological signals. Examples of cell death proteins include Bax and Bim. For example, translational regulators function as markers by controlling the translation of other mRNAs into proteins by recognizing and binding to the tertiary structure of specific RNA. The translation regulators are 5R1, 5R2 (Nat Struct Biol. 1998 Jul; 5(7):543-6), B2 (Nat Struct Mol Biol. 2005 Nov;12(11):952-7), Fox-1 (EMBO J. 2006 Jan 11;25(1):163-73.), GLD-1 (J Mol Biol. 2005 Feb 11;346(1):91-104.), Hfq (EMBO J. 2004 Jan 28;23(2):396-405), HuD (Nat Struct Biol. 2001 Feb;8(2):141-5.), SRP19 (RNA. 2005 Jul;11(7):1043-50), and L1 (Nat Struct Biol. 2003 Feb;10(2):104-8.), L11(Nat Struct Biol. 2000 Oct;7(10):834-7.), L18(Biochem J. 2002 Mar 15;362(Pt 3):553-60), L20(J Biol Chem. 2003 Sep 19;278(38):36522-30.), L23(J Biomol NMR. 2003 Jun;26(2):131-7), L25(EMBO J.1999 Nov 15;18(22):6508-21.)、L30(Nat Struct Biol. 1999 Dec;6(12):1081-3.)、LicT(EMBO J. 2002 Apr 15;21(8):1987-97.)、MS2 coat(FEBS J. 2006 Apr;273(7):1463-75.)、Nova-2(Cell. 2000 Feb 4;100(3):323-32)、Nucleocapsid(J Mol Biol. 2000 Aug 11;301(2):491-511.)、Nucleolin(EMBO J. 2000 Dec 15;19(24):6870-81.)、p19(Cell. 2003 Dec 26;115(7):799-811)、L7Ae(RNA. 2005 Aug;11(8):1192-200.)、PAZ(PiWi Argonaut and Zwille)(Nat Struct Biol. 2003 Dec;10(12):1026-32.)、RnaseIII(Cell. 2006 Jan 27;124(2):355-66)、RR1-38(Nat Struct Biol. 1998 Jul;5(7):543-6.)、S15(EMBO J. 2003 Apr 15;22(8):1898-908.)、S4(J Biol Chem. 1979 Mar 25;254(6):1775-7.)、S8(J Mol Biol. 2001 Aug 10;311(2):311-24.)、SacY(EMBO J. 1997 Aug 15;16(16):5019-29.)、SmpB(J Biochem (Tokyo). 2005 Dec;138(6):729-39)、snRNP U1A(Nat Struct Biol. 2000 Oct;7(10):834-7.)、SRP54(RNA. 2005 Jul;11(7):1043-50)、Tat(Nucleic Acids Res. 1996 Oct 15;24(20):3974-81.)、ThrRS(Nat Struct Biol. 2002 May;9(5):343-7.)、TIS11d(Nat Struct Mol Biol. 2004 Mar;11(3):257-64.), Virp1 (Nucleic Acids Res. 2003 Oct 1;31(19):5534-43.), Vts1P (Nat Struct Mol Biol. 2006 Feb;13(2):177-8.), and λN (Cell. 1998 Apr 17;93(2):289-99.). More preferred translation control factors are MS2 coat protein and L7Ae.

[0032] The nucleic acid sequence of (ii) may encode a localization signal in addition to the marker protein. Examples of the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and the like. Specifically, examples of the localization signal include, but are not limited to, a classical nuclear localization sequence (NLS), an M9 sequence, a mitochondrial targeting sequence (MTS), and an endoplasmic reticulum localization sequence. Such a localization signal is advantageous when the marker protein is visualized by imaging cytometry or the like.

[0033] In the mRNA switch, the nucleic acid sequence of (i) is present on the 5' or 3' side of the nucleic acid sequence of (ii), and (i) and (ii) are operably linked. Detailed aspects of the operably linked structure will be described below for the OFF switch mRNA and the ON switch mRNA. When an mRNA switch having such structural features is introduced into a cell, it becomes possible to control the translation of the nucleic acid sequence of (ii) when an input protein specifically recognizes the nucleic acid sequence of (i).

[0034] A more specific structure of an mRNA molecule that functions as an OFF switch mRNA will be described. The OFF mRNA switch may be a structure in which, starting from the 5' side of an mRNA molecule, 5'-UTR, coding region, and 3'-UTR are linked. When the nucleic acid sequence of (i) is present on the 5' side of the nucleic acid sequence of (ii), the 5'-UTR may be a structure in which, starting from the 5' side, [Cap structure or Cap analog] and [the nucleic acid sequence of (i)] are linked. The Cap structure may be 7-methylguanosine 5' phosphate. The Cap analog is a modified structure that is recognized by the translation initiation factor eIF4E in the same way as the Cap structure, and includes, but is not limited to, Anti-Reverse Cap Analog (ARCA) manufactured by Ambion, m7G(5')ppp(5')G RNA Cap Structure Analog manufactured by New England Biolabs, and CleanCap manufactured by TriLink. Any 5' capping structure for avoiding synthetic mRNA from natural immune responses may be used. The 3' side of the Cap structure or Cap analog, and the 5' side of the nucleic acid sequence of (i), may contain an arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 0 to 30 bases. At least one nucleic acid sequence of (i) may be contained, but two, three, four or more repeats of the nucleic acid sequence of (i) may be contained. The 3' end side of the 5'-UTR, which is the 3' side of the nucleic acid sequence of (i), may contain an arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 10 to 30 bases. These arbitrary nucleic acid sequences are preferably nucleic acid sequences that do not form a secondary structure and do not specifically interact with an input protein or an output protein. It is preferable that the 5'-UTR does not have an AUG as a start codon. For example, when the nucleic acid sequence of (i) contains an AUG, a frame shift can be avoided by adding one or two bases to the end of the sequence. Alternatively, a stop codon sequence may be added outside the nucleic acid sequence of (i) counted in 3-base units from the AUG. Alternatively, one or more AUG bases may be converted to any base as long as they do not affect the interaction with the protein. The coding region comprises the nucleic acid sequence of (ii).The 3'-UTR may contain a polyA tail and may have a binding sequence for a Cas protein (a sequence recognized by any Cas protein) inserted therein. When the nucleic acid sequence of (i) is present on the 3' side of the nucleic acid sequence of (ii), the nucleic acid sequence of (i) may be located at the 3' end or 5' end of the polyA tail, or may be inserted within the polyA tail. The nucleic acid sequence of (i) may also be present on both the 3' and 5' sides of the nucleic acid sequence of (ii).

[0035] The OFF switch mRNA may contain modified bases such as pseudouridine and 5-methylcytidine instead of normal uridine and cytidine to reduce cytotoxicity, but unmodified bases are preferable. The positions of the modified bases can be all or part of the uridine and cytidine independently, and if part of the modified bases, they can be at random positions in any ratio.

[0036] Next, a more specific structure of an mRNA molecule that functions as an ON switch mRNA will be described. An ON switch mRNA may also have a structure in which, from the 5' end of an mRNA molecule, a 5'-UTR, a coding region, and a 3'-UTR are linked. The 5'-UTR of an ON switch mRNA has a structure in which, from the 5' end, [Cap structure or Cap analog], [the nucleic acid sequence of (i)], and [an RNA inverter sequence] are linked.

[0037] The RNA inverter sequence is a sequence that is located on the 5' side of the start codon (coding region) and on the 3' side of the nucleic acid sequence of (i) to reverse translational repression and control translation of an output protein from an mRNA only in the presence of an input protein. The RNA inverter sequence is also referred to as an ON switch cassette and is described in detail in International Publication No. 2014 / 014122, which is incorporated herein by reference. Specifically, the RNA inverter sequence is composed of a sequence including, in order from the 5' side, a mutant open reading frame (bait ORF), an intron, and an IRES (internal ribosome entry site). Here, the bait ORF is a mutant ORF that has a stop codon 320 bases or more away from the 3' end that binds to the intron, among sequences that code for any gene, in order to cause RNA degradation by nonsense-mediated mRNA decay (NMD). In the present invention, the bait ORF may be any coding gene. Examples of bait ORF include, but are not limited to, a sequence in which a stop codon is inserted at the 457th and 466th bases from the 5' side of Renilla luciferase (SEQ ID NO: 64 or SEQ ID NO: 65), or a sequence in which a stop codon is inserted at the 172nd base from the 5' side of EGFP (SEQ ID NO: 66). Introns may have a sequence to which spliceosomes bind, and examples include sequences of 20 or more bases having a GT sequence at the 5' end and an AG sequence at the 3' end. Preferably, it is a human β-globin intron (SEQ ID NO: 67) or a chimeric intron (SEQ ID NO: 68). Examples of bait ORF and intron sequences are shown in Table 2 below.

[0038] [Table 2A] [Table 2B]

[0039] In the ON switch mRNA, as in the OFF switch mRNA, an arbitrary nucleic acid sequence may be included in the design of the 5'-UTR on the 3' side of the Cap structure or Cap analog and on the 5' side of the nucleic acid sequence of (i). An arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 10 to 30 bases, may also be included on the 3' side of the nucleic acid sequence of (i) on the 5' side of the RNA inverter sequence, and on the 3' end side of the 5'-UTR on the 3' side of the RNA inverter sequence. In addition, the design when AUG is included in the sequence, the coding region, the configuration of the 3'-UTR, and the mode of including modified bases may also be the same as those of the OFF switch mRNA.

[0040] [2. How to use mRNA switches] Next, the method of using and the action of this mRNA switch will be described. The mRNA switch can be used in a manner that it is introduced into a cell, and translation is controlled depending on the presence or absence and the amount of an input protein in the cell.

[0041] Here, the "cell" is not particularly limited and may be any cell. For example, it may be a cell collected from a multicellular organism, or may be a cell (including a cell line) that has been artificially manipulated. Preferably, it is a cell derived from a mammal (e.g., human, mouse, monkey, pig, rat, etc.), and most preferably, it is a cell derived from a human. There is no particular limit to the degree of differentiation of the cell or the age of the animal from which the cell is collected, and it may be any of (A) stem cells, (B) progenitor cells, (C) terminally differentiated somatic cells, and (D) other cells.

[0042] (A) Examples of 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, etc. Among these, ES cells and iPS cells are preferred, and iPS cells are particularly preferred.

[0043] (B) Examples of precursor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0044] (C) Examples of somatic cells include keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the tongue surface), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic and storage cells (e.g., hepatic cells), luminal epithelial cells that form the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain (e.g., vascular endothelial cells), ciliated cells with transport function (e.g., airway epithelial cells), cells that secrete 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), neurons and glial cells (e.g., astrocytes) of the central and peripheral nervous systems, pigment cells (e.g., retinal pigment epithelial cells), and their precursor cells (tissue precursor cells).

[0045] (D) Other cells include, for example, cells that have undergone differentiation induction, including precursor cells and somatic cells induced to differentiate from pluripotent stem cells. Also, the cells may be a cell group induced by so-called "direct conversion (also called direct reprogramming or trans-differentiation)" in which somatic cells or precursor cells are directly differentiated into desired cells without going through an undifferentiated state. In addition, the cells may be a cell group that may include cells for which gene editing is desired and cells for which gene editing is not desired, such as a cell group that may include cancer cells and normal cells.

[0046] When the mRNA switch is a synthetic RNA molecule (in most cases, a synthetic mRNA molecule) or a self-replicating RNA, the RNA molecule can be directly introduced into the cell using an introduction method such as lipofection, liposome, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, gene gun, etc. The advantage of introducing a synthetic RNA molecule is that it is not integrated into the genome, and the cell after introducing the mRNA switch can be easily used for medical applications.

[0047] A DNA construct such as an expression vector can be used to introduce the mRNA switch into a cell. In this case, an expression vector encoding the mRNA switch is designed, and the expression vector can be directly introduced into a cell by the same introduction method as described above. The expression vector encoding the sequence of the mRNA switch can be one well known and commonly used in the art, and examples thereof include a virus vector, an artificial chromosome vector, a plasmid vector, and an expression system using a transposon (sometimes called a transposon vector). Examples of the virus vector include a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a Sendai virus vector. Examples of the artificial chromosome vector include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC). Examples of the plasmid vector include mammalian plasmids in general, and may be, for example, an episomal vector. Examples of the transposon vector include an expression vector using a piggyBac transposon. For example, vectors disclosed in U.S. Patent No. 10,378,070, a U.S. patent by the present inventors and incorporated herein by reference, may be used, but are not limited thereto. By introducing an expression vector encoding an mRNA switch into a cell, the mRNA switch transcribed from the expression vector and generated in the cell can function in the same manner as a directly introduced synthetic mRNA molecule. The advantage of introducing an expression vector is that the mRNA switch can be made to function continuously in the cell, and for example, it can be used in artificial cells such as CAR-T. In addition, by incorporating an inducible promoter into the DNA construct, it is possible to induce the expression of the mRNA switch at a desired time or to stop the expression induction. In addition, by performing cloning after the introduction of the expression vector, a cell population with an almost uniform expression level of the mRNA switch can be obtained.

[0048] Furthermore, the DNA construct may be a vector containing a nucleic acid sequence encoding an mRNA switch and a transcription control sequence provided upstream of the nucleic acid sequence encoding the mRNA switch. Such a vector is referred to as a transcription control switch vector in this specification. By providing a transcription control sequence, it becomes possible to control the transcription activity of the vector, particularly to activate transcription, and to control the expression of the mRNA switch encoded by the vector. The transcription control sequence is a sequence that is specifically recognized by a transcription control protein. In addition, the transcription control protein is a protein, a fusion protein, or a protein complex containing these, having a DNA binding site and a transcription control site. The DNA binding site constituting the transcription control protein is a site necessary for the protein itself to directly recognize the transcription control sequence, or a site necessary for recognizing the transcription control sequence via DNA, RNA, or the like, and is a site that specifically recognizes the transcription control switch vector. In addition, the transcription control site is a protein having a transcription promoting activity or a transcription repressing activity, or a variant thereof. Examples of such transcription control proteins include, for example, a complex of a Cas protein or a variant thereof and a corresponding crRNA or sgRNA sequence or a variant thereof. Alternatively, the transcription control protein may be, but is not limited to, a TALEN. The mechanism of transcriptional control may be, for example, a method of directly methylating or demethylating DNA, or acetylating or deacetylating histones on DNA or chromatin, a method of cleaving DNA, or a method of indirectly accumulating a factor having these functions in a transcriptional control sequence, but is not limited to these. When the transcriptional control protein is a complex containing a Cas protein or a variant thereof, the Cas protein or a variant thereof constituting the complex may be the same as or different from the input protein of the mRNA switch encoded by the transcriptional control switch vector. In this case, the transcriptional control protein may be a complex containing an inactivated Cas protein fused to the above-mentioned transcriptional activation factors VP16, VP64, and VP64-p65-Rta (VPR).Alternatively, it may be a complex containing an inactive Cas protein fused to a transcriptional repressor Kruppel associated box domain (KRAB).Specific examples of the transcriptional regulatory protein that can be used include, but are not limited to, complexes containing dCas9-VP64, dCas9-VPR, dCas9-SunTag, dCas9-VP16, dCas9-VP160, dCas9-P300, and dCas9-KRAB.

[0049] The transcription control switch vector may contain, in addition to the desired mRNA switch, a nucleic acid sequence encoding an input RNA that can be used to control the transcription control switch vector, other transcription control switch vectors, or mRNA switch. The input RNA may be a small RNA used for transcription control or translation control. For example, when the transcription control protein is a complex containing a Cas protein or a variant thereof, the input RNA may be a crRNA or sgRNA sequence constituting the complex, or a variant thereof. The input RNA may be a crRNA or sgRNA sequence that specifically recognizes the transcription control sequence of the transcription control switch vector, or a variant thereof. Alternatively, the input RNA may be a crRNA or sgRNA sequence that specifically recognizes the transcription control sequence of a transcription control switch vector different from the transcription control switch vector, or a variant thereof. When the transcription control switch vector contains a nucleic acid sequence encoding an input RNA, it is preferable to provide a nucleic acid sequence that stabilizes the mRNA switch at the 3' of the nucleic acid sequence encoding the mRNA switch. The nucleic acid sequence that stabilizes the mRNA switch may be MALAT1 triplex, etc., but is not limited to a specific sequence. It is preferable that the nucleic acid sequence that stabilizes the mRNA switch contains a nucleic acid sequence encoding an input RNA on the 3' side. It is preferable that the nucleic acid sequence encoding the input RNA further contains a nucleic acid sequence encoding a self-cleaving ribozyme on the 5' and 3' sides.

[0050] The mRNA switch according to the present invention can be used in the form of an mRNA molecule or in the form of a vector in combination with an input protein, and translation control can be achieved in various usage modes. In addition, a vector containing a nucleic acid sequence encoding the mRNA switch can be used in combination with a transcription control protein as a transcription control switch vector, and transcription control and translation control can be achieved simultaneously. Below, the mRNA switch, mRNA switch set, and protein expression control kit containing these that realize this will be described for each mode.

[0051] [A. Translational control of mRNA switches by input proteins] A translation control system consisting of an input / output system that combines an input protein and an mRNA switch will be described. The translation control of the mRNA switch can be rephrased as an expression control system of an output protein. The input protein according to this embodiment may be any of the input proteins described above. The mRNA switch may be an ON switch mRNA or an OFF switch mRNA as long as it has an aptamer sequence that is specifically recognized by the input protein and codes for any output protein.

[0052] In the translational control system of aspect A, the input protein can be introduced into the cell as it is, or in the form of an mRNA encoding the input protein, or a vector encoding the mRNA. The mRNA encoding the input protein is also called a trigger mRNA, and the plasmid vector encoding it is also called a trigger plasmid. The mRNA switch can be introduced into the cell in the form of an mRNA molecule or a vector encoding the mRNA. The mRNA molecule constituting the mRNA switch is also called a switch mRNA, and the vector encoding the mRNA switch is also called a switch vector. When the switch vector is a plasmid vector, it is also called a switch plasmid.

[0053] A protein expression control kit for implementing the translational control system of embodiment A may include at least one component selected from the group consisting of an input protein, a trigger mRNA, and a trigger plasmid, and at least one component selected from the group consisting of an mRNA switch or a switch vector.

[0054] The translation control method of the mRNA switch according to the embodiment A can also be called a method for controlling the expression of an output protein, and includes a step of introducing an mRNA switch or a switch vector into a desired cell. Hereinafter, this step is referred to as an introduction step of the mRNA switch. It also includes a step of introducing an input protein, a trigger mRNA, and a trigger plasmid into the cell. Hereinafter, this step is also referred to as an input step. The introduction step and the input step can be performed simultaneously or at any time difference. By such an input step, the translation state by the mRNA switch or the switch vector can be switched to a state in which a desired output protein is expressed (output present), or a state in which the translation of the mRNA switch or the switch vector is suppressed and the output protein is not expressed (no output). In particular, in the case of the ON switch mRNA, the input step causes the translation of the mRNA switch to be performed to a state in which the output protein is expressed (output present), and in the case of the OFF switch mRNA, the input step causes the translation of the mRNA switch to be suppressed to a state in which the output protein is not expressed (no output).

[0055] By using two or more combinations of input proteins and mRNA switches, a system that independently controls the translation of two or more mRNA switches, i.e., the expression of two or more output proteins, can be realized. In this case, a first input / output system using a combination of a first input protein, a trigger mRNA or a trigger plasmid, and a first mRNA switch or a switch vector, and a second input / output system using a combination of a second input protein, a trigger mRNA or a trigger plasmid, and a second mRNA switch are used. The first mRNA switch has an aptamer sequence that is specifically recognized by the first input protein, and encodes the first output protein. The second mRNA switch has an aptamer sequence that is specifically recognized by the second input protein, and encodes the first output protein. By designing the Cas protein contained in the first input protein and the Cas protein contained in the second input protein to be different Cas proteins, these two input / output systems are orthogonal. That is, the two input / output systems can independently control input / output without interfering with each other, and the expression of the two output proteins is independently controlled.

[0056] [B. Translational control of mRNA switches by input proteins and input inhibitors] In addition to the input / output system of embodiment A, a translation control system (output protein expression control system) using an input inhibitor will be described. The system of embodiment B includes a combination of a first input protein, a trigger mRNA or a trigger plasmid, and a first mRNA switch or a switch vector, as well as a substance that inhibits the recognition of the mRNA switch by the input protein. Such a substance is referred to as an input inhibitor. An example of an input inhibitor is a protein that inactivates the Cas protein contained in the first input protein. Such a protein is referred to as an input inhibitor protein. An example of an input inhibitor protein is AcrIIC2. Another example of an input inhibitor is a drug consisting of a low molecular weight compound.

[0057] The protein expression control kit according to embodiment B may contain an input inhibitor in addition to the components of the translation control kit described in embodiment A. When the input inhibitor is an input inhibitory protein, the kit may contain the input inhibitory protein itself, an mRNA encoding the input inhibitory protein, or a vector encoding the mRNA. When the input inhibitor is a drug, the protein expression control kit according to embodiment B contains the drug.

[0058] The mRNA molecule encoding the input inhibitory protein or the vector encoding the mRNA may be an mRNA that expresses the input inhibitory protein without translational control or a vector encoding the mRNA molecule, i.e., an mRNA that does not have an aptamer sequence or a vector encoding the mRNA.

[0059] Alternatively, the mRNA encoding the input-inhibiting protein may be an mRNA switch. This is referred to as an input-inhibiting mRNA switch. In this case, the input-inhibiting mRNA switch can also be referred to as a second mRNA switch. The second mRNA switch has an aptamer sequence that is specifically recognized by the second input protein, and encodes the input-inhibiting protein as an output protein. Then, by designing the first and second mRNA switches so that the first input protein and the second input protein are different, the first mRNA switch and the second mRNA switch (input-inhibiting mRNA switch) are independently translationally controlled, and the expression of the output protein of the first mRNA switch is controlled.

[0060] In embodiment B, the step of introducing the first mRNA switch or switch vector and the step of inputting the first input protein, trigger mRNA or trigger plasmid can be carried out as described above in embodiment A. By further including a step of introducing an input inhibitor into the cell or contacting the cell with an input inhibitor, it is possible to inhibit the interaction between the first mRNA switch and the first input protein and control the translation state achieved by the first mRNA switch and the first input protein, thereby controlling the expression of the output protein.

[0061] [C. Translational control of mRNA switches by fragmented input proteins] In an input / output system based on a combination of an input protein and an mRNA switch, a translation control system (protein expression control) system for an mRNA switch in the case where the input protein is a fragmented protein is described below. A fragmented protein is also called a split protein. In embodiment C, the input protein is composed of a Cas protein split into multiple fragments or a modified version thereof. These multiple fragments associate under certain conditions to form an association having nucleic acid targeting ability (aptamer recognition ability), and function as an input protein.

[0062] The fragmented input protein can be designed based on the 25 types of Cas proteins listed above and other Cas proteins. It is known that in certain Cas proteins, the nucleic acid targeting ability is restored by fragmenting the protein between specific residues and then associating the fragments, and a Cas protein fragment capable of restoring the nucleic acid targeting ability can be designed based on known information. Specifically, fragmentation between residues 714 and 715 of SpCas9, between residues 535 and 536, between residues 713 and 714, etc. are known, but are not limited to these. A person skilled in the art can identify a fragment capable of restoring the nucleic acid targeting ability for any Cas protein. As long as the nucleic acid targeting ability can be restored, one input protein may be fragmented into two, or may be fragmented into three, four, five, or more.

[0063] When an input protein is split into a first fragment and a second fragment, a heterodimerization domain may be bound to each of the first fragment and the second fragment. The heterodimerization domain bound to the first fragment and the heterodimerization domain bound to the second fragment are domains capable of forming a heterodimer. The heterodimerization domain can increase the efficiency of the assembly during the formation of the assembly. As an example, the heterodimerization domain may be a separated intein, and may be one in which an N intein and a C intein are bound to two fragments of the split input protein, respectively. When the heterodimerization domain is a separated intein, if the first fragment bound to the N intein and the second fragment bound to the C intein are present in the cell at the same time, an assembly is formed. In the assembly, the intein portion is excised and functions as an input protein. Therefore, the action of translation control by the assembly is the same as that of the input protein in embodiment A.

[0064] Another example of the heterodimerization domain is a domain that associates in the presence of a drug consisting of a specific low molecular weight compound, and the iDimerize™ Inducible Heterodimer System (Clontech) can be used. When the heterodimerization domain is based on this system, the fragmented input protein may be a fragment in which the N-terminal fragment of the input protein is fused with a DmrA binding domain, and a fragment in which the C-terminal fragment of the input protein is fused with a DmrC binding domain. These domains associate in the presence of A / C Heterodimerizer (AP21967 ligand) and restore the nucleic acid recognition ability of the input protein.

[0065] In the embodiment C, a first trigger mRNA containing a nucleic acid sequence encoding a first fusion protein containing a first fragment of an input protein and a first heterodimerization domain, and a second trigger mRNA containing a nucleic acid sequence encoding a second fusion protein containing a second fragment of an input protein and a second heterodimerization domain are used. The first and second trigger mRNAs may be mRNAs without an aptamer sequence, or may be mRNA switches. In the case of an mRNA switch, the first trigger mRNA may be an mRNA switch having a nucleic acid sequence encoding a first fusion protein and an aptamer sequence specifically recognized by a predetermined Cas protein. The second trigger mRNA may be an mRNA switch having a nucleic acid sequence encoding a second fusion protein and an aptamer sequence specifically recognized by a predetermined Cas protein. The aptamer sequence of the first trigger mRNA and the aptamer sequence of the second trigger mRNA may be the same or different. Preferably, the aptamer sequence of the first trigger mRNA and the aptamer sequence of the second trigger mRNA are different, and the first trigger mRNA and the second trigger mRNA may be specifically recognized by different Cas proteins. One of the first trigger mRNA and the second trigger mRNA may be an mRNA without an aptamer sequence, and the other may be an mRNA switch.

[0066] In addition, even if the first and second trigger mRNAs are mRNAs having no aptamer sequence or mRNA switches, vectors encoding the mRNAs may be used instead of the first and second trigger mRNAs. The following explanation of embodiment C will be given for the case where a trigger mRNA is used, but a vector encoding the mRNA can also be used instead of the trigger mRNA.

[0067] The protein expression control kit according to embodiment C includes the first and second trigger mRNAs or vectors encoding the trigger mRNAs in addition to the mRNA switch or vector encoding the mRNA described in embodiment A. In addition, the kit may optionally include an agent for promoting heterodimerization.

[0068] In the translation control method (expression control method) using the translation control system (protein expression control system) of the mRNA switch according to aspect C, the introduction step of the mRNA switch can be carried out in the same manner as in aspect A. In this step, the input step includes a step of introducing a first trigger mRNA and a step of introducing a second trigger mRNA. The first and second trigger mRNAs can be introduced into cells simultaneously, or can be introduced at any time lag. In addition, in a system using a drug for promoting heterodimerization, the system includes a step of contacting the drug with cells, and this contact step can also be carried out simultaneously with the introduction step of the mRNA switch and the introduction step of the first and second trigger mRNAs, or can be carried out at any time lag. According to aspect C, after the input step, the trigger mRNA or the vector encoding the mRNA expresses the first fragment of the input protein and the second fragment of the input protein, and when the two become an assembly having nucleic acid targeting ability (aptamer recognition ability), the assembly functions as an input protein, enabling translation control of the mRNA switch. That is, the translation control system of the mRNA switch according to embodiment C (protein expression control system) makes it possible to control input proteins.

[0069] [D. Hierarchical Control] Hierarchical control using an mRNA switch set will be described. The mRNA switch set is composed of a plurality of different mRNA switches or vectors encoding the mRNAs. Specifically, the mRNA switch set includes n types of mRNA switches consisting of the first mRNA switch to the nth mRNA switch. n represents the type of mRNA switch included in the set, and n is selected from integers of 2 to 25. The kth mRNA switch included in the n types of mRNA switches is defined as follows: (A) The kth mRNA switch (i) a nucleic acid sequence specifically recognized by an input protein consisting of a kth protein, including a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein consisting of a (k+1)th protein; The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, the kth protein and the (k+1)th protein are different proteins; k is an integer from 1 to (n-1). Furthermore, the nth mRNA switch included in the n types of mRNA switches is defined as follows. (B) The nth mRNA switch is (i) a nucleic acid sequence that is specifically recognized by an nth protein that is an output protein of an (n-1)th mRNA switch; (ii) a nucleic acid sequence encoding an output protein that is a (n+1) protein; The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, The (n+1)th protein is an arbitrary protein.

[0070] In the case of the kth mRNA switch defined in (A), "k" is a variable for defining one type of mRNA switch included in the n types of mRNA switches. The kth mRNA switch receives input from the kth protein and outputs the (k+1)th protein. The (k+1)th protein output here functions as an input protein for the (k+1)th mRNA switch in the next hierarchy. A switch set consisting of n types of switches includes (n-1) types of mRNA switches defined in (A), from the first mRNA switch to the (n-1)th mRNA switch. The first protein that serves as an input protein for the first RNA switch may be a protein expressed by another mRNA switch not defined in (A) or (B), may be a protein expressed by an mRNA that is not a switch (has no aptamer and is not subject to translational control), or may be any other protein.

[0071] In the above definition, the kth protein and the (k+1)th protein are "different" proteins means that the kth protein and the (k+1)th protein are derived from different Cas proteins. More specifically, the aptamer possessed by the kth mRNA switch must be different from the aptamer possessed by the (k+1)th mRNA switch. For example, when the input protein is a first Cas protein or a variant thereof, the output protein is a second Cas protein or a variant thereof, and the first Cas protein and the second Cas protein must be different.

[0072] The nth mRNA switch defined in (B) is controlled by the nth protein encoded by the (n-1)th mRNA in the previous hierarchy, and outputs the (n+1)th protein. The (n+1)th protein can be designed not to act on other mRNA switches constituting the switch set. Alternatively, the (n+1)th protein can be designed to act as an input protein for the first mRNA switch. Details will be described later.

[0073] In designing such a switch set, a total of (n+1) proteins, from the first protein to the (n+1)th protein, must be selected. Of these, the first protein to the nth protein, which can be input proteins, are preferably all different. Here, the first protein to the nth protein being all different means that the n proteins are all derived from different Cas proteins. Each of these can be selected from the following Cas proteins or variants thereof. SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, Cas14a1.

[0074] For example, when n is 2, the switch set is composed of two types of mRNA switches, and the first mRNA switch includes a nucleic acid sequence specifically recognized by the first protein and a nucleic acid sequence encoding the second protein. The second mRNA switch includes a nucleic acid sequence specifically recognized by the second protein and a nucleic acid sequence encoding the third protein. In order to design this switch set, it is necessary to select three types of proteins, the first protein, the second protein, and the third protein. These three types can be selected from the 25 types of Cas proteins mentioned above, and the third protein does not have to be a Cas protein.

[0075] In one embodiment, the mRNA switch set may be a switch set for forming a cascade circuit. In the mRNA switch set for a cascade circuit, the nth mRNA switch is an mRNA switch dedicated to output, and encodes a protein that does not control the translation of other mRNA switches. The protein that does not control the translation of other mRNA switches may be a marker protein or the like, as described above in detail. By forming a cascade circuit with the switch set according to this embodiment, it is possible to condition the expression of a gene to be expressed (the gene of the (n+1)th protein output by the nth mRNA switch) based on the state of existence of an intracellular substance. More specifically, it is possible to form a circuit in which gene expression occurs with a certain miRNA expression pattern.

[0076] In one embodiment, the mRNA switch set may be a switch set for forming an oscillator circuit. In the mRNA switch set for oscillator circuit, the (n+1)th protein output by the nth mRNA switch is the first protein that becomes the input protein of the first mRNA switch. The switch set according to this embodiment forms an oscillator circuit, making it possible to reprogram cells and control the timing of expression of therapeutic genes.

[0077] As a further application of this hierarchical control, the function of the (k+1)th protein output by the kth mRNA may not be limited to the input protein of the (k+1)th mRNA. That is, a protein output by a certain mRNA switch may be an input protein of two or more other mRNA switches, and a switch set may include an autoregulatory mRNA in which the input protein and output protein are the same.

[0078] [E. Imaging with mRNA switches] The mRNA switch can be used in the RNA imaging method in combination with an input protein. In this embodiment, the input protein is a fusion protein in which a protein that enables imaging is fused to a Cas protein or a variant thereof. The protein that enables imaging may be a protein that can be visualized and quantified by fluorescence, luminescence, color, or by assisting fluorescence, luminescence, or color. Such proteins that can be visualized and quantified can be those exemplified as output proteins. An input protein consisting of a fusion protein fused with a protein that enables imaging is referred to as an imaging input protein.

[0079] In this embodiment, the mRNA switch is an RNA molecule that is desired to be visualized and quantified. The mRNA switch may contain an aptamer sequence that is specifically recognized by the Cas protein contained in the imaging input protein, and the output protein is not particularly limited. In addition, the aptamer sequence that is specifically recognized by the Cas protein is preferably present at the end of the RNA molecule, and may be the 5' end or the 3' end.

[0080] According to the imaging method of this embodiment, a protein that enables imaging can be bound to an RNA molecule using a Cas protein, making it possible to observe the behavior of the RNA molecule.

[0081] [F. Simultaneous control of translation and transcription by a single input protein] Simultaneous control of translation and transcription using an mRNA switch using a single input protein and a vector having a transcription control sequence will be described with reference to FIG. 16. The input protein in this embodiment uses a protein that functions as a part of a transcription control protein. Therefore, it is preferable to use a fusion protein in which an inactivated Cas protein is fused with a transcription activation or transcription repression factor as the input protein. Referring to the center part of panel (a) of FIG. 16, dSpCas9-VPR capable of transcription activation is illustrated as an example of an input protein.

[0082] The mRNA switch includes a nucleic acid sequence (i) that is a crRNA or sgRNA sequence corresponding to a Cas protein contained in an input protein, and a nucleic acid sequence (ii) that encodes a first output protein. The mRNA switch may be an OFF switch or an ON switch. Referring to the left side of panel (a) of FIG. 16, an example of an mRNA switch is an OFF switch mRNA in which the nucleic acid sequence (i) is a gRNA corresponding to dSpCas9-VPR, the first output protein is tagRFP, and translation is suppressed in response to an input protein.

[0083] The vector comprising the transcription control sequence includes, in the 5' to 3' direction, a transcription control sequence, a promoter sequence, and a nucleic acid sequence encoding a second output protein. The transcription control sequence is a nucleic acid sequence that is specifically recognized by a crRNA or sgRNA sequence corresponding to a Cas protein contained in the input protein. The second output protein is a protein different from the first output protein. Referring to the right side of panel (a) of FIG. 16, an example of a vector comprising a transcription control sequence is a vector comprising a binding sequence of a gRNA corresponding to dSpCas9-VPR, and the second output protein is hmAG1.

[0084] The protein expression control kit for implementing the translation control system of embodiment F may include at least one component (first component) selected from the group consisting of an input protein, a trigger mRNA, and a trigger plasmid, at least one component (second component) selected from the group consisting of an mRNA switch or a switch vector, a vector (third component) having a transcription control sequence, and at least one component (fourth component) selected from the group consisting of a crRNA or sgRNA corresponding to the input protein, or a vector encoding these RNAs. The input protein derived from the first component and the small RNA derived from the fourth component form a complex and function as a transcription control protein. In embodiment F, the introduction step of the second component and the third component and the input step of the first component and the fourth component can be carried out as described in embodiment A above.

[0085] [G.Half Subtractor] A system for realizing a half-subtractor circuit using three transcription control switch vectors will be described with reference to Figures 18A, B, and C. In this embodiment, a half-subtractor circuit can be constructed by using two types of input proteins a and b and three types of transcription control switch vectors a, b, and c. Both of the two types of input proteins a and b use proteins that also function as transcription control proteins. With reference to Figure 18A, dSpCas9-VPR is exemplified as the input protein a, and dSaCas9-VPR is exemplified as the input protein b.

[0086] The transcriptional control switch vector a comprises a transcriptional control sequence controlled by a transcriptional control protein comprising a complex of an input protein a and a corresponding crRNA or sgRNA, a nucleic acid sequence encoding an mRNA switch translationally controlled by an input protein b, and a nucleic acid sequence encoding a first output protein. That is, the transcriptional control sequence contained in the transcriptional control switch vector a is a sequence specifically recognized by the crRNA or sgRNA corresponding to the input protein a. The mRNA switch encoded by the transcriptional control switch vector a is a crRNA or sgRNA whose nucleic acid sequence (i) corresponds to the input protein b. Referring to the upper part of FIG. 18A, the transcriptional control switch vector a is a binding sequence of a transcriptional control protein comprising dSpCas9-VPR, and the mRNA switch comprises an sgRNA sequence corresponding to dSaCas9-VPR and a sequence encoding tagBFP.

[0087] The transcription control switch vector b comprises a transcription control sequence controlled by a transcription control protein including a complex of an input protein b and a corresponding crRNA or sgRNA, a nucleic acid sequence encoding an mRNA switch translationally controlled by an input protein a, a nucleic acid sequence encoding a first output protein, and a nucleic acid sequence encoding an sgRNA sequence corresponding to the input protein b. That is, the transcription control sequence contained in the transcription control switch vector b is a sequence specifically recognized by the crRNA or sgRNA corresponding to the input protein b. The mRNA switch encoded by the transcription control switch vector b is a crRNA or sgRNA in which the nucleic acid sequence of (i) corresponds to the input protein a. The transcription control switch vector b comprises a nucleic acid sequence encoding an sgRNA sequence corresponding to the input protein b as an input RNA. Referring to the middle part of FIG. 18A, the transcription control switch vector b is a binding sequence of a transcription control protein including dSaCas9-VPR, and the mRNA switch includes an sgRNA sequence corresponding to dSpCas9-VPR and a sequence encoding tagBFP. In addition, the transcriptional control switch vector b further contains a sequence encoding an sgRNA corresponding to dSaCas9-VPR.

[0088] The transcriptional control switch vector c comprises a transcriptional control sequence controlled by a transcriptional control protein including a complex of an input protein b and a corresponding crRNA or sgRNA, a nucleic acid sequence encoding an mRNA switch translationally controlled by the input protein a, and a nucleic acid sequence encoding a second output protein. Referring to the lower part of Figure 18A, the transcriptional control switch vector c is a binding sequence for a transcriptional control protein including dSaCas9-VPR, and the mRNA switch includes an sgRNA sequence corresponding to dSpCas9-VPR and a sequence encoding hmAG1.

[0089] By combining these input proteins a and b with the transcriptional control switch vectors a, b, and c, when the input protein a is absent and the input protein b is present, both the first output protein and the second protein are translated and expressed. When the input protein a is present and the input protein b is absent, only the first output protein is translated and expressed. In this way, a half-subtractor circuit can be constructed.

[0090] The protein expression control kit for implementing the translation control system of the embodiment G may include at least one component (first component) selected from the group consisting of an input protein a, a trigger mRNA, and a trigger plasmid, at least one component (second component) selected from the group consisting of a crRNA or sgRNA corresponding to the input protein a, or a vector encoding these RNAs, at least one component (third component) selected from the group consisting of an input protein b, a trigger mRNA, and a trigger plasmid, at least one component (fourth component) selected from the group consisting of a crRNA or sgRNA corresponding to the input protein b, or a vector encoding these RNAs, and a transcription control switch vector a (fifth component), b (sixth component), and c (seventh component). The crRNA or sgRNA that binds to the transcription control sequence of the transcription control switch vector c (seventh component) is generated by the transcription control switch vector b (sixth component). In the embodiment G, the introduction process of the transcription control switch vectors a, b, and c and the input process of the first component to the fourth component can be carried out as described in the above embodiment A.

[0091] It is also possible to arbitrarily combine the above-mentioned aspects A to D and F and G. This makes it possible to configure an artificial circuit controlled by a plurality of mRNA switches that can receive input from a plurality of different input proteins.

[0092] In one embodiment, the present invention is a cell containing an mRNA switch or a vector encoding the mRNA. The cell is a cell into which an mRNA switch or a vector encoding the mRNA has been introduced. The functioning of an artificial circuit in a cell containing an mRNA switch is disclosed, for example, in Auslander et al., Nature volume 487, pages 123-127 (2012) and Kitada et al., Science 2018 Feb 9; 359 (6376). The mRNA switch according to the present invention can also be contained in a cell and made to function in the same way. In particular, a desired artificial circuit that functions in a cell can be obtained by containing, in addition to the mRNA switch, nucleic acids and proteins that are components of the system described in the above aspects A to D, F, and G in the cell. Such cells are useful as cell preparations.

[0093] The mRNA switch of the present invention can also be used in a cell-free system. For example, an artificial circuit system can be constructed by attaching the mRNA switch to a desired carrier and drying it to support it. By making the artificial circuit function using paper as a carrier, a desired artificial circuit that functions can be obtained by placing such a carrier carrying the RNA switch under a predetermined translation-enabling condition. Examples of carriers include, but are not limited to, paper, plastic, porous bodies, and fibers. For example, this is disclosed in Pardee et al., Cell 159, 940-954, November 6, 2014. In the present invention, in addition to the mRNA switch, a nucleic acid or protein that is a component of the system described in the above aspects A to E is attached to the carrier to obtain a desired artificial circuit. EXAMPLES

[0094] The present invention will be described in more detail below with reference to examples, which are not intended to limit the present invention.

[0095] [Experimental Method] [Plasmid construction] Construction of switch plasmids First, pAptamerCassette-EGFP was digested with restriction enzymes AgeI and BamHI. Next, the single-stranded synthetic DNA oligos shown in Table 3 were converted into double-stranded DNA by annealing, and inserted into the digested pAptamerCassette-EGFP.

[0096] [Table 3A] [Table 3B] [Table 3C] [Table 3D] [Table 3E] [Table 3F]

[0097] [Preparation of trigger plasmid] First, the ORF of the trigger protein was amplified by PCR using the primer set shown in Table 4. Next, the amplified ORF was cut with an appropriate restriction enzyme and inserted downstream of the CMV promoter of pcDNA3.1-myc-HisA. However, only SpCas9 in Figure 2 used a plasmid (#41815) purchased from Addgene as the trigger. After preparing version 1 of Nc_gRNA_v2 and Cas14a1_sgRNA2, inverse PCR was performed using each iPCR primer set using the KOD -Plus- Mutagenesis Kit (TOYOBO) or Q5 Site-Directed Mutagenesis Kit (NEB), and ligation was performed.

[0098] [Table 4A] [Table 4B]

[0099] [Creation of split Cas9] Inverse PCR was performed using the SpCas9 expression vector as a template to delete the first or second half of the ORF with the primer set shown in Table 5. In addition, when preparing drug-responsive Cas9, DmrA and DmrC were amplified using the primer set shown in Table 5, and then fused to each fragment of split Cas9 using the In-Fusion HD Cloning Kit (Clontech).

[0100] [Table 5A] [Table 5B]

[0101] [Preparation of plasmids for constructing multiple circuits] First, the ORF of each Cas protein was amplified using the primer set shown in Table 6. Next, inverse PCR was performed using each switch plasmid as a template using the primer set shown in Table 6 to amplify a linear plasmid backbone containing a region other than the ORF. Finally, the ORF of the Cas protein was inserted into the plasmid backbone using the In-Fusion HD Cloning Kit (Clontech).

[0102] [Table 6]

[0103] Plasmids for transfection into cultured cells were purified in large quantities using a commercially available Midiprep kit (QIAGEN or Promega).

[0104] [Construction of IVT template] The SpCas9 protein coding region (ORF), 5'-UTR and 3'-UTR sequences for SpCas9 mRNA and reference mRNA were amplified by PCR from plasmids or oligo DNA using appropriate primers. The template plasmids and primers used, and the template oligo DNA and primers used are shown in Table 7.

[0105] [Table 7]

[0106] To prepare IVT templates for synthesizing trigger and reference mRNAs, the 5'-UTR fragment, 3'-UTR fragment, and ORF were linked by PCR amplification using the primer set of T7FwdA and Rev120A shown in Table 8. The reference mRNA refers to an mRNA that is translated without translational control to express the encoded protein.

[0107] [Table 8]

[0108] To prepare an IVT template for switch mRNA synthesis, a region containing 5'-UTR and ORF was amplified using the SpCas9-responsive switch plasmid as a template with the primers in Table 8. The 3'-UTR fragment amplified product was then linked by PCR amplification using the primer set in Table 8. The ORFs used were those listed in SEQ ID NOs: 1 to 25 and SEQ ID NOs: 239 to 252.

[0109] PCR products were purified using the MinElute PCR purification kit (QIAGEN) according to the manual attached to the kit. Products amplified by PCR using plasmid as a template were digested with Dpn I (TOYOBO) at 37°C for 30 minutes before purification.

[0110] [mRNA synthesis and purification] Synthesis of mRNA was performed using MEGAscript T7 Transcription Kit (Ambion). In this reaction, except for the switch mRNA, pseudouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate (TriLink BioTechnologies) were used instead of uridine triphosphate and cytidine triphosphate, respectively. Guanosine triphosphate was used after dilution by 5 times with Anti-Reverse Cap Analog (New England Biolabs). After reacting the reaction mixture at 37°C for 6 hours, TURBO DNase (Ambion) was added and further incubated at 37°C for 30 minutes. The obtained mRNA was purified using FavorPrep Blood / Cultured Cell total RNA extraction clumn (Favorgen Biotech) or Monarch RNA Cleanup kit (New England Biolabs). The purified mRNA was dephosphorylated at the 5' end by incubating at 37°C for 30 minutes using Antarctic Phosphatase (New England Biolabs). The RNA was then purified using the RNeasy MinElute Cleanup Kit (QIAGEN) or Monarch RNA Cleanup kit (New England Biolabs).

[0111] [Cell culture] 293FT cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS, 2 mM L-Glutamine (Invitrogen), 1X MEM Non-Essential Amino Acids (Invitrogen), and 1 mM Sodium Pyruvate (Sigma) at 37°C and 5% CO2.

[0112] [Plasmid transfection] 293FT cells were seeded in 24-well, 96-well, or 384-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. The switch plasmid and trigger plasmid were mixed in Opti-MEM (Invitrogen) so that the mass ratio was 1:4. In split Cas9 tests, plasmids expressing each fragment were introduced in four times the amount of the switch. In drug responsiveness tests, A / C Heterodimerizer (Clontech) was added to the medium at a final concentration of 0.5 uM at least 1 hour before transfection. In multilayer circuit experiments, each plasmid was co-introduced at layer0:1:2:3:4:5 = 1:4:4:16:64:256. In all experiments, iRFP670 expression plasmid was used as a transfection control.

[0113] [mRNA transfection] Cultured cells were seeded on 24-well plates, and the synthesized mRNA was transfected the next day. Transfection was performed using 1 μL of Lipofectamine messengerMAX (Invitrogen) according to the attached manual. 293FT cells were co-transfected with 100 ng of switch mRNA, 100 ng of trigger mRNA, and 100 ng of reference mRNA.

[0114] [Cell Imaging] Fluorescence micrographs were taken using a Cytell Cell Imaging System (GE Healthcare Life Sciences). The brightness and contrast of the fluorescence micrographs were edited using ImageJ software (NIH).

[0115] [Flow cytometry] The day after transfection, cells were detached from the plate and analyzed by flow cytometry through a mesh. In experiments using 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP was detected using FL1 (530 / 30 nm) filters. iRFP670 was detected using FL4 (675 / 12.5 nm) filters. Dead cells and debris were excluded based on forward and side light scattering signals. For calculation of translation efficiency using plasmids, live cells with iRFP670 fluorescence values ​​above a certain level were analyzed.

[0116] [result] [Design of mRNA switches] The mRNA switch was constructed by inserting a nucleic acid sequence (aptamer sequence) that is specifically recognized by an input protein into the 5'-UTR of an mRNA encoding a gene of interest. Here, the switch was designed by regarding the sequence of a known crRNA (CRISPR RNA) or sgRNA (single guide RNA; a chimeric RNA of crRNA and trans-activating crRNA (tracrRNA)) that corresponds to each Cas protein, which is the input protein, as the aptamer sequence. Translation inhibition is triggered when each Cas protein recognizes and binds to the specific crRNA / sgRNA embedded in the mRNA (Figure 1).

[0117] [Functional evaluation of Cas protein-responsive mRNA switches] Based on the above design, a plasmid (switch plasmid) expressing an mRNA switch with GFP as the output protein was prepared and introduced into 293FT cells together with a plasmid (trigger plasmid) expressing the input protein Cas protein and a plasmid (reference plasmid) expressing iRFP670 as a transfection control. Here, we first selected 10 types of Cas proteins that have been reported to have RNA-guided nucleic acid targeting ability in human cells. Figure 2 is a graph showing the translation efficiency of each switch plasmid. The translation efficiency was calculated by first dividing the GFP fluorescence intensity by the iRFP670 fluorescence intensity for each switch, and then dividing the value when the trigger plasmid was introduced by the value when it was not introduced. In addition, the control switch plasmid (No aptamer) was used as a standard for comparison. The results are shown in Figure 2A-J. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). Nine switches other than the NmCas9-responsive mRNA switch showed high translation inhibition (minimum inhibition rate of approximately 80%). We found that mRNA switches can be created with a high probability by using Cas proteins and the corresponding crRNA / sgRNA.

[0118] [Creating an NmCas9-responsive switch by forward engineering (1)] We verified whether the function of the NmCas9-responsive switch, for which translational repression could not be observed, could be improved by an RNA engineering approach. As multiple designs have been reported for the sgRNA of NmCas9, we verified whether gRNAs with these other gRNA sequences or their combined designs could function as switches. In addition to the gRNA sequence (Nm_gRNA) used in the first verification experiment, new designs and gRNA sequences were tested (Nm_gRNA_v1-5). Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). As a result, repression was observed in Nm_gRNA_v3 and v4 (Figure 3). It was shown that even in those with weak translational repression ability, functional improvement can be expected by considering the sequence of the sgRNA to be inserted.

[0119] [SpCas9 response switch suppresses reporter expression by regulating translation] Many Cas proteins are originally used for genome editing targeting intracellular DNA. In addition, because artificial RNA containing a gRNA sequence was co-introduced, it is possible that reporter expression was suppressed by unintended genome editing effects. Therefore, we used SpCas9 to verify whether reporter expression suppression was truly caused by translational repression. We compared the expression levels of the same reporter when four types of SpCas9 were introduced as input proteins: wild type (having a nuclear localization signal (NLS)), mutants for DNA cleavage activity (nickase and DNA cleavage null), and SpCas9 without NLS (Figure 4A). As a result, no significant difference in reporter expression was observed under all conditions. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). Therefore, it is highly likely that reporter expression suppression by SpCas9 is caused by translational repression. In addition, we also verified whether reporter expression suppression was inhibited when an anti-CRISPR protein (AcrIIA4), which inhibits the DNA binding ability of SpCas9, was co-introduced. The input protein used was the same as the wild type (SpCas9 (WT)) in Figure 4A. As a result, the introduction of AcrIIA4 did not significantly affect the suppression of reporter expression (Figure 4B). Therefore, it is supported that the suppression of reporter expression by SpCas9 is not caused by DNA binding of SpCas9, but is an effect due to translational suppression.

[0120] [Verification of translational repression by Cas proteins without cleavage] Some Cas proteins bind to pre-crRNA, which is a sequence of multiple crRNAs, and then excise and use the crRNA appropriately. When creating a switch using such a Cas protein, the binding of the Cas protein to the crRNA in the mRNA may cause the mRNA to be cleaved and degraded, resulting in translational repression. This can be a drawback if you want to achieve translational repression without degradation of the mRNA switch. We examined whether mRNA cleavage is essential for translational repression using such Cas proteins, using AsCas12a (AsCpf1), one of the Cas proteins that has been reported to have pre-crRNA excision activity. It has been reported that the AsCas12a(H800A) mutant can bind to the crRNA itself but does not have the ability to process pre-crRNA. We investigated whether translational repression without mRNA degradation by Cas12a is possible using this mutant. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). As a result, it was suggested that the Cas12a mutant had a slightly lower translation repression efficiency than the wild-type Cas12a, but still had a high translation repression ability (about 80%) (Figure 5). This result suggests that in translation repression by Cas protein bound to crRNA on mRNA, the binding between crRNA and Cas protein itself is important, and the excision activity of crRNA is not necessarily required. In other words, the Cas protein-responsive mRNA switch can be used for both translation repression accompanied by mRNA degradation and translation repression without it.

[0121] [Cas protein-responsive RNA inverter] An mRNA switch is an OFF switch that suppresses its own translation in the presence of a target protein, but an RNA inverter has been developed that converts this OFF switch into an ON switch (induces its own translation in the presence of a target protein). The construction of an RNA inverter can be achieved by inserting an aptamer sequence into the 5'-UTR of an mRNA, just like an OFF switch. An ON switch is constructed by inserting a special RNA inverter sequence that converts the output between the 5'-UTR of the OFF switch and a reporter gene. Therefore, we verified whether the Cas-responsive mRNA switch can also be converted into an ON switch using an RNA inverter. Among the Cas proteins that showed translation suppression, SpCas9, SaCas9, St1Cas9, CjCas9, AsCas12a and its mutant AsCas12a (H800A), and PspCas13b were selected, and an ON switch equipped with crRNA / sgRNA was created. The RNA inverter sequences used were those described in SEQ ID NOs: 64 and 67. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments, n = 2 for PspCas13b only). As a result, we succeeded in constructing an RNA inverter that can promote translation in Cas proteins other than PspCas13b (Figure 6A-6F). Since PspCas13b has the ability to process pre-crRNA, it is thought that this cleavage caused mRNA degradation, making it impossible to promote translation (Figure 6B). On the other hand, translation promotion was observed in AsCas12a, which has the ability to process pre-crRNA like PspCas13b (Figure 6F). This difference is expected to be due to the difference in the cleavage position of RNA when Cas proteins process pre-crRNA. PspCas13b cleaves RNA at the 3' side of the binding region, while AsCas12a is thought to cleave at the 5' side. Therefore, the PspCas13b-crRNA complex is detached from the mRNA, resulting in the production of mRNA with an exposed 5' end. As a result, mRNA degradation is promoted, making it impossible to generate an ON switch in response to PspCas13b. On the other hand, AsCas12a cleaves the 5' end of the mRNA, so it is expected that it will continue to bind to the end of the mRNA.Therefore, it is thought that the degradation of mRNA is suppressed, and as a result, the RNA inverter functions. Based on the above, it is highly likely that it is possible to create a Cas protein-responsive RNA inverter using any Cas protein other than Cas13b, which cleaves the 3' side of the RNA from the binding site.

[0122] [Functional evaluation of Cas protein-responsive mRNA switches using RNA transfection] We verified whether the Cas protein-responsive mRNA switch would function even if mRNA, instead of a plasmid, was directly introduced into cultured cells. Here, we used the SpCas9-responsive mRNA switch as a representative example. We co-introduced the switch mRNA, trigger mRNA expressing SpCas9 (input protein), and reference mRNA (iRFP670 mRNA) to confirm whether a series of mRNAs had been introduced into the cells into 293FT cells. The upper panel of Figure 7A shows the fluorescence change of the switch with and without the introduction of the trigger mRNA plotted by flow cytometry. It was confirmed that the population shifted downward due to the introduction of the trigger mRNA. The lower panel of Figure 7A is a histogram showing the difference in the translation efficiency of the mRNA with and without the introduction of the trigger mRNA. In Figure 7B, the translation efficiency was calculated by first dividing the fluorescence intensity of EGFP by the fluorescence intensity of iRFP670, and then dividing the value when the trigger mRNA was introduced by the value when it was not introduced. It was observed that the introduction of SpCas9 specifically suppressed the translation from the switch mRNA. Therefore, it was demonstrated that the Cas protein-responsive mRNA switch also functions via the mRNA introduction method.

[0123] [Expansion of Cas protein-responsive mRNA switches] In addition to the Cas proteins tested in Figure 2, 15 more Cas proteins were examined to see whether they function as translational repressors of mRNA switches. In addition to FnCas9, St3Cas9, LbCas12a, FnCas12a, and MbCas12a, which have nucleic acid targeting ability in human cells, we also used CdCas9, ClCas9, NcCs9, SpaCas9, and PlCas9, which are thought to have no or very low activity, as well as the recently reported Cas12b family, Cas14a1, and CasX (PlmCasX). When multiple sgRNAs were reported for each Cas protein, the effect was verified using each sgRNA sequence. As a result, it was shown that 13 of these 15 Cas proteins could achieve translational repression by using appropriate crRNA / sgRNA as an aptamer (Figure 8A-O). These results suggest that the approach of embedding crRNA / sgRNA into the 5'-UTR to switch off Cas proteins may be applicable with a high degree of certainty to Cas proteins that will be newly discovered in the future.

[0124] [Expansion of Cas protein-responsive translation ON switches] In addition to the additionally expanded mRNA switch, we further expanded the translation ON switch based on the knowledge gained from the development of the ON switch using the RNA inverter sequence mentioned above. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). We were able to develop ON switches for all Cas proteins tested (Figure 9A-G). In addition, we found that some Cas proteins, such as the CasRx response switch, showed a high translation ON effect by using an RNA inverter even though the translation repression effect was inferior to others (Figure 9A). Therefore, even Cas proteins that are not promising as translation repression switches can become useful translation control switches by using an RNA inverter sequence.

[0125] [Translation programming based on protein engineering 1] We verified whether translational control can be programmed using existing engineering of Cas proteins. Here, we used SpCas9, which is often used for modification, and examined whether it is possible to achieve NAND-gate type translational control, which suppresses translation only when two input molecules (input A, B) are present at the same time. To achieve NAND-gate type translational control, we attempted to use split Cas9, which is a Cas9 protein divided into two regions. It is known that when SpCas9 is split between residues 713 and 714, its nucleic acid targeting ability is restored if the fragments are assembled. Here, we introduced a split intein into each fragment to increase the assembly efficiency. An intein is the name of a protein portion that is excised by a phenomenon called protein splicing. Proteins containing inteins autonomously excise their own intein sites and then recombine the remaining portions. Among them, split inteins are contained as N-inteins or C-inteins in two proteins that are translated as separate proteins. When these two proteins are assembled via an intein, the intein portion is excised and a protein in which the two proteins are neatly fused is generated. In other words, in this case, when both SpCas9(N-term) and SpCas9(C-term) are present in the cell, full-length SpCas9 is generated, which causes translational repression (Fig. 10A, 10B). To verify the above hypothesis, we prepared each split protein expression plasmid and co-transfected it into 293FT cells together with the switch plasmid and reference plasmid. As a result, as intended, only when both Cas9 fragments were introduced ([1,1]), translational repression equivalent to that achieved when full-length Cas9(WT) was introduced was achieved (Fig. 10C). In other words, the use of intein-containing split Cas9 can achieve NAND-gate type translational control (Fig. 10B, 10C).

[0126] [Translation programming based on protein engineering 2] If the function of the mRNA switch can be controlled by drugs, it will be easy to manipulate the timing and duration of translational control not only at the cultured cell level but also when used in living organisms. A system has been reported in which split Cas9 associates with a drug-induced split Cas9 to restore nucleic acid targeting ability. We thought that by applying this mechanism, we could construct a system that causes translational repression only when a drug is added. Here, we introduced the commercially available iDimerize Inducible Heterodimer System. We created split Cas9 by fusing the DmrA-binding domain and the DmrC-binding domain to the C-terminus or N-terminus of each fragment of SpCas9 (SpCas9(N-term) and SpCas9(C-term)) split between residues 174 and 175 (Split1), between residues 535 and 536 (Split2), or between residues 713 and 714 (Split3) (Figure 11A, Split3 is shown as a representative example). After adding A / C Heterodimerizer (drug) to the medium, 293FT cells were transfected with the switch plasmid, split Cas9 expression plasmid or full-length Cas9 plasmid, and reference plasmid. As a result, translation from the mRNA switch was significantly suppressed in the presence of split Cas9 only when the drug was added in Split 2 and 3 (Figure 11B). The results so far (Figures 10 and 11) show that translation programming using the mRNA switch can be successfully achieved by applying existing protein engineering knowledge for Cas9.

[0127] [Translation control by novel CRISPR system] In recent years, new anti-CRISPR proteins that inhibit the binding of Cas proteins to gRNA have been reported. The anti-CRISPR protein AcrIIC2 against NmCas9 is known to bind to Cas9 and inhibit its binding to gRNA. It has also been reported that AcrIIC2 can bind to some Cas9 family members such as SaCas9. Therefore, we thought that a new translation control system could be constructed by using AcrIIC2. In other words, we aimed to construct a system that can release translational repression by Cas9 protein when AcrIIC2 is present. Our results so far have shown that the NmCas9-responsive switch does not show sufficient translational repression ability. On the other hand, it has been suggested that SaCas9 can repress translation very efficiently. Here, we attempted to construct a translational control system by utilizing the ability of AcrIIC2 to bind to SaCas9. 200 ng of SaCas9 plasmid and 100 ng of iRFP670 plasmid were introduced into 293FT cells for 100 ng of switch plasmid. At this time, 0-10 times the amount of AcrIIC2 expression plasmid was also co-introduced with the SaCas9 plasmid. Error bars indicate the mean ± standard deviation (n = 3, three independent experiments). The results suggest that AcrIIC2 relieves translational repression by SaCas9 (Figure 12). Therefore, it is thought that the translational control method using Cas proteins can also be used with various CRISPR systems that will be discovered in the future.

[0128] [Cas protein response OFF switch mRNA orthogonality] When constructing a circuit combining Cas protein-responsive OFF switch mRNA, we verified its orthogonality. First, we verified the orthogonality between the nine types of Cas protein-responsive mRNA switches that showed translational repression ability in Figures 2A-C, E-J. As a result, crosstalk was confirmed between St1Cas9 and SaCas9, and between PguCas13b and RanCas13b, but orthogonality was shown for the other seven types (Figures 13A and 13B). The results of the translation efficiency in Figure 13B displayed as numerical values ​​are shown in Table 9.

[0129] [Table 9]

[0130] Furthermore, we verified the orthogonality between 15 types of protein-responsive mRNA switches, including 7 of the newly expanded Cas protein-responsive OFF switch mRNAs in Figure 6 and the previously developed L7Ae-responsive mRNA switch. Crosstalk was observed between Cas12a, but no significant crosstalk was observed in other combinations (Figure 13C). Therefore, a set with orthogonality between 12 types of protein-responsive mRNA switches, the largest number ever, was developed.

[0131] [Construction of multi-layer circuits] We verified whether it is possible to construct a multilayer circuit by combining Cas protein-responsive OFF switch mRNAs. A multilayer circuit is constructed by hierarchically combining multiple OFF switch mRNAs. An input protein that triggers a switch is expressed as an output protein from an OFF switch that responds to another trigger protein. Since a protein output from a switch becomes an input protein for a switch one layer downstream, the expression of the output gene (here, GFP) changes from ON to OFF to ON with each layer (Figure 14A). Here, a multilayer circuit was constructed using five switches that showed strong translational repression in Figure 2. Figure 14B shows fluorescence microscopy images at each layer level, histograms obtained from flow cytometry analysis, and calculated output levels. Although the overall responsiveness worsens with increasing layer level, the intended output change (ON→OFF→ON→OFF→ON→OFF) was observed.

[0132] [Plasmid construction] [Construction of switch plasmid] pAptamerCassette-tagRFP2 was cleaved with restriction enzymes AgeI and BamHI. Next, single-stranded synthetic DNA oligos shown in SEQ ID NOs: 69 and 70, or SEQ ID NOs: 73 and 74 were annealed to form double-stranded DNA, which was then inserted into the cleaved pAptamerCassette-tagRFP2.

[0133] [Construction of VPR fusion trigger plasmid] To prepare a SaCas9 expression vector (pcDNA3.1+-dSaCas9-VPR) fused with VPR, a mutation was first introduced into the SaCas9 expression plasmid (pcDNA3.1+-SaCas9) to eliminate nuclease activity (replacement of D at position 10 with A and N at position 580 with A). The KOD Mutagenesis Kit (Takara) was used for the mutation introduction. The primers shown in Table 10 were used. Then, the plasmid backbone containing the ORF of SaCas9 was amplified by inverse PCR. Finally, the VPR region amplified by PCR was inserted into the N-terminal side of the SaCas9 ORF using the In-Fusion HD Cloning Kit (Clontech). The sequences of the primers used are shown in Table 10.

[0134] [Table 10]

[0135] [Preparation of reporter plasmid for transcriptional regulation] The transcriptional control reporter plasmid pTRE-Tight-hmAG1 was constructed as follows: A DNA fragment containing hmAG1 was amplified by PCR from p5'RTM-hmAG1 (deltapA)-ABHD12Bexon13 (Unpublished) using the forward primer (SEQ ID NO: 261) and reverse primer (SEQ ID NO: 262) shown in Table 11 below. The DNA fragment was inserted between the EcoRI and EcoRV sites of pTRE-Tight (Clontech) to generate pTRE-Tight-hmAG1.

[0136] The gRNA expression plasmid pHL-gRNA[TRE]-iRFP-RIH was prepared as follows. First, pHL-gRNA[EGFP]-mEF1α-mRFP-RIH was prepared from pHL-gRNA[DMD]-mEF1α-mRFP-RIH (provided by Dr. Akitsu Hotta). That is, a DNA fragment containing the target gene EGFP was amplified by PCR using a forward primer (SEQ ID NO: 263) and a reverse primer (SEQ ID NO: 265). The DNA fragment was inserted between the BamHI site and the EcoRI site of pHL-gRNA[DMD]-mEF1α-mRFP-RIH to generate pHL-gRNA[EGFP]-mEF1α-mRFP-RIH.

[0137] Next, pHL-gRNA[TRE]-mEF1α-mRFP-RIH was generated from pHL-gRNA[EGFP]-mEF1α-mRFP-RIH. A DNA fragment containing the target gene TRE was amplified by PCR using a forward primer (SEQ ID NO: 264) and a reverse primer (SEQ ID NO: 265). 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.

[0138] Finally, pHL-gRNA[TRE]-mEF1α-iRFP-RIH was generated from pHL-gRNA[TRE]-mEF1α-mRFP-RIH. A DNA fragment containing iRFP670 was amplified by PCR from piRFP670-N1 (Addgene, plasmid #45457, Shcherbakova DM, Verkhusha VV. (2013) Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10: 751-754) using forward primer (SEQ ID NO: 266) and reverse primer (SEQ ID NO: 267). The DNA fragment was inserted between the EcoRV and AvrII sites of pHL-gRNA[TRE]-mEF1α-mRFP-RIH to generate pHL-gRNA[TRE]-mEF1α-iRFP-RIH.

[0139] The primer sets used are shown in Table 11. The target sequences are underlined. [Table 11]

[0140] [Preparation of plasmid for constructing half-subtractor] First, the expression plasmid for the SaCas9 response switch (pGluc_Sa_gRNA-tagBFP) was amplified by inverse PCR using the primers shown in Table 12. Then, the plasmid backbone amplified by NEBuilder HiFi DNA assembly was linked to the single-stranded DNA oligo shown in Table 12 to replace the CMV promoter with a minimal CMV promoter, and a plasmid (pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP) was prepared in which the binding site for SpCas9 gRNA was introduced upstream of the minimal CMV promoter.

[0141] Next, MALAT1 triplex, Hammerhead ribozyme (HH ribozyme), SaCas9 gRNA, and HDV ribozyme were inserted into the 3'-UTR of pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP. To do this, the plasmid backbone was first amplified by inverse PCR using pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP as a template. The amplified plasmid backbone and double-stranded DNA containing MALAT1 triplex, HH ribozyme, SaCas9 gRNA, and HDV ribozyme were then ligated using the In-Fusion HD Cloning Kit (Clontech). Finally, this plasmid was cut with XbaI and AgeI and ligated with double-stranded DNA containing the SaCas9 gRNA binding sequence, the minimal CMV promoter, and the SpCas9 response switch sequence to create a plasmid (pSa_IgRNA_a-CMVmin-Gluc_Sp_gRNA-tagBFP-Triplex-HHR-Sa_gRNA[TRE]-HDVR) with the SaCas9 gRNA binding sequence, the minimal CMV promoter, the SpCas9 response switch sequence, and the SaCas9 gRNA in the 3'-UTR. The sequences of the primers used are shown in Table 12.

[0142] The number of SpCas9 gRNA binding sites was doubled for the SaCas9 response switch expression plasmid (pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP) that introduced the minimal CMV promoter and SpCas9 gRNA binding site described above (pSp_IgRNA_ax2-CMVmin-Gluc_Sa_gRNA-tagBFP). First, this plasmid was cleaved with SpeI and EcoRI. Next, double-stranded DNA containing the SpCas9 gRNA binding site was amplified by PCR and cleaved with XbaI and EcoRI. Finally, the plasmid backbone and DNA fragment were ligated.

[0143] The sequences of the primers used are shown in Table 12. [Table 12A] [Table 12B]

[0144] To prepare the input gRNA expression vectors (pHL-Sp_IgRNA_a-iRFP-RIH and pHL-Sa_IgRNA_a-iRFP-RIH), the single-stranded oligo DNA shown in Table 12 was annealed to form double-stranded DNA, which was then ligated to pHL-gRNA[TRE]-iRFP-RIH cleaved with BamHI and EcoRI.

[0145] To create a plasmid vector (pTRE-Tight-Gluc_Sp_gRNA-hmAG1) expressing a switch in which transcription is activated by VPR-fused SaCas9 and translation of hmAG1 is suppressed by SpCas9, the backbone of the hmAG1 expression vector was first amplified by inverse PCR. Next, the region containing SpCas9 gRNA was amplified by PCR. Finally, the plasmid backbone and the amplified DNA fragment were ligated using the In-Fusion HD Cloning Kit (Clontech), and the SpCas9 gRNA sequence was inserted into the 5' side of hmAG1. The sequences of the primers used are shown in Table 12.

[0146] [Preparation of plasmids for constructing AND circuits] This is in accordance with the preparation of the plasmid for constructing multiple circuits. First, the ORF of each Cas protein was amplified using the primer set shown in Table 13. Next, using the primer set used in the preparation of the plasmid for constructing multiple circuits, inverse PCR was performed using each switch plasmid as a template to amplify a linear plasmid backbone containing regions other than the ORF. Finally, the ORF of the Cas protein was inserted into the plasmid backbone using the In-Fusion HD Cloning Kit (Clontech).

[0147] [Table 13]

[0148] Plasmids for transfection into cultured cells were purified in large quantities using a commercially available Midiprep kit (QIAGEN or Promega).

[0149] [Plasmid transfection] [Check the orthogonality of the OFF switch and the ON switch] 293FT cells were seeded in 384-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. The switch plasmid and trigger plasmid were mixed in Opti-MEM (Invitrogen) at a mass ratio of 1:4. An iRFP670 expression plasmid was used as a transfection control.

[0150] [Coregulation of translation and transcription] 293FT cells were seeded in 24-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. 100 ng of switch plasmid (pGluc-Sp_gRNA-tagRFP), 400 ng of trigger plasmid (Sp_dCas9-VPR (Addgene: Plasmid #63798) or pcDNA3.1+-myc-HisA (control)), 100 ng of gRNA expression plasmid (pHL-gRNA[TRE]-iRFP-RIH), and 100 ng of transcriptional control reporter plasmid (pTRE-Tight-hmAG1) were mixed in Opti-MEM (Invitrogen). 100 ng of tagBFP expression plasmid (pAptamerCassette-tagBFP) was used as a transfection control.

[0151] [AND Gate] 293FT cells were seeded in 96-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. 6.25 ng of switch plasmid, 50 ng of each trigger plasmid, and 25 ng of each mediator plasmid were mixed in Opti-MEM (Invitrogen). 25 ng of iRFP670 expression plasmid was also used as a transfection control.

[0152] [Half Subtractor] 293FT cells were seeded in 24-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. Trigger plasmid (400 ng each), hmAG1 expression plasmid (400 ng), TagBFP expression plasmid (800 ng or 400 ng each), and gRNA expression plasmid (100 ng each) were mixed in Opti-MEM (Invitrogen).

[0153] [Creating an NmCas9-responsive switch by forward engineering (2)] 293FT cells were seeded in 24-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. The switch plasmid and trigger plasmid were mixed in Opti-MEM (Invitrogen) at a mass ratio of 1:4. An iRFP670 expression plasmid was used as a transfection control.

[0154] [Simultaneous activation of ON and OFF switches] 293FT cells were seeded in 24-well plates 24 hours before transfection. Plasmids were introduced using Lipofectamine2000 (Invitrogen) according to the attached manual. 100 ng of each switch plasmid and 400 ng of trigger plasmid (pcDNA3.1-SaCas9 or pcDNA3.1+-myc-HisA (control)) were mixed in Opti-MEM (Invitrogen). 100 ng of iRFP670 expression plasmid was used as a transfection control.

[0155] [Cell Imaging] Fluorescence micrographs were taken using a Cytell Cell Imaging System (GE Healthcare Life Sciences). Brightness and contrast of the fluorescence micrographs were edited using ImageJ software (NIH). Image quantification was performed using ImageJ.

[0156] [Flow cytometry] The day after transfection, cells were detached from the plate and analyzed by flow cytometry through a mesh. For experiments using 24-well and 96-well plates, Accuri C6 (BD Biosciences) was used. EGFP was detected using FL1 (530 / 30 nm) filters. iRFP670 was detected using FL4 (675 / 12.5 nm) filters. Dead cells and debris were excluded based on forward and side light scattering signals. For calculation of translation efficiency using the plasmid, live cells with iRFP670 fluorescence values ​​above a certain level were analyzed.

[0157] [result] [Checking the orthogonality of the OFF switch] The orthogonality of 29 types of OFF switch mRNA was tested. Panel (a) of Figure 15A is a fluorescent photograph showing the results of the orthogonality test. The mRNA switches are 25 types of Cas-responsive switches, including the newly developed AaCas12b-responsive mRNA switch, three types of protein-responsive switches created in the past, and one type of control switch. The BsCas12b switch was not included. Panel (b) shows 13 types of Cas-responsive mRNA switches that showed clear orthogonality among the tested switches. Figure 15B is a heat map showing the image quantification results for these 13 types of Cas-responsive mRNA switches.

[0158] [Coregulation of translation and transcription] Using SpCas9-VPR, we tested whether translational repression (OFF switch mRNA) and transcriptional activation can be controlled simultaneously. The left schematic diagram of Figure 16 panel (a) shows an mRNA switch comprising a nucleic acid sequence specifically recognized by dSpCas9 and a nucleic acid sequence encoding the output protein RFP. This mRNA was designed as a translation regulator. The central schematic diagram shows dSpCas9-VPR. The right schematic diagram shows a vector comprising a gRNA binding site that specifically binds to the gRNA corresponding to dSpCas9-VPR, a CMV promoter, and an hmAG1 gene sequence that is an output protein, as well as a complex of dSpCas9-VPR and gRNA (transcriptional control protein) that activates the transcription of such DNA. This vector was designed as a transcription regulator.

[0159] Panel (b) of Figure 16 shows photographs of cells with (+) and without (-) dSpCas9-VPR introduced. Translation (red fluorescence) was suppressed and transcription (green fluorescence) was activated only when the dSpCas9-VPR protein was introduced.

[0160] [AND Gate] We tested whether a logic circuit can be constructed using the Cas-responsive mRNA switch. Here, we tested an AND gate that can be constructed by combining three types of Cas-responsive mRNA switches (modules). Figure 17 shows the scheme of the AND gate. The AND gate is a circuit in which the output (EGFP) is expressed only when two types of inputs are present at the same time. The Cas-responsive mRNA switch in the upper left of Figure 17 is an mRNA molecule that has a nucleic acid sequence that is specifically recognized by Cas protein A (Input A) and a nucleic acid sequence that codes for Cas protein C (Mediator) as an output protein. The Cas-responsive mRNA switch in the lower left is an mRNA molecule that has a nucleic acid sequence that is specifically recognized by Cas protein B (Input B) and a nucleic acid sequence that codes for Cas protein C (Mediator) as an output protein. The Cas-responsive mRNA switch on the right is an mRNA molecule that has a nucleic acid sequence that is specifically recognized by Cas protein C (Mediator) and a nucleic acid sequence that codes for GFP as an output protein.

[0161] To test the feasibility of constructing a circuit with a Cas-responsive mRNA switch, we comprehensively combined six types of Cas-responsive mRNA switches and tested 60 different arrangements. Table 14 shows the truth table of the AND gate. Table 15 shows the results of a heat map of the arrangement of each Cas protein and the output gene expression (columns marked 00, 10, 01, 11), as well as the performance of the circuit (θ: the smaller the value, the more likely it is to behave like an AND gate). In Table 15, Pgu stands for PguCas13b, Mb stands for MbCas12a, Sa stands for SaCas9, Ak stands for AkCas12b, Psp stands for PspCas13b, and Nc stands for NcCas9.

[0162] [Table 14]

[0163] [Table 15A] [Table 15B]

[0164] [Half Subtractor] By simultaneously utilizing transcriptional and translational control by Cas proteins, a half-subtractor was constructed using only two modules. Figure 18A shows a scheme of the half-subtractor. The upper left side shows a complex of SpCas9-VPR and SpCas9-gRNA (A), and the right side shows a vector equipped with a target sequence of SpCas9-gRNA (A) and encoding a SaCas-responsive tagBFP mRNA switch, and the SaCas-responsive tagBFP mRNA switch generated by transcriptional activation of this vector. The middle left side shows a complex of SaCas9-VPR and SaCas9-gRNA (B), and the right side shows a binding sequence of SaCas9-gRNA (B), a SpCas-responsive tagBFP mRNA switch, and a vector encoding SaCas9-gRNA (C), and the SpCas-responsive tagBFP mRNA switch (upper) and SaCas9-gRNA (C) (lower) generated by transcriptional activation of this vector. The lower left side shows a complex of SaCas9-VPR and SaCas9-gRNA(C), and the right side shows a vector equipped with a binding sequence for SaCas9-gRNA(C) and encoding an SpCas-responsive hmAG1 mRNA switch, and the SaCas-responsive hmAG1 mRNA switch that is generated upon transcriptional activation of this vector.

[0165] FIG. 18B shows the truth value of the half subtractor. FIG. 18C shows a fluorescent micrograph of the cell. From FIG. 18C, as intended, according to the input pattern, when Input 1 is 1 and Input 2 is 0, and when Input 1 is 0 and Input 2 is 1, expression of tagBFP was confirmed, and when Input 1 is 0 and Input 2 is 1, expression of hmAG1 was confirmed. Conventionally, a technique for constructing a half subtractor by combining four types of modules is known, but in the present invention, it was possible to make it function even if the number of modules introduced into the cell was small. In other words, it is considered that it is possible to save the modules (biological parts) required for constructing a large number of half subtractors, and further to reduce the use of internal resources when introduced into the cell.

[0166] [Creating an NmCas9-responsive switch by forward engineering (2)] For the creation of the NmCas9 response switch by forward engineering (1), two new gRNA sequences were further created and tested (Nm_gRNA_v1-7). The translational repression efficiency is shown in Figure 19. We successfully identified v7, which has a higher translational repression effect.

[0167] [Checking the orthogonality of the ON switch] The orthogonality of 26 types of ON switch mRNA was tested. Figure 20 shows the fluorescent photographs showing the results of the orthogonality test. The mRNA switches used were 26 types of Cas-responsive ON switch mRNA, including the newly developed AaCas12b-responsive ON switch mRNA.

[0168] [Simultaneous activation of ON and OFF switches] We tested whether the translation OFF switch (red fluorescence) and the translation ON switch (green fluorescence) could be driven simultaneously for the SaCas9 response switch. Figure 21 panel (a) shows a cell photograph, and panel (b) shows the quantitative results by flow cytometry. The Control ON switch and Control OFF switch are mRNAs that do not have the nucleic acid sequence (aptamer sequence) of (i) and are not affected by the input protein, and the Reference was a transfection control. Figure 21 shows that it is possible to drive the ON switch and the OFF switch simultaneously using a single input protein.

Claims

1. (i) a nucleic acid sequence that is specifically recognized by an input protein, including a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein; Including, The artificial mRNA molecule is an artificial mRNA molecule in which (i) is present on the 5' or 3' side of (ii), and (i) and (ii) are operably linked; The nucleic acid sequence of (i) is a crRNA or sgRNA sequence of the input protein, or a variant thereof; The mRNA switch, wherein the Cas protein is one Cas protein selected from the group consisting of SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, and Cas14a1.

2. The mRNA switch according to claim 1, wherein the artificial mRNA molecule further comprises an RNA inverter sequence between the nucleic acid sequences of (i) and (ii), the RNA inverter sequence consisting of a nucleic acid sequence comprising a bait open reading frame, an intron and an internal ribosome entry site.

3. A vector comprising a nucleic acid sequence encoding the mRNA switch of claim 1 or 2.

4. The vector according to claim 3 , further comprising a transcriptional control sequence located upstream of the nucleic acid sequence encoding the mRNA switch, the transcriptional control sequence being a sequence that is specifically recognized by a transcriptional control protein.

5. The vector of claim 4, wherein the transcriptional control protein comprises a Cas protein or a variant thereof, and further comprises a nucleic acid sequence encoding a small RNA used for transcriptional or translational control, which is provided downstream of the nucleic acid sequence encoding the mRNA switch.

6. A cell comprising an mRNA switch according to claim 1 or 2, or a vector according to any one of claims 3 to 5.

7. (a) an mRNA switch according to claim 1 or 2 or a vector encoding said mRNA switch; and (b) the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA; A kit for controlling protein expression comprising the steps of:

8. (a) the mRNA switch of claim 1 or 2 or a vector encoding said mRNA switch; (b) a first trigger mRNA comprising a nucleic acid sequence encoding a first fusion protein comprising a first fragment of the input protein and a first heterodimerization domain, or a vector encoding the first trigger mRNA; and (c) a second trigger mRNA comprising a nucleic acid sequence encoding a second fusion protein comprising a second fragment of the input protein of (b) and a second heterodimerization domain, or a vector encoding the second trigger mRNA. A protein expression control kit.

9. The expression control kit of claim 8 , further comprising an agent that promotes heterodimerization by the first and second heterodimerization domains.

10. The protein expression control kit according to any one of claims 7 to 9, further comprising an input inhibitor that inhibits recognition of the nucleic acid sequence of (i) by the input protein.

11. An mRNA switch set comprising n types of mRNA switches consisting of a first mRNA switch to an nth mRNA switch or a vector encoding the mRNA switches, wherein n is selected from an integer of 2 to 25; (A) The kth mRNA switch is (i) a nucleic acid sequence specifically recognized by an input protein consisting of a kth protein, including a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein consisting of a (k+1)th protein; The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, The nucleic acid sequence of (i) is a crRNA or sgRNA sequence of the input protein, or a variant thereof; the Cas protein or a variant thereof contained in the k and (k+1) proteins is one Cas protein or a variant thereof selected from the group consisting of SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, and Cas14a1; the kth protein and the (k+1)th protein are different proteins; k is an integer from 1 to (n-1); (B) The nth mRNA switch is (i) a nucleic acid sequence that is specifically recognized by an nth protein that is an output protein of an (n-1)th mRNA switch; (ii) a nucleic acid sequence encoding an output protein that is a (n+1)th protein; The (i) is present on the 5' or 3' side of the (ii), and the (i) and (ii) are operably linked to each other in an mRNA molecule, An mRNA switch set, wherein the (n+1)th protein is any protein.

12. The mRNA switch set of claim 11 , wherein the first protein to the nth protein are all different proteins.

13. 12. The switch set of claim 11, wherein the (n+1) protein is a first input protein.

14. (a) an mRNA switch according to claim 1 or 2 or a vector encoding said mRNA switch; and (b) the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA; A method for regulating protein expression (excluding methods for surgery, therapy, or diagnosis of humans), comprising the step of introducing the above into a cell.

15. (a) the mRNA switch of claim 1 or 2 or a vector encoding said mRNA switch; (b) a first trigger mRNA comprising a nucleic acid sequence encoding a first fusion protein comprising a first fragment of the input protein and a first heterodimerization domain, or a vector encoding the first trigger mRNA; and (c) a second trigger mRNA comprising a nucleic acid sequence encoding a second fusion protein comprising a second fragment of the input protein of (b) and a second heterodimerization domain, or a vector encoding the second trigger mRNA. A method for regulating protein expression (excluding methods for surgery, therapy, or diagnosis of humans), comprising the step of introducing the above into a cell.

16. The method for controlling expression of the protein according to claim 15 (excluding methods for surgery, treatment or diagnosis of humans), further comprising the step of contacting the cells with an agent that promotes heterodimerization by the first and second heterodimerization domains after the introducing step.

17. The method for controlling expression of a protein according to any one of claims 14 to 16 (excluding methods for surgery, therapy, or diagnosis of humans), further comprising the step of introducing into a cell an input-inhibiting protein, an input-inhibiting mRNA encoding the input-inhibiting protein, or a vector encoding the input-inhibiting mRNA, which specifically inhibits recognition of the nucleic acid sequence of (i) by the input protein.

18. A method for controlling protein expression (excluding methods for surgery, therapy, or diagnosis of humans), comprising the step of introducing the mRNA switch set according to any one of claims 11 to 13 into a cell, tissue, or individual.

19. (a) (i) a nucleic acid sequence that is specifically recognized by an input protein comprising a Cas protein or a variant thereof; (ii) a nucleic acid sequence encoding an output protein; Including, A nucleic acid sequence encoding an mRNA switch consisting of an artificial mRNA molecule in which (i) is present on the 5' or 3' side of (ii) and (i) and (ii) are operably linked; A transcriptional control sequence that is specifically recognized by a transcriptional control protein and is provided upstream of a nucleic acid sequence that encodes the mRNA switch; A vector comprising: The transcriptional regulatory protein comprises a Cas protein or a variant thereof, and the vector further comprises a nucleic acid sequence encoding a crRNA or sgRNA that specifically recognizes the transcriptional regulatory sequence, the nucleic acid sequence being provided downstream of the nucleic acid sequence encoding the mRNA switch. and (b) a transcription control protein comprising a Cas protein or a variant thereof corresponding to a nucleic acid sequence encoding a crRNA or sgRNA that specifically recognizes the transcription control sequence of (a), a transcription activity control mRNA encoding the transcription control protein, or a vector encoding the transcription activity control mRNA. A kit for controlling protein expression comprising the steps of:

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