WO2021112136A1 - Mrna switch, and method for regulating expression of protein using same - Google Patents

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

mRNA switch and protein expression control method using it

The present invention relates to an mRNA switch and a method for controlling protein expression using the mRNA switch.

In recent years, in gene therapy and cell therapy, research has been conducted with the aim of improving the therapeutic effect and reducing side effects by "programming" the behavior of cells using DNA and RNA molecules introduced from outside the cell. The programmed cells are expected to detect signals in the environment and autonomously determine and output the appropriate therapeutic molecule selection and the timing, duration, intensity, etc. of exerting it. However, in order to realize such a precise control mechanism, a logic that performs complicated information processing like a computer by creating a plurality of modules that control transcription and translation and combining the plurality of modules in a complex manner. It is necessary to construct an arithmetic circuit (translation control circuit). In this respect, the translation control circuit has an advantage that it is safer than the circuit by transcription control because there is substantially no risk of insertion into the genome. In addition, since it is not necessary to go through the transcription step, the response to the input is faster than the circuit based on the transcription control, and it is possible to construct a mechanism that can respond more quickly to the environmental signal, which attracts attention. ing.

An mRNA switch is known as a translation control module. The mRNA switch is an mRNA having an RNA sequence (aptamer) that binds to a specific molecule (input molecule), and the translation state of the protein (output protein) encoded by itself when the input molecule binds to the aptamer. Is an artificial mRNA that changes. There are an OFF switch that suppresses the translation of the output protein by binding the input molecule and an ON switch that induces the translation of the output protein. This switching is mainly (1) steric hindrance due to the input molecule / aptamer complex. , Or (2) caused by cleavage of the mRNA by the input molecule.

As an example of (1), the 5'UTR has a kink turn sequence specifically recognized by L7Ae (a constituent protein of the ribosome large subunit of archaea) developed by the present inventors, and L7Ae expression. An OFF switch (Patent Document 1) in which an L7Ae-kincturn complex is formed on the 5'UTR and translation is suppressed can be mentioned. Examples of (2) include an OFF / ON switch having a target sequence of miRNA and being degraded in the presence of the miRNA to suppress or release the expression suppression (Patent Document 2). Furthermore, it has a pre-crRNA sequence of 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 its expression. An OFF / ON switch that releases the suppression of expression has also been reported (Non-Patent Documents 1 and 2, Patent Document 3, etc.).

Then, by the present inventors, three types of mRNA switches are introduced into cells, the output protein expressed from the first mRNA switch is the input protein of the second mRNA switch, and the output protein of the second mRNA switch is the third. It has been shown that a highly accurate translation control mechanism in which the expression of the third output protein is doubly controlled can be constructed by functioning as an input protein of the mRNA of (Patent Document 4, Non-Patent Document 3). ..

International Publication No. 2016/040395 International Publication No. 2015/105172 U.S. Patent Application Publication No. 2019/0032054 International Publication No. 2016/040395

Borchardt, E. K. et al. RNA 21, 1921-1930 (2015) Zhong, G., Wang, H., Li, Y., Tran, M. H. & Farzan, M. Nat. Chem. Biol. 13, 839-841 (2017) Matsuura, S., et al. Nat. Communi. 9, 4847-4854 (2018)

However, in order to realize a more precise translation control mechanism, the translation of one or more final output proteins needs to be controlled by a module consisting of more RNA-protein interactions. For that purpose, it is necessary to prepare as many translation control modules as are required for the desired gene circuit, which are highly versatile and have high orthogonality without interfering with each other, but there are too few modules that meet this requirement ( Only a few) was a problem.

(1) In the mRNA switch based on the steric hindrance of the input molecule / aptamer complex, the stability of the complex is very important as well as the specificity of sequence recognition. Therefore, the input molecule and the aptamer satisfying these requirements Finding a combination was not easy. In addition, (2) in an mRNA switch using 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 poses a problem in terms of versatility. It was. Furthermore, since the mRNA switch using the Cas protein as an input molecule utilizes a reaction in which the Cas protein converts (processes) its own pre-crRNA into crRNA, the Cas protein that can be used as an input molecule is very limited. .. This is because the Cas protein that has RNA-cleaving activity in a heterologous cell and is responsible for the processing reaction is very limited.

Thus, it was very difficult to develop a translation control module with versatility and orthogonality, which hindered the construction of a precise translation control circuit.

As a result of diligent studies, the present inventors have found that by using crRNA or sgRNA of Cas protein as an aptamer, Cas protein can be used as an input molecule regardless of the presence or absence of RNA cleavage activity. This is a method of utilizing the Cas protein not as an enzyme that cleaves RNA in a sequence-dependent manner, but simply as an RNA-binding protein, whereby a highly stable complex containing the Cas protein and the aptamer on the mRNA. It became clear that it is possible to form. Based on these findings, the present inventors have developed a novel mRNA switch, and further found that a precise translation control circuit can be constructed in human cells by combining these mRNA switches in a complex manner. It came to be completed.

That is, the present invention includes the following aspects.
[1] (i) A nucleic acid sequence specifically recognized by an input protein containing a Cas protein or a variant thereof, and
(Ii) Containing a nucleic acid sequence encoding an output protein
An mRNA switch comprising an artificial mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
[2] The mRNA switch according to [1], wherein the nucleic acid sequence of (i) is a crRNA or sgRNA sequence of the input protein or a variant thereof.
[3] The Cas proteins are SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, FnCas12a, LbCas12a The mRNA switch according to [1] or [2], which is one Cas protein selected from the group consisting of PguCas13b, RanCas13b, CasRx, PlmCasX, and Cas14a1.
[4] The artificial mRNA molecule further contains an RNA inverter sequence between the nucleic acid sequences of (i) and (ii), and the RNA inverter sequence contains a bait open reading frame, an intron and an internal ribosome entry site. The mRNA switch according to any one of [1] to [3], which comprises a nucleic acid sequence containing the same.
[5] A vector containing a nucleic acid sequence encoding the mRNA switch according to any one of [1] to [4].
[6] The above-described [5], which further comprises a transcription control sequence provided on the upstream side of the nucleic acid sequence encoding the mRNA switch, and the transcription control sequence is a sequence specifically recognized by the transcription control protein. vector.
[7] A nucleic acid sequence in which the transcriptional control protein contains a Cas protein or a variant thereof and is provided downstream of the nucleic acid sequence encoding the mRNA switch and encodes a small RNA used for transcriptional control or translational control. The vector according to [6], further comprising.
[8] A cell containing the mRNA switch according to any one of [1] to [4] or the vector according to 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 protein expression control kit 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 containing a nucleic acid sequence encoding a first fusion protein containing a first fragment of the input protein and a first heterodimerized 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) above and a second heterodimerized domain or said second. A protein expression control kit containing a vector encoding a trigger mRNA of.
[11] The expression control kit according to [10], further comprising an agent that promotes heterodimerization by the first and second heterodimerization domains.
[12] The protein expression control kit according to any one of [9] to [11], further comprising an input inhibitor that inhibits the recognition of the nucleic acid sequence of (i) by the input protein.
[13] An mRNA switch set including n kinds of mRNA switches consisting of a first mRNA switch to an nth mRNA switch or a vector encoding the mRNA switch, in which 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 the kth protein, including the Cas protein or a variant thereof,
(Ii) Containing a nucleic acid sequence encoding an output protein consisting of the (k + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
The kth protein and the (k + 1) th protein are different proteins,
k is an integer from 1 to (n-1) and
(B) The nth mRNA switch is
(I) A nucleic acid sequence specifically recognized by the nth protein, which is the output protein of the (n-1) th mRNA switch, and
(Ii) Containing a nucleic acid sequence encoding an output protein, which is the (n + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
An mRNA switch set in which the (n + 1) th protein is an arbitrary protein.
[14] The mRNA switch set according to [13], wherein the first to nth proteins are all different proteins.
[15] The switch set according to [13], wherein the first (n + 1) protein is the first input protein.
[16] The Cas protein or a variant thereof contained in the k and (k + 1) proteins is SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, As. 1 Cas protein selected from the group consisting of FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, Cas15a1 The mRNA switch set according to any one of the above.
[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, which comprises a step of introducing the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA into a cell.
[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 containing a nucleic acid sequence encoding a first fusion protein containing a first fragment of the input protein and a first heterodimerized 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) above and a second heterodimerized domain or said second. A method for controlling protein expression, which comprises the step of introducing a vector encoding the trigger mRNA of the protein into a cell.
[19] The protein expression control according to [18], further comprising contacting the cells with a drug that promotes heterodimerization by the first and second heterodimerization domains after the introduction step. Method.
[20] An input-inhibiting protein, an input-inhibiting mRNA encoding the input-inhibiting protein, or a vector encoding the input-inhibiting mRNA that specifically inhibits the recognition of the nucleic acid sequence of (i) by the input protein is applied to the cell. The method for controlling expression of a protein according to any one of [17] to [19], further comprising a step of introducing the protein.
[21] A method for controlling protein expression, which comprises the step of introducing the mRNA switch set according to any one of [13] to [16] into cells.
[22] (a) A crRNA or sgRNA in which the transcriptional regulatory protein contains a Cas protein or a variant thereof and is provided downstream of the nucleic acid sequence encoding the mRNA switch and specifically recognizes the transcriptional regulatory sequence. The vector according to [6], further comprising the encoding nucleic acid sequence; and (b) the Cas protein corresponding to the crRNA or sgRNA encoding the transcriptional regulatory sequence of (a) above. A protein expression control kit comprising a transcriptional control protein containing a variant, a transcriptional activity control mRNA encoding the transcriptional control protein, or a vector encoding the transcriptional activity control mRNA.

According to the present invention, it is possible to obtain a switch nucleic acid having a translation control module having orthogonality and not interfering with other modules. Furthermore, by combining a plurality of the translation control modules and by combining one or more transcription control modules, it is possible to construct a precise translation control circuit and a gene expression control circuit in eukaryotic cells. The mRNA switch according to the present invention enables safer and more efficient cell programming and has various applications.

FIG. 1 is a diagram schematically showing translation control by an input protein and an mRNA switch according to an embodiment of the present invention, and the upper left figure specifically recognizes an mRNA switch whose aptamer sequence is sgRNA and the sgRNA. The lower left figure shows the state in which the translation of the output protein is suppressed by the input protein. The upper right figure shows the combination of the mRNA switch whose aptamer sequence is crRNA and the input protein that specifically recognizes the crRNA, and the lower left figure shows the state in which the translation of the output protein is suppressed by the input protein. FIG. 2A shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by SpCas9 in the presence of the input protein SpCas9 and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2B shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by SaCas9 in the presence of the input protein SaCas9 and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2C shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by CjCas9 in the presence of the input protein CjCas9 and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2D shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by NmCas9 in the presence of the input protein NmCas9 and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2E shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by St1Cas9 in the presence of the input protein St1Cas9 and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2F shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by AsCas12a in the presence of the input protein AsCas12a and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2G has an aptamer sequence specifically recognized by PspCas13b in the presence of the input protein PspCas13b, and encodes an output protein without an aptamer and an OFF switch mRNA (Switch) encoding the output protein. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2H has an aptamer sequence specifically recognized by PguCas13b in the presence of the input protein PguCas13b, and encodes an output protein without an aptamer and an OFF switch mRNA (Switch) encoding the output protein. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2I has an aptamer sequence specifically recognized by RanCas13b in the presence of the input protein RanCas13b, and encodes an output protein without an aptamer and an OFF switch mRNA (Switch) encoding the output protein. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 2J shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by CasRx in the presence of the input protein CasRx and encoding an output protein, and an output protein without an aptamer. It is a graph which compared the translation efficiency with a control mRNA (No aptamer). FIG. 3 shows six types of mRNA switches (Nm_gRNA, Nm_gRNA_v1, Nm_gRNA_v2, Nm_gRNA_v3, Nm_gRNA_v4, Nm_gRNA_v5) having an aptamer sequence specifically recognized by NmCas9 and different aptamer sequences for the mRNA switch encoding the output protein. It is a graph prepared and compared with the control mRNA (No aptamer) and its translation efficiency in the presence of NmCas9. FIG. 4A shows wild-type SpCas9 [SpCas9 (WT)], a mutant for DNA cleavage activity (nickase) [SpCas9 (D10A), to confirm whether the suppression of mRNA switch expression is truly caused by translational suppression. )], Mutant (DNA cleavage null) [SpCas9 (D10A_H840A)] for DNA cleavage activity, SpCas9 [SpCas9 (ΔNLS)] without NLS are used as input proteins, and are specific to each of them in the presence of each input protein. The results of confirming the translation efficiency of the mRNA switch (Switch) and the control mRNA (No aptamer) that are recognized in a specific manner are shown. FIG. 4B shows an mRNA switch (Switch) that specifically recognizes SpCas9 in order to confirm whether the anti-CRISPR protein (AcrIIA4) that inhibits the DNA binding ability of SpCas9 inhibits the expression suppression of the mRNA switch. , The results of confirming the translation efficiency of control mRNA (No aptamer) in the presence and absence of AcrIIA4 are shown. FIG. 5 shows mRNA specifically recognized by wild-type AsCas12a (AsCpf1) [Cpf1 WT] and AsCas12a (H800A) mutant [Cpf1 (H800A)] to confirm whether mRNA cleavage is essential for translational repression. The results of confirming the translation efficiency of the switch (As_crRNA) and the control mRNA (No aptamer) in the presence of each input protein are shown. FIG. 6A shows an ON switch mRNA (SpCas9-responsive) having an aptamer sequence specifically recognized by SpCas9 in the presence of the input protein SpCas9 and encoding an output protein, and an inverter sequence having no aptamer sequence. It is a graph comparing the translation efficiency with control mRNA (Control) which is a nucleic acid having. FIG. 6B shows the translation efficiency between the ON switch mRNA (PspCas13b-responsive), which has an aptamer sequence specifically recognized by PspCas13b and encodes the output protein, and the control mRNA (Control) in the presence of the input protein PspCas13b. It is a graph comparing. FIG. 6C shows the translation efficiency between the ON switch mRNA (SaCas9-responsive), which has an aptamer sequence specifically recognized by SaCas9 and encodes the output protein, and the control mRNA (Control) in the presence of the input protein SaCas9. It is a graph comparing. FIG. 6D shows the translation efficiency between the ON switch mRNA (CjCas9-responsive), which has an aptamer sequence specifically recognized by CjCas9 and encodes the output protein, and the control mRNA (Control) in the presence of the input protein CjCas9. It is a graph comparing. FIG. 6E shows the translation efficiency between the ON switch mRNA (St1Cas9-responsive), which has an aptamer sequence specifically recognized by St1Cas9 and encodes the output protein, and the control mRNA (Control) in the presence of the input protein St1Cas9. It is a graph comparing. FIG. 6F has an aptamer sequence specifically recognized by AsCas12a and AsCas12a (H800A) in the presence of the input proteins AsCas12a and AsCas12a (H800A), respectively, and an ON switch mRNA (AsCas12a-responsive and) encoding the output protein. It is a graph comparing the translation efficiency between AsCas12a (H800A) -responsive) and control mRNA (Control). FIG. 7A is a diagram in which the fluorescence change of the switch depending on the presence or absence of the introduction of the trigger mRNA expressing the input protein is plotted by flow cytometry, the upper figure is a dot plot in each case of the presence or absence of the trigger mRNA introduction, and the lower figure is the trigger. It is a histogram which showed the distribution of the numerical value of the fluorescence intensity ratio EGFP / iRFP670 in each case of the presence or absence of mRNA introduction. FIG. 7B is a graph showing the translation efficiency of the control mRNA (No aptamer) and the mRNA switch (Sp_gRNA) at each trigger mRNA introduction amount. FIG. 8A has an aptamer sequence specifically recognized by FnCas9 in the presence of the input protein FnCas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8B shows an OFF switch mRNA (Switch 1) having an aptamer sequence specifically recognized by FnCpf1 in the presence of the input protein FnCpf1 and encoding the output protein, and an output protein without an aptamer. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8C shows an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by LbCpf1 in the presence of the input protein LbCpf1 and encodes an output protein, and an output protein without an aptamer. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8D shows an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by MbCpf1 in the presence of the input protein MbCpf1 and encodes an output protein, and an output protein without an aptamer. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8E has an aptamer sequence specifically recognized by AkCas12b in the presence of the input protein AkCas12b, does not have two OFF switch mRNAs (Switch 1, Switch 2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8F shows an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by BvCas12b in the presence of the input protein BvCas12b and encodes an output protein, and an output protein without an aptamer. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8G has an aptamer sequence specifically recognized by BsCas12b in the presence of the input protein BsCas12b, has an OFF switch mRNA (Switch1) that encodes the output protein, and has no aptamer and encodes the output protein. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8H has an aptamer sequence specifically recognized by PlmCasX in the presence of the input protein PlmCasX, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8I has an aptamer sequence specifically recognized by CdCas9 in the presence of the input protein CdCas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8J shows an OFF switch mRNA (Switch 1) that has an aptamer sequence specifically recognized by ClCas9 in the presence of the input protein ClCas9 and encodes an output protein, and does not have an aptamer and encodes an output protein. It is a graph comparing the translation efficiency with the control mRNA (No aptamer). FIG. 8K has an aptamer sequence specifically recognized by NcCas9 in the presence of the input protein NcCas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8L has an aptamer sequence specifically recognized by PlCas9 in the presence of the input protein PlCas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8M has an aptamer sequence specifically recognized by SpaCas9 in the presence of the input protein SpaCas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8N has an aptamer sequence specifically recognized by St3Cas9 in the presence of the input protein St3Cas9, does not have two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 8O has an aptamer sequence specifically recognized by Cas14a1 in the presence of the input protein Cas14a1, has two OFF switch mRNAs (Switch1, Switch2) encoding the output protein, and does not have an aptamer. It is a graph which compared the translation efficiency with the control mRNA (No aptamer) which encodes an output protein. FIG. 9A shows the translation efficiency between the ON switch mRNA (CasRx-responsive), which has an aptamer sequence specifically recognized by CasRx and encodes the output protein, and the control mRNA (Control) in the presence of the input protein CasRx. It is a graph comparing. FIG. 9B shows the translation efficiency between the ON switch mRNA (PguCas13b-responsive), which has an aptamer sequence specifically recognized by PguCas13b and encodes the output protein, and the control mRNA (Control) in the presence of the input protein PguCas13b. It is a graph comparing. FIG. 9C shows the translation efficiency between the ON switch mRNA (AkCas12b-responsive), which has an aptamer sequence specifically recognized by AkCas12b and encodes the output protein, and the control mRNA (Control) in the presence of the input protein AkCas12b. It is a graph comparing. FIG. 9D shows the translation efficiency between the ON switch mRNA (BvCas12b-responsive), which has an aptamer sequence specifically recognized by BvCas12b and encodes the output protein, and the control mRNA (Control) in the presence of the input protein BvCas12b. It is a graph comparing. FIG. 9E shows the translation efficiency between the ON switch mRNA (PlmCasX-responsive), which has an aptamer sequence specifically recognized by PlmCasX and encodes the output protein, and the control mRNA (Control) in the presence of the input protein PlmCasX. It is a graph comparing. FIG. 9F shows the translation efficiency between the ON switch mRNA (LbCas12a-responsive), which has an aptamer sequence specifically recognized by LbCas12a and encodes the output protein, and the control mRNA (Control) in the presence of the input protein LbCas12a. It is a graph comparing. FIG. 9G has an aptamer sequence specifically recognized by FnCas12a in the presence of the input protein FnCas12a, and the translation efficiency between the ON switch mRNA (FnCas12a-responsive) encoding the output protein and the control mRNA (Control). It is a graph comparing. FIG. 10A shows that when both Cas9 fragments SpCas9 (N-term) and SpCas9 (C-term) are present in the cell, full-length SpCas9 is produced, causing translational repression of the mRNA switch. It is a conceptual diagram which shows. In FIG. 10B, when SpCas9 (N-term) and SpCas9 (C-term) are present, there is an input (1), and when SpCas9 (C-term) is not present, there is no input (0), and the output protein is expressed without translation suppression. It is a table showing the experimental results, where the output is present (1), and the output protein is not expressed due to translation inhibition as no output (0). FIG. 10C is a graph showing the translation efficiency of each input of FIG. 10B and the use of unseparated wild-type Cas (WT). In FIG. 11A, split Cas9 in which a DmrA or DmrC binding domain is fused to the C-terminal or N-terminal of each fragment of SpCas9 (N-term) and SpCas9 (C-term) is prepared, and the full length is obtained only when a drug is added. It is a conceptual diagram showing that SpCas9 of the above is generated and the translational repression of the mRNA switch is caused. Split1-3 in FIG. 11B has an aptamer sequence specifically recognized by SpCas9, and has an OFF switch mRNA (Switch) that encodes an output protein and a control mRNA (No.) that does not have an aptamer and encodes an output protein. For each of aptamer), for each of Split1-3, the translation efficiency was shown when the drug was added (Drug +) and when the drug was not added (Drug-) in the presence of both fragments, and WT showed the same OFF switch. Graph showing translation efficiency when a drug is added (Drug +) and when a drug is not added (Drug-) in the presence of wild-type SpCas9 to mRNA (Switch) and control mRNA (No aptamer), respectively. Is. FIG. 12 shows an OFF switch mRNA (Switch) having an aptamer sequence specifically recognized by SaCas9 and encoding an output protein, and a control mRNA (No aptamer) having no aptamer and encoding an output protein. The translation efficiency is shown when the plasmid vector encoding AcrIIC2 is not added (0 ng) or when the plasmid vector encoding AcrIIC2 of 200 ng, 400 ng, 1000 ng, 2000 ng is added, and the translation suppression by SaCas9 is suppressed by AcrIIC2. Indicates that it will be released. FIG. 13A shows translation inhibition between 9 Cas proteins of SpCas9, SaCas9, CjCas9, St1Cas9, AsCas12a, PspCas13b, PguCas13b, RanCas13b, CasRx and 9 mRNA switches having an aptamer sequence specifically recognized for each. It is a fluorescence photograph which shows the result of confirming the orthogonality of. Unintended translation suppression occurred for the combinations corresponding to the white squares, but orthogonality was confirmed for the other combinations. FIG. 13B is a diagram showing the results of FIG. 13A in shades based on translation efficiency. FIG. 13C shows 14 types of Cas proteins, 14 types of mRNA switches having an aptamer sequence specifically recognized for each, and L7Ae protein known as a protein-RNA binding motif (RNP motif), and L7Ae. It is a fluorescent photograph showing the result of confirming the orthogonality of the translation suppression of the Box CD sequence. FIG. 14A is a diagram conceptually showing the configuration of a multi-layer circuit in which a switch set composed of six types of OFF switch mRNAs is hierarchically combined. FIG. 14B is a diagram showing fluorescence micrographs in each layer, histograms obtained from flow cytometry analysis, and calculated output levels. The panel (a) of FIG. 15A is a fluorescence photograph showing the orthogonality test results for 29 types of OFF switch mRNAs, and the panel (b) shows the clear orthogonality of the Cas response OFF switch mRNA switches tested. These are the 13 types of manifestations shown. FIG. 15B is a heat map showing the results of image quantification for the 13 types of Cas response OFF switch mRNAs shown in FIG. 15A and panel (b). FIG. 16 shows the results of testing whether translation (OFF switch mRNA) and transcriptional activation of DNA constructs can be controlled at the same time using SpCas9. Panel (a) outlines the mechanism, and panel (b) shows the outline of the mechanism. A cell photograph is shown. FIG. 17 shows a scheme of an AND gate circuit that can be produced by combining three types of Cas response OFF switch mRNAs. FIG. 18A shows a scheme of a half subtractor configured using only two modules that simultaneously utilize transcriptional regulation and translational regulation by Cas protein. FIG. 18B shows the truth table of the half subtractor. FIG. 18C shows a fluorescence micrograph of the cells corresponding to the output. In FIG. 19, for OFF switch mRNA having an aptamer sequence specifically recognized by NmCas9 and encoding an output protein, Nm_gRNA_v6 and Nm_gRNA_v7 were prepared in addition to FIG. 3, and NmCas9 was prepared together with a control mRNA (No aptamer). It is a graph comparing the translation efficiency in the presence of. FIG. 20 is a fluorescence photograph showing the results of an orthogonality test for 26 types of ON switch mRNA. FIG. 21 shows the results of a simultaneous drive test of SaCas9-responsive translation OFF switch mRNA (red fluorescence) and translation ON switch mRNA (green fluorescence). Panel (a) shows a cell photograph, and panel (b) shows flow cytometry. The quantitative result by is shown.

Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the embodiments described below.

[1. mRNA switch]
According to the first embodiment, the present invention relates to an mRNA switch, which is a mRNA switch.
(I) Nucleic acid sequences specifically recognized by input proteins, including Cas proteins or variants thereof,
(Ii) Containing a nucleic acid sequence encoding an output protein, said (i) is present on the 5'or 3'side of (ii) and said (i) and (ii) are operably linked. There is.

The mRNA switch in the present embodiment refers to an artificial mRNA molecule that encodes a specific output protein and whose translation of the output protein is controlled in a specific response to a specific input protein. Translation is controlled in response to a specific input protein specifically depending on whether the specific input protein is present or not, and the translation state (translation is being performed or translation is being performed) of the output protein. It means that the state where translation is not performed) is reversed. In the present embodiment, since the state in which translation is not performed is caused by translation suppression, it may be referred to as a translation suppression state. In addition, changing the degree of translation or translational repression (translation efficiency) according to the amount of a specific input protein is also included in "control". In the present specification, an mRNA switch in which translation is performed in the presence of a specific input protein and translation is suppressed in the 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 the absence is referred to as an OFF switch mRNA.

The input protein is a protein that specifically recognizes an mRNA switch, and includes at least a Cas protein or a variant thereof. The Cas protein may be any Cas protein, for example, SpCas9 (Cas9 derived from Streptococcus pyogenes, also known as SpyCas9, SEQ ID NO: 1), SaCas9 (Cas9 derived from Staphylococcus aureus, also known as SauCas9, SEQ ID NO: 2), CjCas9. (Cas9 derived from Campylobacter jejuni, also known as CjeCas9, SEQ ID NO: 3), NmCas9 (Cas9 derived from Neisseria meningitidis, also known as NmeCas9, SEQ ID NO: 4), St1Cas9 (Cas9 derived from Streptococcus thermophilus, alias Sth1Cas9, SEQ ID NO: 5), FnCas9 (Cas9 derived from Francisella novicida, also known as FnoCas9, SEQ ID NO: 6), CdCas9 (Cas9 derived from Corynebacterium diphtheriae, also known as CdiCas9, SEQ ID NO: 7), ClCas9 (Cas9 derived from Campylobacter lari CF89-12, SEQ ID NO: 8) , PlCas9 (Cas9 derived from Parvibaculum lavamentivorans, SEQ ID NO: 9), NcCas9 (Cas9 derived from Neisseria cinerea, SEQ ID NO: 10), SpaCas9 (Cas9 derived from Streptococcus pasteurianus, SEQ ID NO: 11), St3Cas9 (Streptococcus derived from Streptococcus Cas9, also known as Sth3Cas9, SEQ ID NO: 12), AsCas12a (Acidaminococcus sp. BV3L6 derived from Cas12a, alias AsCpf1, SEQ ID NO: 13), FnCas12a (Francisella novelida U112 derived from Cas12a, alias FnCpf1, alias 14), LbCa Cas12a derived from bacterium ND2006, also known as LbCpf1, SEQ ID NO: 15), MbCas12a (Cas12a derived from Moraxella bovoculi 237, also known as MbCpf1, alias 16), AkCas12b (also known as Cas12b derived from Alicyclobacillus kakegawensis, alias AkC 2c1, SEQ ID NO: 17), BvCas12b (Cas12b derived from Bacillus sp. V3-13, also known as BvC2c1, SEQ ID NO: 18), BsCas12b (Cas12 derived from Bacillus sp. 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 (derived from Ruminococcus flavefaci) Select from Cas protein, also known as RfxCas13d, SEQ ID NO: 23), PlmCasX (CasX derived from Planctomycetes, also known as PlmCas12e, SEQ ID NO: 24), Cas14a1 (Cas14a derived from uncultured archaeon, also known as Cas14a.1, SEQ ID NO: 25), AaCas12b. be able to. Cpf1 displayed as a part of the alias is an abbreviation for CRISPR-associated endonuclease in Prevotella and Francisella 1. The Cas protein can be appropriately selected according to the purpose and use of the mRNA switch. In the present invention, the Cas protein as an input protein may or may not have DNA cleaving activity.

The input protein may be the Cas protein itself, but may be a variant thereof. The variant may be, for example, a fusion of Cas protein and an additional protein. The additional protein may be one that does not inhibit the recognition ability of the mRNA switch by the Cas protein, and is a transcriptional activator such as VP16 or VP64, VP64-p65-Rta (VPR), or a transcriptional repressor Kruppel associated box. Domain (KRAB), DNA methylase DNMT3A, DNA demethylase TET1, histon acetylase LSD1 and p300, RNA degrading enzyme CNOT7 and DDX6, translation factor eIF family, virus-derived protein VPg, RNA modifier ADAR, DNMT, It may be a functional protein such as METTL, WTAP, FTO, ALKBH5, or fluorescent protein.

The Cas protein variant that functions as an input protein may be an aggregate in which Cas proteins divided into a plurality of fragments are associated, or may be a molecule in which a molecule such as a protein used for association is further bound. The aggregate may be an aggregate having the ability to recognize the mRNA switch. The Cas protein variant may also be a Cas protein fragment from which the portion of the Cas protein that is not involved in RNA sequence recognition has been removed. The Cas protein fragment can also be referred to as a miniaturized Cas protein.

The mRNA switch is typically an RNA molecule. In certain embodiments, the mRNA switch may be a synthetic mRNA molecule. The synthetic mRNA molecule is not particularly limited, but may be, for example, an mRNA molecule synthesized in vitro. Synthetic mRNA can be introduced into cells as it is in the form of mRNA molecule and used for translation control. In the present specification, an mRNA switch composed 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 SpCas9 responsive mRNA.

In another embodiment, the mRNA switch may be an mRNA produced by being transcribed from a DNA construct in a cell, and the DNA construct may be a vector or the like. Hereinafter, the structure and sequencing of RNA molecules constituting the mRNA switch will be described.

(I) Nucleic acid sequence specifically recognized by an input protein containing a Cas protein or a variant thereof The mRNA switch contains a nucleic acid sequence specifically recognized by an input protein containing a Cas protein or a variant thereof. In the present specification, the nucleic acid sequence of (i) may be referred to as an aptamer sequence.

The nucleic acid sequence of (i) is not particularly limited as long as it is specifically recognized by an input protein containing a Cas protein or a variant thereof and enables translational control, but typically, the Cas protein is used. The corresponding crRNA or sgRNA sequence. The nucleic acid sequence of (i) may be a variant of a crRNA or sgRNA sequence, and the variant means a variant that retains a specific binding ability to Cas protein. Combinations of a Cas protein with its corresponding crRNA or sgRNA sequence are 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).
When pre-crRNA corresponding to Cas protein is used as the nucleic acid sequence of (i), the mRNA switch may be cleaved in the pre-crRNA region, which may not be preferable. This is because if the mRNA switch is cleaved, reversible translation control may not be possible depending on the presence or absence of the input protein.

Specific combinations of the Cas protein (Target protein) and the crRNA or sgRNA sequence can be exemplified by those shown in Table 1 below, but are not limited thereto. Switch name represents the name of the mRNA switch.

(Ii) Nucleic acid sequence encoding the output protein The mRNA switch contains a nucleic acid sequence encoding the output protein. The nucleic acid sequence encoding the output protein can be appropriately determined by those skilled in the art according to the desired output protein. The output protein is not particularly limited and may be any protein.

In certain embodiments, the output protein may be a Cas protein or a variant thereof. The Cas protein or a variant thereof may be similar to that described in the definition of input protein and can be selected from similar options. In one mRNA switch molecule, the output protein may be the same as or different from the input protein.

In another embodiment, the output protein may be anti-CRISPR, which inactivates the Cas protein. Anti-CRISPRs capable of inactivating spCas9 include, but are not limited to, AcrIIA2, AcrIIA4, AcrIIA5, AcrIIA7, AcrIIA8, AcrIIA9, and AcrIIA10. Examples of anti-CRISPR that inactivates NmCas9 include AcrIIC1, AcrIIC2, AcrIIC3, AcrIIC4, and AcrIIA5. Examples of anti-CRISPR that inactivates CjCas9 include AcrIIC1 and anti-CRISPR that inactivates St1Cas9. Examples of CRISPR include AcrIIA5 and AcrIIA6, and examples of anti-CRISPR that inactivates SaCas9 include AcrIIC2 and AcrIIA5.

In another embodiment, the output protein may be a marker protein. A marker protein is a protein that is expressed from an mRNA switch, functions as a marker in a cell, and can identify the cell. As an example, the marker protein may be a protein that can be visualized and quantified by fluorescence, luminescence, coloration, or by assisting fluorescence, luminescence, or coloration. Fluorescent proteins include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (eg hmAG1), ZsGreen, EmGFP, Green fluorescent proteins such as EGFP, GFP2, HyPer, etc .; Yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana; Orange fluorescent proteins such as KusabiraOrange (eg, hmKO2), mOrange. ; Red fluorescent protein such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg hdKeimaRed), mRasberry, mPlum, etc. Examples include, but are not limited to, external fluorescent proteins.

Aequorin can be exemplified as a photoprotein, but the luminescent protein is not limited to this. In addition, examples of proteins that assist fluorescence, luminescence, or color development include, but are not limited to, enzymes that decompose fluorescence, luminescence, or color development precursors such as luciferase, phosphatase, peroxidase, and β-lactamase. Here, in the present invention, when a protein that assists fluorescence, luminescence, or color development is used as an output protein, the corresponding precursor is brought into contact with the cell into which the mRNA switch is introduced, and the corresponding precursor is introduced into the cell. By doing so, fluorescence, light emission or color development can be observed.

Another example of marker proteins is proteins that directly affect cell function. Examples thereof include cell proliferation proteins, cell killing proteins, cell signaling factors, drug resistance genes, transcriptional regulators, translational regulators, differentiation regulators, reprogramming inducers, RNA-binding protein factors, chromatin regulators, and membrane proteins. However, it is not limited to these. For example, a cell proliferation protein functions as a marker by proliferating only the cells expressing it and identifying the proliferated cells. The cell-killing protein kills the cell itself by causing cell death of the cell expressing it, and functions as a marker indicating the life or death of the cell. The cell signal factor functions as a marker when the cell expressing it emits a specific biological signal and identifies this signal. Examples of cell-killing proteins include Bax or Bim. Translation regulators, for example, function as markers by recognizing and binding to the tertiary structure of a particular RNA to control the translation of other mRNAs into proteins. Translation regulators include 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), 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.), MS2coat (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.) Is illustrated. More preferred translational regulators are MS2coatprotein, L7Ae.

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, and specifically, a classical nuclear localization sequence (NLS), M9. Sequences, mitochondrial target sequences (MTS), endoplasmic reticulum translocation sequences, but are not limited to these. Such a localization signal is advantageous when the marker protein is visualized by imaging cytometry or the like.

In the mRNA switch, the nucleic acid sequence of (i) exists on the 5'side or 3'side of the nucleic acid sequence of (ii), and the above (i) and (ii) are operably linked. A detailed embodiment of the operably linked manner will be described later for each of the OFF switch mRNA and the ON switch mRNA. When an mRNA switch having such structural characteristics is introduced into a cell, if the input protein specifically recognizes the nucleic acid sequence of (i), it is possible to control the translation of the nucleic acid sequence of (ii). It becomes.

The more specific structure of the mRNA molecule that functions as an OFF switch mRNA will be explained. The OFF mRNA switch may have a structure in which the 5'-UTR, the coding region, and the 3'-UTR are linked in order from the 5'side of the mRNA molecule. When the nucleic acid sequence of (i) is present on the 5'side of the nucleic acid sequence of (ii), the 5'-UTR is [Cap structure or Cap analog] and [nucleic acid sequence of (i)] in order from the 5'side. May be a connected structure. The Cap structure may be 7-methylguanosine 5'phosphate. The Cap analog is a modified structure recognized by eIF4E, which is a translation initiation factor, like the Cap structure, and is m7G (5') ppp (5') manufactured by Ambion's Anti-Reverse Cap Analog (ARCA) and New England Biolabs. ') GRNACapStructureAnalog, CleanCap made by TriLink, etc., but are not limited to these, and may be any 5'capping structure for avoiding synthetic mRNA from the innate immune response. Even if the cap structure or the 3'side of the Cap analog and the 5'side of the nucleic acid sequence of (i) contain, for example, an arbitrary nucleic acid sequence of about 0 to 50 bases, preferably about 0 to 30 bases. Good. The nucleic acid sequence of (i) may include at least one, but may include 2 repeats, 3 repeats, 4 repeats, or more repetitions of the nucleic acid sequence of (i). The 3'end side of the 5'-UTR, which is the 3'side of the nucleic acid sequence of (i), may contain, for example, an arbitrary nucleic acid sequence of 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 input proteins and output proteins. It is preferable that there is no AUG as a start codon in the 5'-UTR. For example, when AUG is contained in the nucleic acid sequence of (i), a frame shift can be avoided by adding one or two bases at the end of the sequence. Alternatively, a stop codon sequence may be added outside the nucleic acid sequence of (i) counted in units of 3 bases from the above-mentioned AUG. Alternatively, one or more bases of AUG can be converted into any base and used as long as it does not affect the interaction with the protein. The coding region contains the nucleic acid sequence of (ii). The 3'-UTR contains a poly A tail, and a Cas protein binding sequence (a sequence recognized by any Cas protein) may be inserted. When the nucleic acid sequence of (i) is located on the 3'side of the nucleic acid sequence of (ii), the nucleic acid sequence of (i) is placed at the 5'end even if it is placed at the 3'end of the poly A tail. May be inserted in the poly A tail. Further, the nucleic acid sequence of (i) may be present on both the 3'side and the 5'side of the nucleic acid sequence of (ii).

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

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

The RNA inverter sequence is located on the 5'side of the start codon (coding region) and on the 3'side of the nucleic acid sequence of (i), reversing translational repression and from mRNA only in the presence of the input protein. A sequence that can be controlled to translate the output protein. RNA inverter sequences, also referred to as ON switch cassettes, are detailed in WO 2014/014122, which is hereby incorporated by reference. Specifically, the RNA inverter sequence consists of a sequence containing a mutant open reading frame (bait ORF), an intron, and an IRES (internal ribosome entry site) in order from the 5'side. Here, the bait ORF is a stop codon that is 320 bases or more away from the 3'end that binds to an intron in the sequence encoding an arbitrary gene in order to cause RNA degradation by the nonsense-mediated mRNA decay mechanism (NMD). It is a mutant ORF having. In the present invention, the bait ORF may be any coding gene. The bait ORF is not particularly limited, but is 457th from the 5'side of Renilla luciferase, a sequence in which a stop codon is inserted at the 466th base (SEQ ID NO: 64 or SEQ ID NO: 65), or 172nd from the 5'side of EGFP. A sequence in which a stop codon is inserted into the base of (SEQ ID NO: 66) is exemplified. Further, the intron may have a sequence to which spliceosomes bind, and examples thereof include a sequence of 20 bases or more having a GT sequence on the 5'end side and an AG sequence on the 3'end side. Preferably, it is a human β-globin intron (SEQ ID NO: 67) or a chimeric intron (SEQ ID NO: 68). Table 2 below shows an example of the arrangement of the bait ORF and intron.

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

[2. How to use mRNA switch]
Next, the usage and operation of this mRNA switch will be described. The mRNA switch can be used in a mode of introduction into a cell, and translation is controlled depending on the presence / absence and abundance of the input protein in the cell.

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 a cell (including a cell line) that has been artificially manipulated. It is preferably a cell derived from a mammal (for example, human, mouse, monkey, pig, rat, etc.), and most preferably a cell derived from human. There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and it can be any of (A) stem cells, (B) progenitor cells, (C) final differentiated somatic cells, and (D) other cells. You may.

(A) Examples of stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, and sperm stem cells (“GS cells”). , Embryonic germ cells (“EG cells”), induced pluripotent stem (iPS) cells and the like. Of these, ES cells and iPS cells are preferable, and iPS cells are particularly preferable.

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

(C) Somatic cells include, for example, keratinizing epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells on the surface of the tongue), exocrine gland epithelial cells (eg, mammary cells), hormone secretion. Cells (eg, adrenal medulla cells), cells for metabolism and storage (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner canal (eg, type I alveolar cells) E.g., vascular endothelial cells), ciliated cells capable of carrying (eg, airway epithelial cells), extracellular matrix secretory cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood Immune system cells (eg, T lymphocytes), sensory cells (eg, rod cells), central / peripheral nervous system nerve cells and glial cells (eg, stellate glial cells), pigment cells (eg, retinal pigment epithelium) Cells) and their precursor cells (tissue precursor cells) and the like.

(D) Other cells include, for example, cells that have undergone differentiation induction, and also include progenitor cells and somatic cells that have undergone differentiation induction from pluripotent stem cells. Further, it may be a cell group induced by so-called "direct reprogramming (also referred to as trans-differentiation)" in which somatic cells or progenitor cells are directly differentiated into desired cells without undergoing an undifferentiated state. .. In addition, it may be a cell group that can include cells for which gene editing is desired, such as a cell group that can contain cancer cells and normal cells, and a cell group that can include cells for which gene editing is not desired.

Introduction of the mRNA switch into cells is performed when the mRNA switch is a synthetic RNA molecule (often a synthetic mRNA molecule) or a self-replicating RNA, for example, lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation. RNA molecules can be introduced directly into cells using introduction methods such as the method, DEAE dextran method, microinjection method, and gene gun method. The advantage of introducing a synthetic RNA molecule is that it does not integrate into the genome and the cells after introducing the mRNA switch can be easily used for medical applications.

A DNA construct such as an expression vector can also be used to introduce the mRNA switch into cells. In this case, an expression vector encoding an mRNA switch can be designed, and the expression vector can be directly introduced into cells by the same introduction method as described above. As the expression vector encoding the sequence of the mRNA switch, those commonly used in the art can be used, and for example, an expression system using a viral vector, an artificial chromosome vector, a plasmid vector, or a transposon (sometimes called a transposon vector). ) Etc. can be mentioned. Examples of the viral vector include a retrovirus vector, a lentiviral 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), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid vector, a general mammalian plasmid can be used, and for example, an episomal vector may be used. Examples of the transposon vector include an expression vector using the piggyBac transposon. For example, the vectors disclosed in U.S. Pat. No. 10,378,070, which are U.S. patents by the inventors and which are incorporated herein by reference, can 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 function continuously in the cell, and for example, it can be used for 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 or stop the expression induction at a desired time. Furthermore, by cloning after the introduction of the expression vector, there is an advantage that a cell population having almost the same expression level of the mRNA switch can be obtained.

Further, the DNA construct may be a vector containing a nucleic acid sequence encoding an mRNA switch and a transcription control sequence provided on the upstream side of the nucleic acid sequence encoding the mRNA switch. Such a vector is referred to herein as a transcriptional control switch vector. By providing the transcription control sequence, it is possible to control the transcription activity of the vector, particularly activation of transcription, and to control the expression of the mRNA switch encoded by the vector. A transcriptional regulatory sequence is a sequence that is specifically recognized by a transcriptional regulatory protein. The transcriptional regulatory protein is a protein having a DNA binding site and a transcriptional regulatory site, a fusion protein, or a protein complex containing these. The DNA binding site that constitutes a transcriptional regulatory protein is a site that is necessary for the protein itself to directly recognize the transcriptional regulatory sequence, or a site that is necessary for recognizing the transcriptional regulatory sequence via DNA, RNA, etc., and is transcribed. It is a site that specifically recognizes the control switch vector. The transcription control site is a protein having a transcription promoting activity or a transcription inhibitory activity, or a variant thereof. Examples of such a transcriptional regulatory protein include a complex of a Cas protein or a variant thereof and a corresponding crRNA or sgRNA sequence or a variant thereof. Alternatively, the transcriptional regulatory protein may be, but is not limited to, TALEN. In addition, the mechanism of action of transcriptional regulation has these actions, for example, by directly applying DNA methylation or demethylation, histone acetylation or deacetylation to DNA or chromatin, or by cleaving DNA. A method of indirectly accumulating factors in a transcription control sequence may be used, but the method is not limited thereto. When the transcriptional regulatory protein is a complex containing the Cas protein or a variant thereof, the Cas protein or a variant thereof constituting the complex is different even if it is the same as the input protein of the mRNA switch encoded by the transcriptional regulatory switch vector. May be. In this case, the transcriptional regulatory protein may be a complex containing an inactivated Cas protein fused to the above-mentioned transcriptional activators VP16, VP64, and VP64-p65-Rta (VPR). Alternatively, it may be a complex containing an inactivated Cas protein fused to the Kruppel associated box domain (KRAB), which is a transcriptional repressor. Specifically, as the transcriptional regulatory protein, a complex containing dCas9-VP64, dCas9-VPR, dCas9-SunTag, dCas9-VP16, dCas9-VP160, dCas9-P300, and dCas9-KRAB can be used. Not limited to.

The transcription control switch vector may contain, in addition to the desired mRNA switch, the transcription control switch vector or other transcription control switch vector, or a nucleic acid sequence encoding an input RNA that can be used to control the mRNA switch. The input RNA may be a small RNA used for transcriptional or translational regulation. For example, when the transcriptional regulatory protein is a complex containing 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 transcriptional regulatory sequence of the transcriptional regulatory switch vector, or a variant thereof. Alternatively, the input RNA may be a crRNA or sgRNA sequence that specifically recognizes the transcriptional regulatory sequence of a transcriptional regulatory switch vector different from the transcriptional regulatory switch vector, or a variant thereof. When the transcription control switch vector contains a nucleic acid sequence encoding an input RNA, it is preferable that 3'of the nucleic acid sequence encoding the mRNA switch includes a nucleic acid sequence that stabilizes the mRNA switch. The nucleic acid sequence that stabilizes the mRNA switch may be MALAT1 triplet or the like, but is not limited to a specific sequence. Then, it is preferable to include the nucleic acid sequence encoding the input RNA on the 3'side of the nucleic acid sequence that stabilizes the mRNA switch. It is preferred that the 5'and 3'sides of the nucleic acid sequence encoding the input RNA further include a nucleic acid sequence encoding a self-cleaving ribozyme.

The mRNA switch according to the present invention can be used in combination with an input protein in the form of an mRNA molecule or in the form of a vector, and translation control can be performed according to various usage modes. Further, the vector containing the nucleic acid sequence encoding the mRNA switch can be used in combination with the transcription control protein by using the transcription control switch vector, and the transcription control and the translation control can be performed at the same time. Hereinafter, for each aspect, an mRNA switch, an mRNA switch set, and a protein expression control kit containing these will be described.

[A. Translation control of mRNA switch by input protein]
A translation control system consisting of an input / output system consisting of a combination of an input protein and an mRNA switch will be described. The translation control of the mRNA switch can be rephrased as the expression control system of the output protein. The input protein according to this embodiment may be any input protein described above. Further, the mRNA switch may be an ON switch mRNA or an OFF switch mRNA as long as it has an aptamer sequence specifically recognized by the input protein and encodes an arbitrary output protein.

In the translation control system of Aspect A, the input protein can be introduced into the cell as it is in the protein state, or can be introduced into the cell 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 this is also called a trigger plasmid. In addition, the mRNA switch can be introduced into cells in the form of an mRNA molecule or a vector encoding the mRNA. The mRNA molecule that constitutes an mRNA switch is also called a switch mRNA, and the vector that encodes an mRNA switch is also called a switch vector. When the switch vector is a plasmid vector, it is also called a switch plasmid.

The protein expression control kit for carrying out the translational control system of Aspect A is selected from the group consisting of at least one component selected from the group consisting of an input protein, a trigger mRNA, a trigger plasmid, and an mRNA switch or a switch vector. It may contain at least one component.

The translation control method of the mRNA switch according to the aspect A can also be said to be a method of controlling the expression of the output protein, and includes a step of introducing the mRNA switch or the switch vector into a desired cell. Hereinafter, it is referred to as an introduction step of the mRNA switch. It also includes the step of introducing the input protein, trigger mRNA, and trigger plasmid into the cell. Hereinafter, it is also referred to as an input process. The introduction step and the input step can be carried out at the same time or at an arbitrary time difference. By such an input step, the translation state by the mRNA switch or switch vector can be switched, and the desired output protein is expressed (with output), or the mRNA switch or switch vector is translationally suppressed and the output protein is expressed. It can be in a state where it is not performed (no output). In particular, in the ON switch mRNA, the translation of the mRNA switch is performed by the input process and the output protein is expressed (with output), and in the OFF switch mRNA, the translation of the mRNA switch is suppressed by the input process and the output protein is released. It can be in a state where it is not expressed (no output).

By using two or more combinations of input proteins and mRNA switches, it is possible to realize a system that independently controls the translation of two or more mRNA switches, that is, the expression of two or more output proteins. In this case, a first input / output system, a second input protein, a trigger mRNA or a trigger plasmid, and a second, a combination of the first input protein, the trigger mRNA or the trigger plasmid, and the first mRNA switch or switch vector. A second input / output system is used by combining the mRNA switches of. The first mRNA switch comprises an aptamer sequence specifically recognized by the first input protein and encodes the first output protein. The second mRNA switch comprises an aptamer sequence 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 have orthogonality. That is, the two input / output systems can control input / output independently without interfering with each other, and the expression of the two output proteins is controlled independently.

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

The protein expression control kit according to aspect B can include an input inhibitor in addition to the components of the translation control kit described in aspect A. When the input inhibitor is an input inhibitor protein, the kit may include the input inhibitor protein itself, an mRNA encoding the input inhibitor protein, or a vector encoding the mRNA. When the input inhibitor is a drug, the protein expression control kit according to embodiment B includes the drug.

The mRNA molecule encoding the input-inhibiting protein or the vector encoding the mRNA may be an mRNA expressing the input-inhibiting protein or a vector encoding the mRNA molecule without being subject to translation control. That is, it may be an mRNA having no aptamer sequence or a vector encoding the mRNA.

Alternatively, the mRNA encoding the input inhibitory 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 comprises an aptamer sequence specifically recognized by the second input protein and, as an output protein, encodes an input inhibitory 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 used. ) Is independently translated and regulated, and the expression of the output protein of the first mRNA switch is regulated.

Also in aspect 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 in the previous aspect A. By further including the step of introducing an input inhibitor into the cell or contacting the input inhibitor, the interaction between the first mRNA switch and the first input protein is inhibited, and the first mRNA switch and the first mRNA switch are used. , The translational state achieved with the first input protein can be controlled, thereby controlling the expression of the output protein.

[C. Translational regulation of mRNA switches by fragmented input proteins]
In an input / output system using a combination of an input protein and an mRNA switch, a translation control system (protein expression control) system of the mRNA switch when the input protein is a fragmented protein will be described. Fragmented proteins are also referred to as split proteins. In aspect C, the input protein is composed of Cas protein or a variant thereof divided into a plurality of fragments. The plurality of fragments associate under predetermined conditions to form an aggregate having a nucleic acid targeting ability (aptamer recognition ability) and function as an input protein.

Fragmented input proteins can be designed based on the 25 Cas proteins listed above and other Cas proteins. In certain Cas proteins, it is known that the nucleic acid targeting ability is restored by partitioning between specific residues and then associating each fragment, and the nucleic acid targeting ability is restored based on known information. Possible Cas protein fragments can be designed. Specifically, the division between the 714th and 715th residues of SpCas9, the division between the 535th and 536th residues, and the division between the 713th and 714th residues. Division is known, but it is not limited to these. One of ordinary skill in the art can identify fragments of any Cas protein that can restore nucleic acid targeting ability. One input protein may be fragmented into two, or 3, 4, 5 or more, as long as the nucleic acid targeting ability can be restored.

When one input protein is divided into a first fragment and a second fragment, a heterodimerized domain may be bound to each of the first fragment and the second fragment. The heterodimerized domain bound to the first fragment and the heterodimerized domain bound to the second fragment are domains capable of forming a heterodimer. The heterodimerized domain can increase the efficiency of association during assembly formation. The heterodimerized domain may, for example, be a segregated intein, with N intein and C intein bound to two fragments of the disrupted input protein, respectively. When the heterodimerized domain is the isolated intein, if the first fragment to which the N intein is bound and the second fragment to which the C intein is bound are simultaneously present in the cell, they form an aggregate. .. In the aggregate, the intein moiety is excised and functions as an input protein. Therefore, the action of translation control by the aggregate is similar to the action of the input protein in aspect A.

Another example of a heterodimerized domain is a domain that causes association in the presence of a drug consisting of a specific small molecule compound, and iDimerize ™ Inducible Heterodimer System (Clontech) can be used. When the heterodimerized domain is according to the system, the fragmented input protein is a fragment in which the N-terminal fragment of the input protein is fused with the DmrA-binding domain and a fragment in which the C-terminal fragment of the input protein is fused with the DmrC-binding domain. It may be there. These domains associate in the presence of A / C Heterodimerizer (AP21967 ligand) to restore the nucleic acid recognition capacity of the input protein.

In aspect C, 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 heterodimerized domain, and a second fragment of the input protein. A second trigger mRNA containing a nucleic acid sequence encoding a second fusion protein containing a second heterodimerized domain is used. The first and second trigger mRNAs may be mRNAs having no 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 having an aptamer sequence specifically recognized for a predetermined Cas protein. Further, the second trigger mRNA may be an mRNA switch having a nucleic acid sequence encoding a second fusion protein and having an aptamer sequence specifically recognized for a predetermined Cas protein. The aptamer sequence contained in the first trigger mRNA and the aptamer sequence contained in the second trigger mRNA may be the same or different. Preferably, the aptamer sequence possessed by the first trigger mRNA is different from the aptamer sequence possessed by the second trigger mRNA, and the first trigger mRNA and the second trigger mRNA are specifically recognized by different Cas proteins. It may be the mode to be performed. Of the first trigger mRNA and the second trigger mRNA, one may be an mRNA having no aptamer sequence and the other may be an mRNA switch.

Whether the first or second trigger mRNA is an mRNA having no aptamer sequence or an mRNA switch, it is a vector encoding the mRNA in place of the first or second trigger mRNA. You may. Hereinafter, the description in the aspect C describes the case where the trigger mRNA is used, but instead of the trigger mRNA, a vector encoding the mRNA can also be used in the same manner.

The protein expression control kit according to Aspect C includes the first and second trigger mRNAs or the vector encoding the trigger mRNA in addition to the mRNA switch described in Aspect A or the vector encoding the mRNA. In addition to these, a drug for optionally promoting heterodimerization may be included in the kit.

In the translation control method (expression control method) using the translation control system (protein expression control system) of the mRNA switch according to the aspect C, the introduction step of the mRNA switch can be carried out in the same manner as in the 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 at the same time, or can be introduced at arbitrary time lag. Further, in a system using a drug for promoting heterodimerization, a step of bringing the drug into contact with cells is included, and this contact step also includes a step of introducing an mRNA switch and a step of introducing first and second trigger mRNAs. It can be carried out at the same time, or it can be carried out at an arbitrary time difference. According to Aspect C, after the input step, the trigger mRNA or the vector encoding the mRNA expresses a first fragment of the input protein and a second fragment of the input protein, both of which have nucleic acid targeting ability (aptamer recognition). When it becomes an aggregate with the ability), the aggregate functions as an input protein, and it becomes possible to control the translation of the mRNA switch. That is, the translation control system (protein expression control system) of the mRNA switch according to the aspect C can control the input protein.

[D. Hierarchical control]
Hierarchical control using an mRNA switch set will be described. An mRNA switch set consists of a plurality of different mRNA switches or vectors encoding the mRNA. Specifically, the mRNA switch set includes n types of mRNA switches consisting of a first mRNA switch to an nth mRNA switch. n represents the type of mRNA switch included in the set, and n is selected from an integer of 2 to 25. The k-th mRNA switch included in the n kinds of mRNA switches is defined as follows.
(A) The kth mRNA switch is
(I) A nucleic acid sequence specifically recognized by an input protein consisting of the kth protein, including the Cas protein or a variant thereof,
(Ii) Containing a nucleic acid sequence encoding an output protein consisting of the (k + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
The kth protein and the (k + 1) th protein are different proteins,
k is an integer from 1 to (n-1).
Further, the nth mRNA switch included in the n kinds of mRNA switches is defined as follows.
(B) The nth mRNA switch is
(I) A nucleic acid sequence specifically recognized by the nth protein, which is the output protein of the (n-1) th mRNA switch, and
(Ii) Containing a nucleic acid sequence encoding an output protein, which is the (n + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
The (n + 1) th protein is any protein.

"K" in the case of the k-th mRNA switch defined in (A) is a variable for defining one kind of mRNA switch included in n kinds of mRNA switches. The k-th mRNA switch receives an input by the k-th 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 (k + 1) mRNA switch in the next layer. The switch set consisting of n types of switches includes (n-1) types of mRNA switches defined in (A), including the first mRNA switch, ..., Up to the (n-1) th mRNA switch. Is done. The first protein serving as the input protein of the first RNA switch may be a protein expressed by another mRNA switch not defined in (A) and (B), and is not a switch (has no aptamer). It may be a protein expressing mRNA (which is not subject to translation control), or it may be a protein other than that.

In the above definition, the fact that the kth protein and the (k + 1) th protein are "different" means that the kth protein and the (k + 1) th protein are derived from different Cas proteins. More specifically, the aptamer of the k-th mRNA switch must be different from the aptamer of the (k + 1) mRNA switch, for example, if the input protein is the first Cas protein or a variant thereof. In some cases, the output protein is a second Cas protein or a variant thereof, and the first Cas protein and the second Cas protein need to be different.

The nth mRNA switch defined in (B) is controlled by the nth protein encoded by the previous layer (n-1) mRNA, and outputs the (n + 1) th protein. The (n + 1) th protein can also be designed to have no effect on the other mRNA switches that make up 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.

In the design of such a switch set, it is necessary to select a total (n + 1) type of protein from the first protein to the (n + 1) th protein. Of these, the first to nth proteins, which can be input proteins, are preferably all different. Here, the fact that the first protein to the nth protein are all different means that the n kinds of proteins are all derived from different Cas proteins. Then, each 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.

For example, when n is 2, the switch set is composed of two types of mRNA switches, and the first mRNA switch is a nucleic acid sequence specifically recognized by the first protein and a nucleic acid encoding the second protein. Includes sequences. The second mRNA switch comprises 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 kinds of proteins, a first protein, a second protein, and a third protein. These three types can be selected from the above-mentioned 25 types of Cas proteins, and the third protein does not have to be a Cas protein.

In some embodiments, the mRNA switch set may be a switch set for configuring a cascade circuit. In the mRNA switch set for a cascade circuit, the nth mRNA switch is an output-only mRNA switch, and encodes a protein that does not translate and control other mRNA switches. The protein that does not translate and control other mRNA switches may be the marker protein or the like described in detail above. When the switch set according to this embodiment constitutes a cascade circuit, the expression of the gene to be expressed (the gene of the (n + 1) protein output by the nth mRNA switch) is conditioned on the presence of the intracellular substance. Can be attached. More specifically, it is possible to construct a circuit in which gene expression occurs in a specific miRNA expression pattern.

In some embodiments, the mRNA switch set may be a switch set for configuring an oscillator circuit. In the mRNA switch set for an oscillator circuit, the (n + 1) th protein output by the nth mRNA switch is the first protein that serves as the input protein of the first mRNA switch. When the switch set according to this embodiment constitutes an oscillator circuit, it is possible to reprogram cells, control the expression timing of therapeutic effect genes, and the like.

As a further application form of the 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, the protein output by one mRNA switch may be the input protein of two or more other mRNA switches, and there may be a switch set containing a self-regulating mRNA in which the input protein and the output protein are the same.

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

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

According to the imaging method according to this aspect, a protein that enables imaging can be bound to an RNA molecule using a Cas protein, and the behavior of the RNA molecule can be observed.

[F. Simultaneous regulation 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. As the input protein according to this embodiment, a protein that functions as a part of the transcriptional regulatory protein is used. Therefore, it is preferable to use a fusion protein in which a transcriptional activation or transcriptional repressor is fused with an inactivated Cas protein as an input protein. With reference to the central part of the panel (a) of FIG. 16, dSpCas9-VPR capable of transcriptional activation is exemplified as an example of the input protein.

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

The vector comprising the transcription control sequence contains the transcription control sequence, the promoter sequence, and the nucleic acid sequence encoding the second output protein in the 5'to 3'direction. The transcription control sequence is a nucleic acid sequence specifically recognized by the crRNA or sgRNA sequence corresponding to the Cas protein contained in the input protein. The second output protein is a protein different from the first output protein. With reference to the right side of the panel (a) of FIG. 16, as a vector having a transcription control sequence, a vector having a gRNA binding sequence corresponding to dSpCas9-VPR and having a second output protein of hmAG1 is exemplified.

The protein expression control kit for carrying out the translation control system of Aspect F consists of at least one component (first component) selected from the group consisting of an input protein, a trigger mRNA, and a trigger plasmid, and an mRNA switch or switch vector. A group consisting of at least one component (second component) selected from the group, a vector having a transcription control sequence (third component), crRNA or sgRNA corresponding to the input protein, or a vector encoding these RNAs. It may contain at least one component (fourth component) selected from. The input protein derived from the first component and the low molecular weight RNA derived from the fourth component form a complex and function as a transcription control protein. Also in the F aspect, 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 the previous aspect A.

[G. Half subtractor]
A system for realizing a half subtractor circuit using three transcription control switch vectors will be described with reference to FIGS. 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 input proteins a and b use proteins that also function as transcription control proteins. With reference to FIG. 18A, dSpCas9-VPR is exemplified as the input protein a and dSaCas9-VPR is exemplified as the input protein b.

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

The transcription control switch vector b is a nucleic acid encoding a transcription control sequence controlled by a transcription control protein containing a complex of an input protein b and a corresponding crRNA or sgRNA, and an mRNA switch translationally controlled by the input protein a. It comprises a sequence, 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 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 includes, as an input RNA, a nucleic acid sequence encoding an sgRNA sequence corresponding to the input protein b. Referring to the middle part of FIG. 18A, the transcription control switch vector b is a binding sequence of a transcription control protein whose transcription control sequence contains dSaCas9-VPR, and the mRNA switch encodes the sgRNA sequence corresponding to dSpCas9-VPR and tagBFP. Includes arrays. In addition, the transcription control switch vector b further contains a sequence encoding an sgRNA corresponding to dSaCas9-VPR.

The transcription control switch vector c is a nucleic acid encoding a transcription control sequence controlled by a transcription control protein containing a complex of an input protein b and a corresponding crRNA or sgRNA, and an mRNA switch translationally controlled by the input protein a. It comprises a sequence and a nucleic acid sequence encoding a second output protein. Referring to the lower part of FIG. 18A, the transcription control switch vector c is a binding sequence of a transcription control protein whose transcription control sequence contains dSaCas9-VPR, and the mRNA switch encodes the sgRNA sequence corresponding to dSpCas9-VPR and hmAG1. Includes arrays.

By combining these input proteins a and b with the transcription control switch vectors a, b and c, when the input protein a does not exist and the input protein b exists, the first output protein and the second protein Both 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, the half subtractor circuit can be constructed.

The protein expression control kit for carrying out the translation control system of Aspect G corresponds to at least one component (first component) selected from the group consisting of input protein a, trigger mRNA, and trigger plasmid, and input protein a. At least one component (second component) selected from the group consisting of crRNA or sgRNA, or a vector encoding these RNAs, and at least one selected from the group consisting of input protein b, trigger mRNA, and trigger plasmid. At least one component (fourth component) selected from the group consisting of a component (third component), crRNA or sgRNA corresponding to the input protein b, or a vector encoding these RNAs, and a transcription control switch vector a ( The fifth component), b (sixth component), and c (seventh component) may be included. The crRNA or sgRNA that binds to the transcription control sequence of the transcription control switch vector c (7th component) is generated by the transcription control switch vector b (6th component). Also in aspect G, the steps of introducing the transcription control switch vectors a, b, and c and the steps of inputting the first to fourth components can be carried out as described in the previous aspect A.

Any combination of the above aspects A to D and F, G is also possible. This makes it possible to construct an artificial circuit controlled by a plurality of mRNA switches capable of inputting by a plurality of different input proteins.

The present invention, in certain embodiments, is a cell comprising 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 artificial circuits in cells containing mRNA switches is disclosed in, for example, Australia et al., Nature volume 487, pages 123-127 (2012) and Kitada et al., Science 2018 Feb 9; 359 (6376). Has been done. Similarly, the mRNA switch according to the present invention can be contained in cells to function. In particular, in addition to the mRNA switch, the desired artificial circuit that functions inside the cell can be obtained by incorporating the nucleic acid or protein that is a component of the system described in the above aspects A to D, F, and G into the cell. Can be done. Such cells are useful as cell preparations.

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 in which an mRNA switch is attached to a desired carrier, dried and supported. For the artificial circuit to function using paper as a carrier, a desired artificial circuit that functions can be obtained by placing the carrier on which such an RNA switch is supported under predetermined translatable conditions. Examples of the carrier include, but are not limited to, paper, plastic, porous material, fiber and the like. For example, it is disclosed in Pardee et al., Cell 159, 940-954, November 6, 2014. In the present invention, a desired artificial circuit can be obtained by attaching a nucleic acid or protein which is a component of the system described in the above aspects A to E to a carrier in addition to the mRNA switch.

Hereinafter, a more detailed description will be given using an embodiment of the present invention. The following examples are not limited to the present invention.

[experimental method]
[Plasid construction]
Preparation of switch plasmid First, pAptamerCassette-EGFP was cleaved with restriction enzymes AgeI and BamHI. Next, the single-stranded synthetic DNA oligo shown in Table 3 was converted to double-stranded DNA by annealing, and this was inserted into the cleaved pAptamerCassette-EGFP.

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

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

[Preparation of plasmid for multi-circuit construction]
First, the ORF of each Cas protein was amplified using the primer set shown in Table 6. Subsequently, using the primer sets shown in Table 6, inverse PCR was performed using each switch plasmid as a template, and a linear plasmid backbone containing a region other than the ORF was amplified. Finally, the ORF of the Cas protein was inserted into the plasmid backbone using the In-Fusion HD Cloning Kit (Clontech).

  

  

The plasmid for transfection into cultured cells was mass-purified using a commercially available Midiprep kit (QIAGEN or Promega).

[Building IVT template]
The 5'-UTR and 3'-UTR sequences for the production of the SpCas9 protein coding region (ORF), SpCas9 mRNA and reference mRNA were PCR amplified from plasmids or oligo DNA using appropriate primers. Table 7 shows the plasmid used as a template and the primers used, and the oligo DNA used as a template and the primers used.

To prepare IVT templates for trigger mRNA and reference mRNA synthesis, 5'-UTR fragments, 3'-UTR fragments, and ORFs were ligated by PCR amplification using the T7FwdA and Rev120A primer sets shown in Table 8. Reference mRNA refers to mRNA that expresses a protein that is translated and encoded without being subject to translational control.

In order to prepare an IVT template for switch mRNA synthesis, the region containing 5'-UTR and ORF was amplified using the SpCas9 response switch plasmid as a template using the primers shown in Table 8. The 3'-UTR fragment amplification products were then ligated by PCR amplification using the primer set in Table 8. The ORFs listed in SEQ ID NOs: 1 to 25 and SEQ ID NOs: 239 to 252 were used.

The PCR product was purified using MinElute PCR purification kit (QIAGEN) according to the manual attached to the kit. The product PCR-amplified using the plasmid as a template was incubated with DpnI (TOYOBO) at 37 ° C for 30 minutes before purification to digest the plasmid.

[Synthesis and purification of mRNA]
mRNA synthesis was performed using the MEGA script T7 Transcription Kit (Ambion). In this reaction, except for switch mRNA, pseudouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate (TriLink Bio Technologies) were used instead of uridine triphosphate and cytidine triphosphate, respectively. did. The guanosine triphosphate used was diluted 5-fold 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 the mixture was further subjected to constant temperature treatment 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 constant temperature treatment at 37 ° C for 30 minutes using Antarctic Phosphatase (New England Biolabs). It was then purified using the RNeasy MinElute Cleanup Kit (QIAGEN) or Monarch RNA Cleanup kit (New England Biolabs).

[Cell culture]
293FT cells used 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, 5% CO _It was cultured under two conditions.

[Plasmid transfection]
293FT cells were seeded in 24-well, 96-well or 384-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. The switch plasmid and the trigger plasmid were mixed in Opti-MEM (Invitrogen) so as to have a mass ratio of 1: 4. In the split Cas9 test, a 4-fold amount of plasmid expressing each fragment was introduced into the switch. In the drug responsiveness test, A / C Heterodimerizer (Clontech) was added to the medium so that the final concentration was 0.5 uM at least 1 hour before transfection. In the multi-layer circuit experiment, each plasmid was co-introduced so that layer 0: 1: 2: 3: 4: 5 = 1: 4: 4: 16: 64: 256. The iRFP670 expression plasmid was used as a transfection control in all experiments.

[MRNA transfection]
The cultured cells were seeded on a 24-well plate, and the synthesized mRNA was introduced the next day. The introduction was performed using 1 μL of Lipofectamine messengerMAX (Invitrogen) according to the attached manual. Switch mRNA 100 ng, trigger mRNA 100 ng, and reference mRNA 100 ng were co-introduced into 293FT cells.

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

[Flow cytometry]
The day after transfection, cells were separated from the plate, passed through a mesh and analyzed by flow cytometry. In experiments with 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP was detected by FL1 (530/30 nm) filters. iRFP670 was detected by FL4 (675 / 12.5 nm) filters. Dead cells and debris were excluded by anterior and lateral light scattering signals. For the calculation of translation efficiency using a plasmid, among living cells, those having a fluorescence value of iRFP670 above a certain level were analyzed.

[result]
[MRNA switch design]
The mRNA switch was constructed by inserting a nucleic acid sequence (aptamer sequence) specifically recognized by an input protein into the 5'-UTR of mRNA encoding an arbitrary gene. Here, the sequence of a known crRNA (CRISPR RNA) or sgRNA (single guide RNA; chimeric RNA of crRNA and trans-activating crRNA (tracrRNA)) corresponding to each Cas protein that is an input protein is regarded as an aptamer sequence. Designed the switch. Each Cas protein recognizes and binds to a unique crRNA / sgRNA embedded in mRNA, which causes translational repression (Fig. 1).

[Functional evaluation of Cas protein-responsive mRNA switch]
Based on the above design, a plasmid (switch plasmid) expressing an mRNA switch using GFP as an output protein was prepared, and a plasmid (trigger plasmid) expressing the Cas protein, which is an input protein, and iRFP670, which is a transfection control, were expressed. It was introduced into 293FT cells together with the plasmid (reference plasmid) to be used. Here, first, 10 types of Cas proteins that have been reported to have RNA-induced nucleic acid targeting ability in human cells were selected. FIG. 2 is a graph showing the translation efficiency of each switch plasmid. The translation efficiency was determined by first dividing the fluorescence intensity of GFP by the fluorescence intensity of iRFP670 for each switch, and then dividing the value at the time of introduction of the trigger plasmid by the value at the time of non-introduction. In addition, the control switch plasmid (No aptamer) was used as a reference for comparison. The results are shown in FIGS. 2A to 2J. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). Nine switches other than the NmCas9-responsive mRNA switch showed high translational repression (minimum repression rate of about 80%). It was found that mRNA switches can be produced with high probability by using Cas protein and the corresponding crRNA / sgRNA.

[Manufacturing of NmCas9 response switch by forward engineering (1)]
For the NmCas9 response switch for which translational repression could not be observed, we examined whether its function could be improved from an RNA engineering approach. Since multiple designs of NmCas9 sgRNA have been reported, we examined whether gRNAs with these different gRNA sequences or their composite designs could function as switches. In addition to the gRNA sequence (Nm_gRNA) used in the first validation experiment, a new design and gRNA sequence was tested (Nm_gRNA_v1-5). Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). As a result, inhibition was observed in Nm_gRNA_v3 and v4 (Fig. 3). It was shown that even if the translation inhibitory ability is weak, functional improvement can be expected by examining the sequence of the sgRNA to be inserted.

[SpCas9 response switch suppresses reporter expression by translation control]
Many Cas proteins are originally used for genome editing targeting intracellular DNA. In addition, since artificial RNA containing a gRNA sequence is co-introduced, the expression of the reporter may be suppressed by an unintended genome editing effect. Therefore, we used SpCas9 to verify whether the expression suppression of the reporter was truly caused by translational suppression. The same for wild-type (with nuclear localization signal (NLS)), mutants for DNA-cleaving activity (nickase and null for DNA-cleaving), and SpCas9 without NLS, respectively, introduced as input proteins. The expression levels of the reporters were compared (Fig. 4A). As a result, no significant difference in reporter expression was observed under all conditions. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). Therefore, the suppression of reporter expression by SpCas9 is likely to be caused by translational repression. Furthermore, we also examined whether co-introduction of the anti-CRISPR protein (AcrIIA4), which inhibits the DNA binding ability of SpCas9, inhibits the suppression of reporter expression. As the input protein, the same protein as the wild type (SpCas9 (WT)) shown in FIG. 4A was used. As a result, the introduction of AcrIIA4 did not significantly affect the suppression of reporter expression (Fig. 4B). Therefore, the suppression of reporter expression by SpCas9 is not caused by the DNA binding of SpCas9, and it is supported that it is an effect by translation suppression.

[Verification of translation inhibitory effect of Cas protein without cleavage]
Some Cas proteins bind to a pre-crRNA in which a plurality of crRNAs are linked, and the crRNAs are appropriately excised and used by themselves. When a switch is prepared using such a Cas protein, the binding of the Cas protein to the crRNA in the mRNA may cleave / degrade the mRNA, resulting in translational repression. This can be a drawback if you want to achieve translational repression without degradation of the mRNA switch. Whether or not mRNA cleavage is essential for translational repression was examined using AsCas12a (AsCpf1), which is one of the Cas proteins reported to have pre-crRNA cleavage activity. It has been reported that the AsCas12a (H800A) mutant can bind to crRNA itself but has no pre-crRNA processing ability. We investigated whether this mutant could be used to suppress translation without mRNA degradation by Cas12a. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). As a result, it was suggested that the Cas12a mutant has a high translational repression ability (about 80%), although the translational repression efficiency is slightly lower than that of the wild-type Cas12a (Fig. 5). This result suggests that the binding of crRNA to Cas protein itself is important in the translational repression by Cas protein bound to crRNA on mRNA, and does not necessarily require the cleavage activity of crRNA. That is, the Cas protein-responsive mRNA switch can be used for both translational repression with and without mRNA degradation.

[Cas protein response RNA inverter]
The mRNA switch is an OFF switch that suppresses its own translation in the presence of the target protein, and an RNA inverter that converts this OFF switch into an ON switch (induces its own translation in the presence of the target protein) has been developed. Construction of an RNA inverter can be achieved by inserting an aptamer sequence on the 5'-UTR of mRNA, similar to the OFF switch. The ON switch is constructed by inserting a special RNA inverter sequence that converts the output between the 5'-UTR of the OFF switch and the reporter gene. Therefore, it was verified whether the Cas-responsive mRNA switch could be converted to an ON switch using an RNA inverter in the same manner. SpCas9, SaCas9, St1Cas9, CjCas9, AsCas12a and its mutant AsCas12a (H800A), and PspCas13b were selected from the Cas proteins that showed translational repression, and an ON switch equipped with crRNA / sgRNA was prepared. As the RNA inverter sequence, those shown in SEQ ID NOs: 64 and 67 were used. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments, pspCas13b only n = 2). As a result, we succeeded in constructing an RNA inverter capable of promoting translation in Cas proteins other than PspCas13b (FIGS. 6A to 6F). Since PspCas13b has pre-crRNA processing ability, it is considered that this cleavage causes mRNA degradation and makes it impossible to promote translation (Fig. 6B). On the other hand, translation promotion was observed in AsCas12a, which has a pre-crRNA processing ability similar to PspCas13b (Fig. 6F). This difference is expected to be due to the difference in RNA cleavage positions when the Cas protein processes pre-crRNA. PspCas13b is thought to cleave RNA on the 3'side of the binding region, whereas AsCas12a cleaves the 5'side. Therefore, the PspCas13b-crRNA complex is cleaved from the mRNA, resulting in an exposed mRNA at the 5'end. As a result, mRNA degradation is promoted, and an ON switch for PspCas13b response cannot be prepared. On the other hand, AsCas12a cleaves the 5'side of the mRNA, so it is expected that it will continue to bind to the end of the mRNA. Therefore, it is considered that the degradation of mRNA is suppressed, and as a result, the RNA inverter functions. From the above, it is highly possible that a Cas protein-responsive RNA inverter can be produced except for a Cas protein that cleaves the 3'side of RNA from a binding site such as Cas13b.

[Functional evaluation of Cas protein-responsive mRNA switch by RNA transfer method]
We verified whether the Cas protein-responsive mRNA switch works even if mRNA is directly introduced into cultured cells instead of plasmid. Here, a SpCas9-responsive mRNA switch was used as a representative. A switch mRNA, a trigger mRNA expressing SpCas9 (input protein), and a reference mRNA (iRFP670 mRNA) for confirming whether a series of mRNAs were introduced into the cells were co-introduced into 293FT cells. The upper panel of FIG. 7A is a flow cytometric plot of changes in switch fluorescence with and without trigger mRNA introduction. It was confirmed that the population moved downward by the introduction of the trigger mRNA. The lower panel of FIG. 7A is a histogram showing the difference in the translation efficiency of mRNA with and without the introduction of trigger mRNA. In FIG. 7B, the translation efficiency was determined by first dividing the fluorescence intensity of EGFP by the fluorescence intensity of iRFP670, and then dividing the value at the time of introducing the trigger mRNA by the value at the time of non-introduction. It was observed that the introduction of SpCas9 specifically suppressed translation from switch mRNA. Therefore, it was shown that the Cas protein-responsive mRNA switch also functions by the mRNA transfer method.

[Expansion of Cas protein response mRNA switch]
In addition to the Cas protein tested in FIG. 2, it was verified whether 15 kinds of Cas proteins function as translational suppressors of the mRNA switch. Here, in addition to FnCas9, St3Cas9, LbCas12a, FnCas12a, MbCas12a, which have nucleic acid targeting ability in human cells, CdCas9, ClCas9, NcCs9, SpaCas9, PlCas9, which are considered to be inactive or very low, and recently reported. The Cas12b family, Cas14a1 and CasX (PlmCasX), were used. If multiple sgRNAs were reported for each Cas protein, each sgRNA sequence was used to validate the effect. As a result, it was shown that 13 of these 15 Cas proteins can achieve translational repression by using appropriate crRNA / sgRNA as an aptamer (FIGS. 8A to O). From this result, it was suggested that the approach of turning off the switch by embedding crRNA / sgRNA in 5'-UTR can be applied with high probability to the Cas protein newly discovered in the future.

[Expansion of Cas protein response translation ON switch]
In addition to the additionally expanded mRNA switch, the translation ON switch was further expanded based on the knowledge gained from the development of the ON switch using the RNA inverter sequence described above. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). An ON switch could be developed for all Cas proteins tested (FIGS. 9A-G). In addition, some CasRx response switches, such as CasRx response switches, were found to show a high translation ON effect by using an RNA inverter, even if the translation suppression effect was inferior to that of others (Fig. 9A). Therefore, as a translation suppression switch, even a less promising Cas protein can be a useful translation control switch by using an RNA inverter sequence.

[Translation programming based on protein engineering 1]
We tested whether translation control could be programmed using existing engineering for Cas proteins. Here, using SpCas9, which is often used for modification, we examined whether NAND-gate type translation control that suppresses translation only when two input molecules (input A and B) are present at the same time is possible. In order to realize NAND-gate type translation control, we tried to use split Cas9, which is a Cas9 protein divided into two regions. It is known that when SpCas9 is divided between the 713th residue and the 714th residue, the nucleic acid targeting ability is restored when each fragment is associated. Here, in order to improve the meeting efficiency, a separate intein was introduced in each fragment. Intein is the name of a protein portion that is excised by a phenomenon called protein splicing. Proteins containing intein autonomously excise their intein sites and then rebind the remaining parts. Among them, isolated intein is contained as N-intein or C-intein in two proteins translated as separate proteins. When these two proteins are associated via intein, the intein portion is excised to produce a protein in which the two proteins are neatly fused. That is, 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, Fig. 10A, FIG. 10B). To substantiate the above hypothesis, each split protein expression plasmid was prepared and co-introduced into 293FT cells with a switch plasmid and a reference plasmid. As a result, as intended, only when both Cas9 fragments were introduced ([1,1]), translation suppression equivalent to that when full-length Cas9 (WT) was introduced was realized (Fig. 10C). That is, NAND gated translation control can be achieved by using split Cas9 containing inteins (FIGS. 10B, 10C).

[Translation programming based on protein engineering 2]
If the function control of the mRNA switch can be achieved by the drug, the timing and duration of translation control can be easily manipulated not only at the cultured cell level but also in biological use. So far, a system has been reported in which split Cas9 is associated by drug induction to restore nucleic acid targeting ability. By applying this mechanism, we thought that it would be possible to construct a system that induces translation suppression only when a drug is added. Here, a commercially available iDimerize Inducible Heterodimer System was introduced. Split between the 174th and 175th residues (Split1), between the 535th and 536th residues (Split2), or between the 713th and 714th residues (Split3) Split Cas9 fused to the DmrA and DmrC binding domains was prepared at the C-terminal or N-terminal of each fragment of SpCas9 (SpCas9 (N-term) and SpCas9 (C-term)) (Fig. 11A, Split 3 as a representative example). Described). 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 remarkably suppressed in the presence of split Cas9 only when the drug was added in Splits 2 and 3 (Fig. 11B). The results so far (FIGS. 10 and 11) have shown that translation programming using mRNA switches can be successfully achieved by applying existing protein engineering findings for Cas9.

[Translation control by new CRISPR system]
In recent years, as a new anti-CRISPR protein, one that inhibits the binding between Cas protein and gRNA has been reported. The anti-CRISPR protein AcrIIC2 for NmCas9 is known to bind to Cas9 and inhibit its binding to gRNA. AcrIIC2 has also been reported to be able to bind to several Cas9 families, 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 the translational repression by Cas9 protein in the presence of AcrIIC2. From our results so far, the NmCas9 response switch did not show sufficient translational repression ability. On the other hand, it has been suggested that SaCas9 can suppress translation very efficiently. Here, we tried to build a translation control system by utilizing the fact that AcrIIC2 can also bind to SaCas9. For 100 ng of switch plasmid, 200 ng of SaCas9 plasmid and 100 ng of iRFP670 plasmid were introduced into 293FT cells. At this time, a 0-10 times amount of the AcrIIC2 expression plasmid was also co-introduced with respect to the SaCas9 plasmid. Error bars indicate mean ± standard deviation (n = 3, 3 independent experiments). As a result, it was suggested that AcrIIC2 released the translational repression by SaCas9 (Fig. 12). Therefore, it is considered that the translation control method using Cas protein can also utilize various CRISPR systems that will be discovered in the future.

[Orthogonality between Cas protein response OFF switch mRNA]
The orthogonality was verified when constructing a circuit combining the Cas protein response OFF switch mRNA. First, the orthogonality between the nine types of Cas protein-responsive mRNA switches showing translational repressive ability was verified in FIGS. 2A to C and E to 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 (FIGS. 13A and 13B). Table 9 shows the results of numerically displaying the translation efficiency of FIG. 13B.

Furthermore, in Fig. 6, the orthogonality between 7 types of newly expanded Cas protein response OFF switch mRNA and a total of 15 types of protein response mRNA switches including the previously developed L7Ae response mRNA switch was verified. Crosstalk was observed between Cas12a, but no significant crosstalk was observed in the other combinations (Fig. 13C). Therefore, a set having orthogonality between 12 kinds of protein-responsive mRNA switches, which is the largest number ever, has been developed.

[Construction of multi-layer circuit]
We verified whether it is possible to construct a multi-layer circuit as a circuit that combines Cas protein response OFF switch mRNA. A multi-layer circuit is constructed by hierarchically combining multiple OFF switch mRNAs. An input protein that triggers one switch is expressed as an output protein from an OFF switch that responds to another trigger protein. Since the protein output from a certain switch becomes the input protein of the switch one layer downstream, the expression of the output gene (here, GFP) changes from ON to OFF to ON with each layer (FIG. 14A). .. Here, a multi-layer circuit was constructed using the five switches that showed strong translation suppression in Fig. 2. FIG. 14B shows fluorescence micrographs at each layer level, histograms obtained from flow cytometric analysis, and calculated output levels. As the layer level increased, the overall responsiveness deteriorated, but the output change (ON → OFF → ON → OFF → ON → OFF) was observed as intended.

[Plasid construction]
[Preparation of switch plasmid]
pAptamerCassette-tagRFP2 was cleaved with restriction enzymes AgeI and BamHI. Next, the single-stranded synthetic DNA oligos shown in SEQ ID NOs: 69, 70, or SEQ ID NOs: 73, 74 were converted into double-stranded DNA by annealing, and the DNA was inserted into the cleaved pAptamerCassette-tagRFP2.

[Preparation of VPR fusion trigger plasmid]
In order 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 delete nuclease activity (10). Replace the second D with A and the 580th N with A). The KOD Mutagenesis Kit (Takara) was used for mutagenesis. The primers shown in Table 10 were used. Then, the plasmid backbone containing the ORF of SaCas9 was amplified by inverse PCR. Finally, the PCR-amplified VPR region 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.

[Preparation of reporter plasmid for transcription control]
The reporter plasmid pTRE-Tight-hmAG1 for transcription control was prepared as follows. The 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. did. The DNA fragment was inserted between the EcoRI site and the EcoRV site of pTRE-Tight (Clontech) to generate pTRE-Tight-hmAG1.

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, the DNA fragment containing the target gene EGFP was amplified by PCR using the forward primer (SEQ ID NO: 263) and the 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.

Subsequently, pHL-gRNA [TRE] -mEF1α-mRFP-RIH was prepared from pHL-gRNA [EGFP] -mEF1α-mRFP-RIH. That is, the 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 site and the EcoRI site of pHL-gRNA [EGFP] -mEF1α-mRFP-RIH to generate pHL-gRNA [TRE] -mEF1α-mRFP-RIH.

Finally, pHL-gRNA [TRE] -mEF1α-iRFP-RIH was prepared from pHL-gRNA [TRE] -mEF1α-mRFP-RIH. In other words, the DNA fragment containing iRFP670 is 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) Amplification was performed by PCR using (SEQ ID NO: 266) and reverse plasmid (SEQ ID NO: 267). The DNA fragment was inserted between the EcoRV site and the AvrII site of pHL-gRNA [TRE] -mEF1α-mRFP-RIH to generate pHL-gRNA [TRE] -mEF1α-iRFP-RIH.

The primer sets used are shown in Table 11. Target sequences are underlined.

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

Next, MALAT1 triplet, 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 this end, the plasmid backbone was first amplified by inverse PCR using pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP as a template. Subsequently, the amplified plasmid backbone and double-stranded DNA containing MALAT1 triplet, HH ribozyme, SaCas9 gRNA, and HDV ribozyme were ligated using In-Fusion HD Cloning Kit (Clontech). Finally, by cleaving this plasmid with XbaI and AgeI and ligating it with a double-stranded DNA having the SaCas9 gRNA binding sequence, the minimum CMV promoter, and the SpCas9 response switch sequence, the SaCas9 gRNA binding sequence, the minimum CMV promoter, A plasmid (pSa_IgRNA_a-CMVmin-Gluc_Sp_gRNA-tagBFP-Triplex-HHR-Sa_gRNA [TRE] -HDVR) having a SpCas9 response switch sequence and a SaCas9 gRNA in 3'-UTR was prepared. The sequences of the primers used are shown in Table 12.

For the SaCas9 response switch expression plasmid (pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP) into which the above-mentioned minimum CMV promoter and SpCas9 gRNA binding site were introduced, the number of SpCas9 gRNA binding sites was doubled (pSp_IgRNA_ax2-CMVmin-Gluc_Sa_gRNA-tagBFP). First, this plasmid was cleaved with SpeI and EcoRI. Next, the double-stranded DNA containing the SpCas9 gRNA binding site was amplified by PCR and cleaved by XbaI and EcoRI. Finally, the plasmid backbone and DNA fragment were ligated.

The sequences of the primers used are shown in Table 12.

In order 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 converted to double-stranded DNA by annealing, and it was converted into double-stranded DNA by annealing, which was converted into double-stranded DNA by BamHI and EcoRI. It was ligated with pHL-gRNA [TRE] -iRFP-RIH cleaved in.

In order to prepare a plasmid vector (pTRE-Tight-Gluc_Sp_gRNA-hmAG1) that expresses a switch in which transcription is activated by SaCas9 fused with VPR and translation of hmAG1 is suppressed by SpCas9, the hmAG1 expression vector was first subjected to inverse PCR. Amplified the backbone. 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 on the 5'side of hmAG1. The sequences of the primers used are shown in Table 12.

[Preparation of plasmid for AND circuit construction]
Conforms to the preparation of plasmids for building multiple circuits. First, the ORF of each Cas protein was amplified using the primer set shown in Table 13. Subsequently, using the primer set used in the preparation of the plasmid for constructing a multiple circuit, inverse PCR was performed using each switch plasmid as a template, and the linear plasmid backbone containing the region other than the ORF was amplified. Finally, the ORF of the Cas protein was inserted into the plasmid backbone using the In-Fusion HD Cloning Kit (Clontech).

The plasmid for transfection into cultured cells was mass-purified using a commercially available Midiprep kit (QIAGEN or Promega).

[Plasmid transfection]
[Confirmation of orthogonality between OFF switch and ON switch]
293FT cells were seeded in 384-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. The switch plasmid and the trigger plasmid were mixed in Opti-MEM (Invitrogen) so as to have a mass ratio of 1: 4. The iRFP670 expression plasmid was used as a transfection control.

[Simultaneous control of translation and transcription]
293FT cells were seeded in 24-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. 100 ng switch plasmid (pGluc-Sp_gRNA-tagRFP) and 400 ng trigger plasmid (Sp_dCas9-VPR (Addgene: plasmid # 63798) or pcDNA3.1 + -myc-HisA (control)), 100 ng 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). A 100 ng tagBFP expression plasmid (pAptamerCassette-tagBFP) was also used as a transfection control.

[AND gate]
293FT cells were seeded in 96-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. A 6.25 ng switch plasmid, a 50 ng trigger plasmid each, and a 25 ng mediator plasmid each were mixed in Opti-MEM (Invitrogen). A 25 ng iRFP670 expression plasmid was also used as a transfection control.

[Half subtractor]
293FT cells were seeded in 24-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. Trigger plasmids (400 ng each), hmAG1 expression plasmids 400 ng, TagBFP expression plasmids (800 ng or 400 ng each), and gRNA expression plasmids (100 ng each) were mixed in Opti-MEM (Invitrogen).

[Manufacturing of NmCas9 response switch by forward engineering (2)]
293FT cells were seeded in 24-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. The switch plasmid and the trigger plasmid were mixed in Opti-MEM (Invitrogen) so as to have a mass ratio of 1: 4. The iRFP670 expression plasmid was used as a transfection control.

[Simultaneous drive of ON switch and OFF switch]
293FT cells were seeded in 24-well plates 24 hours prior to transfection. The plasmid was introduced using Lipofectamine 2000 (Invitrogen) according to the accompanying manual. Each 100 ng switch plasmid and 400 ng trigger plasmid (pcDNA3.1-SaCas9 or pcDNA3.1 + -myc-HisA (control)) were mixed in Opti-MEM (Invitrogen). A 100 ng iRFP670 expression plasmid was also used as a transfection control.

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

[Flow cytometry]
The day after transfection, cells were separated from the plate, passed through a mesh and analyzed by flow cytometry. Accuri C6 (BD Biosciences) was used in experiments with 24-well and 96-well plates. EGFP was detected by FL1 (530/30 nm) filters. iRFP670 was detected by FL4 (675 / 12.5 nm) filters. Dead cells and debris were excluded by anterior and lateral light scattering signals. For the calculation of translation efficiency using a plasmid, among living cells, those having a fluorescence value of iRFP670 above a certain level were analyzed.

[result]
[Checking the orthogonality of the OFF switch]
The orthogonality of 29 types of OFF switch mRNA was tested. The panel (a) of FIG. 15A is a fluorescence photograph showing the results of the orthogonality test. The mRNA switches are 25 types of Cas response switches including the newly developed AaCas12b response mRNA switch, 3 types of protein response type switches prepared in the past, and 1 type of control switch. BsCas12b switch was not included. Panel (b) is a manifestation of 13 of the Cas-responsive mRNA switches tested that showed clear orthogonality. FIG. 15B is a heat map showing the image quantification results of the 13 types of Cas response mRNA switches.

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

FIG. 16 panel (b) shows cell photographs when dSpCas9-VPR was introduced (+) and when dSpCas9-VPR was not introduced (-). Translation (red fluorescence) was suppressed and transcription (green fluorescence) was activated only when the dSpCas9-VPR protein was introduced.

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

In order to test the constructability in the circuit of the Cas response mRNA switch, 6 types of Cas response mRNA switches were comprehensively combined and 60 arrangements were tested. Table 14 shows the truth table of AND gates. Table 15 shows the heat map of the arrangement of each Cas protein and the expression of the output gene (columns written as 00, 10, 01, 11), and the circuit performance (θ: The smaller the value, the more AND gate-like behavior. The result of summarizing) is shown. In Table 15, Pgu is an abbreviation for PguCas13b, Mb is an abbreviation for MbCas12a, Sa is SaCas9, Ak is an abbreviation for AkCas12b, Psp is an abbreviation for PspCas13b, and Nc is an abbreviation for NcCas9.

[Half subtractor]
By making full use of transcription control and translation control by Cas protein at the same time, a half subtractor was constructed using only two modules. FIG. 18A shows the scheme of the half subtractor. The upper left side represents the complex of SpCas9-VPR and SpCas9-gRNA (A), and the right side contains the target sequence of SpCas9-gRNA (A), and the vector encoding the SaCas responsive tagBFP mRNA switch and this vector are transcribed. The SaCas responsive tagBFP mRNA switch produced by activation is shown. The left side of the middle row represents the complex of SaCas9-VPR and SaCas9-gRNA (B), and the right side has the binding sequence of SaCas9-gRNA (B) and encodes the SpCas-responsive tagBFP mRNA switch and SaCas9-gRNA (C). The vector and the SpCas-responsive tagBFP mRNA switch (top) and SaCas9-gRNA (C) (bottom) produced by transcriptional activation of this vector are shown. The lower left side represents the complex of SaCas9-VPR and SaCas9-gRNA (C), and the right side has a binding sequence of SaCas9-gRNA (C), a vector encoding a SpCas-responsive hmAG1 mRNA switch, and this vector has transcriptional activity. It shows a SaCas-responsive hmAG1 mRNA switch that is generated by the conversion.

FIG. 18B shows the truth value of the half subtractor. FIG. 18C shows a fluorescence micrograph of the cells. From FIG. 18C, as intended, when Input 1 is 1 and Input 2 is 0, and when Input 1 is 0 and Input 2 is 1, tagBFP expression is confirmed, and Input 1 is 0. When Input 2 was 1, expression of hmAG1 was confirmed. Conventionally, a technique for constructing a half subtractor by combining four types of modules has been known, but in the present invention, it was possible to make it function even if the number of modules introduced into cells is small. In other words, it is thought that the modules (biological components) required to construct more half-subtractors can be saved, and the use of internal resources at the time of cell introduction can be reduced.

[Manufacturing of NmCas9 response switch by forward engineering (2)]
In contrast to the preparation of the NmCas9 response switch by forward engineering (1), two additional gRNA sequences were prepared and tested (Nm_gRNA_v1-7). The translation suppression efficiency is shown in FIG. We succeeded in identifying v7, which has a higher translation suppression effect.

[Confirmation of ON switch orthogonality]
The orthogonality of 26 types of ON switch mRNA was tested. FIG. 20 is a fluorescence photograph showing the results of the orthogonality test. As the mRNA switch, 26 types of Cas response ON switch mRNA including the newly developed AaCas12b response ON switch mRNA were used.

[Simultaneous drive of ON switch and OFF switch]
For the SaCas9 response switch, it was tested whether the translation OFF switch (red fluorescence) and the translation ON switch (green fluorescence) could be driven at the same time. FIG. 21 Panel (a) shows a cell photograph, and Panel (b) shows a quantitative result by flow cytometry. The Control ON switch or Control OFF switch was an mRNA having no nucleic acid sequence (aptamer sequence) of (i) and was not affected by the input protein, and the Reference used a transfection control. From FIG. 21, it was shown that it is possible to drive the ON switch and the OFF switch at the same time using a single input protein.

Claims (22)

(I) Nucleic acid sequences specifically recognized by input proteins, including Cas proteins or variants thereof,
(Ii) Containing a nucleic acid sequence encoding an output protein
An mRNA switch comprising an artificial mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked. The mRNA switch according to claim 1, wherein the nucleic acid sequence of (i) is a crRNA or sgRNA sequence of the input protein or a variant thereof. The Cas protein, SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b The mRNA switch according to claim 1 or 2, which is one Cas protein selected from the group consisting of, CasRx, PlmCasX, and Cas14a1. The artificial mRNA molecule further comprises an RNA inverter sequence between the nucleic acid sequences of (i) and (ii), and the RNA inverter sequence is a nucleic acid sequence containing a bait open reading frame, an intron and an internal ribosome entry site. The mRNA switch according to any one of claims 1 to 3, comprising. A vector containing a nucleic acid sequence encoding the mRNA switch according to any one of claims 1 to 4. The vector according to claim 5, further comprising a transcription control sequence provided on the upstream side of the nucleic acid sequence encoding the mRNA switch, wherein the transcription control sequence is a sequence specifically recognized by the transcription control protein. The transcriptional regulatory protein comprises a Cas protein or a variant thereof, further comprising 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. The vector according to claim 6. A cell containing the mRNA switch according to any one of claims 1 to 4 or the vector according to any one of claims 5 to 7. (A) The mRNA switch according to any one of claims 1 to 4 or a vector encoding the mRNA switch; and
(B) A protein expression control kit comprising the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA. (A) The mRNA switch according to any one of claims 1 to 4 or a vector encoding the mRNA switch;
(B) A first trigger mRNA containing a nucleic acid sequence encoding a first fusion protein containing a first fragment of the input protein and a first heterodimerized 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) above and a second heterodimerized domain or said second. A protein expression control kit containing a vector encoding a trigger mRNA of. The expression control kit according to claim 10, further comprising an agent that promotes heterodimerization by the first and second heterodimerization domains. The protein expression control kit according to any one of claims 9 to 11, further comprising an input inhibitor that inhibits the recognition of the nucleic acid sequence of (i) by the input protein. An mRNA switch set containing n types of mRNA switches consisting of a first mRNA switch to an nth mRNA switch or a vector encoding the mRNA switch, in which 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 the kth protein, including the Cas protein or a variant thereof,
(Ii) Containing a nucleic acid sequence encoding an output protein consisting of the (k + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
The kth protein and the (k + 1) th protein are different proteins,
k is an integer from 1 to (n-1) and
(B) The nth mRNA switch is
(I) A nucleic acid sequence specifically recognized by the nth protein, which is the output protein of the (n-1) th mRNA switch, and
(Ii) Containing a nucleic acid sequence encoding an output protein, which is the (n + 1) th protein.
An mRNA molecule in which the (i) is present on the 5'or 3'side of (ii) and the (i) and (ii) are operably linked.
An mRNA switch set in which the (n + 1) th protein is an arbitrary protein. The mRNA switch set according to claim 13, wherein the first protein to the nth protein are all different proteins. The switch set according to claim 13, wherein the first (n + 1) protein is the first input protein. The Cas protein or a variant thereof contained in the k-th and (k + 1) proteins is SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12, AsCas12. , MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmacasX, Cas14a1. The mRNA switch set described in. (A) The mRNA switch according to any one of claims 1 to 4 or a vector encoding the mRNA switch; and
(B) A method for controlling protein expression, which comprises a step of introducing the input protein, a trigger mRNA encoding the input protein, or a vector encoding the trigger mRNA into a cell. (A) The mRNA switch according to any one of claims 1 to 4 or a vector encoding the mRNA switch;
(B) A first trigger mRNA containing a nucleic acid sequence encoding a first fusion protein containing a first fragment of the input protein and a first heterodimerized 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) above and a second heterodimerized domain or said second. A method for controlling protein expression, which comprises the step of introducing a vector encoding the trigger mRNA of the protein into a cell. The method for controlling expression of a protein according to claim 18, further comprising a step of contacting the cells with a drug that promotes heterodimerization by the first and second heterodimerization domains after the introduction step. A step of introducing 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 of (i) by the input protein, into a cell. The method for controlling expression of a protein according to any one of claims 17 to 19, further comprising. A method for controlling protein expression, which comprises the step of introducing the mRNA switch set according to any one of claims 13 to 16 into cells, tissues, or individuals. (A) A nucleic acid encoding a crRNA or sgRNA in which the transcriptional regulatory protein contains a Cas protein or a variant thereof and is provided downstream of the nucleic acid sequence encoding the mRNA switch and specifically recognizes the transcriptional regulatory sequence. The vector according to claim 6, further comprising a sequence; and (b) a Cas protein or a variant thereof corresponding to a nucleic acid sequence encoding a crRNA or sgRNA that specifically recognizes the transcriptional regulatory sequence of (a). A protein expression control kit comprising a transcriptional regulatory protein comprising, a transcriptional activity regulatory mRNA encoding the transcriptional regulatory protein, or a vector encoding the transcriptional activity regulatory mRNA.

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