WO2021112136A1 - Commutateur d'arnm et procédé de régulation de l'expression d'une protéine l'utilisant - Google Patents

Commutateur d'arnm et procédé de régulation de l'expression d'une protéine l'utilisant Download PDF

Info

Publication number
WO2021112136A1
WO2021112136A1 PCT/JP2020/044905 JP2020044905W WO2021112136A1 WO 2021112136 A1 WO2021112136 A1 WO 2021112136A1 JP 2020044905 W JP2020044905 W JP 2020044905W WO 2021112136 A1 WO2021112136 A1 WO 2021112136A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
mrna
switch
input
nucleic acid
Prior art date
Application number
PCT/JP2020/044905
Other languages
English (en)
Japanese (ja)
Inventor
博英 齊藤
俊輔 川崎
萌 弘澤
紘貴 小野
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2021562691A priority Critical patent/JPWO2021112136A1/ja
Publication of WO2021112136A1 publication Critical patent/WO2021112136A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention relates to an mRNA switch and a method for controlling protein expression using the mRNA switch.
  • 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.
  • 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.
  • 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).
  • Non-Patent Documents 1 and 2, Patent Document 3, etc. disclose 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.).
  • the output protein expressed from the first mRNA switch is the input protein of the second mRNA switch
  • 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). ..
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention includes the following aspects.
  • 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 Cas proteins are SpCas9, SaCas9, CjCas9, NmCas9, St1Cas9, FnCas9, CdCas9, ClCas9, PlCas9, NcCas9, SpaCas9, St3Cas9, AsCas12a, FnCas12a, LbCas12a, FnCas12a, LbCas12a
  • 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.
  • 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.
  • [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.
  • 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.
  • 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 switch set in which the (n + 1) th protein is an arbitrary protein.
  • 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.
  • Cas protein selected from the group consisting of FnCas12a, LbCas12a, MbCas12a, AkCas12b, AaCas12b, BvCas12b, BsCas12b, PspCas13b, PguCas13b, RanCas13b, CasRx, PlmCasX, Cas15a1
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • FIG. 1 is a diagram schematically showing
  • 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).
  • Nm_gRNA Nm_gRNA_v1, Nm_gRNA_v2, Nm_gRNA_v3, Nm_gRNA_v4, Nm_gRNA_v5
  • mRNA switches Nm_gRNA, Nm_gRNA_v1, Nm_gRNA_v2, Nm_gRNA_v3, Nm_gRNA_v4, Nm_gRNA_v5
  • It is a graph prepared and compared with the control mRNA (No aptamer) and its translation efficiency in the presence of NmCas9.
  • SpCas9 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 ( ⁇ NLS)] without NLS are used as input proteins, and are specific to each of them in the presence of each input protein.
  • 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.
  • AcrIIA4 anti-CRISPR protein
  • 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.
  • As_crRNA wild-type AsCas12a
  • H800A AsCas12a mutant [Cpf1 (H800A)]
  • 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.
  • 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.
  • 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.
  • 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. 1 shows an OFF switch mRNA 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.
  • 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. 1 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
  • 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. 1 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
  • 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.
  • 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. 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. 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
  • 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.
  • 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. 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 PguCas13b. It is a graph comparing.
  • FIG. 9B shows the translation efficiency between the ON switch
  • 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. 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. 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. 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.
  • 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.
  • 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.
  • N-term N-term
  • C-term SpCas9
  • FIG. 11B shows 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.
  • Switch OFF switch mRNA
  • 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.
  • 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.
  • 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).
  • 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.
  • the present invention relates to an mRNA switch, which is a mRNA switch.
  • 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".
  • 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.
  • 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.
  • 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.
  • FnCas12a Ferancisella novelida U112 derived from Cas12a, alias FnCpf1, alia
  • 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 Cas14a1
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the input protein is SpCas9
  • SpCas9 responsive mRNA when the input protein is SpCas9, it may be referred to as SpCas9 responsive mRNA.
  • 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.
  • the structure and sequencing of RNA molecules constituting the mRNA switch will be described.
  • nucleic acid sequence specifically recognized by an input protein containing a Cas protein or a variant thereof contains a nucleic acid sequence specifically recognized by an input protein containing a Cas protein or a variant thereof.
  • 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 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).
  • 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.
  • Switch name represents the name of the mRNA switch.
  • nucleic acid sequence encoding the output protein 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.
  • 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.
  • the output protein may be the same as or different from the input protein.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 ⁇ -lactamase.
  • 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.
  • marker proteins 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.
  • 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.
  • the nucleic acid sequence of (ii) may encode a localization signal in addition to the marker protein.
  • 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.
  • a localization signal is advantageous when the marker protein is visualized by imaging cytometry or the like.
  • 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.
  • 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.
  • 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.
  • a frame shift can be avoided by adding one or two bases at the end of the sequence.
  • 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.
  • 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.
  • nucleic acid sequence of (i) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • it is a human ⁇ -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.
  • 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.
  • 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.
  • 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.
  • the "cell” is not particularly limited and may be any cell.
  • 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.
  • a mammal for example, human, mouse, monkey, pig, rat, etc.
  • 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.
  • ES embryonic stem
  • ntES embryonic stem
  • GS cells sperm stem cells
  • EG cells Embryonic germ cells
  • iPS induced pluripotent stem
  • 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.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • 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.
  • 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
  • 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
  • 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.
  • T lymphocytes eg, T lymphocytes
  • sensory cells eg, rod cells
  • central / peripheral nervous system nerve cells and glial cells eg, stellate glial cells
  • 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.
  • RNA molecules can be introduced directly into cells using introduction methods such as the method, DEAE dextran method, microinjection method, and gene gun method.
  • introduction methods such as the method, DEAE dextran method, microinjection method, and gene gun method.
  • a DNA construct such as an expression vector can also be used to introduce the mRNA switch into cells.
  • 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.
  • 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.
  • the viral vector include a retrovirus vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, and a Sendai virus vector.
  • the artificial chromosome vector examples include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like.
  • the plasmid vector a general mammalian plasmid can be used, and for example, an episomal vector may be used.
  • the transposon vector include an expression vector using the piggyBac transposon.
  • 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. ..
  • 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.
  • 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.
  • 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.
  • 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.
  • a vector is referred to herein as a transcriptional control switch 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.
  • the transcriptional regulatory protein may be, but is not limited to, TALEN.
  • 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.
  • 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.
  • 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).
  • VPR transcriptional activators
  • it may be a complex containing an inactivated Cas protein fused to the Kruppel associated box domain (KRAB), which is a transcriptional repressor.
  • KRAB Kruppel associated box domain
  • 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.
  • 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.
  • 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.
  • the transcription control switch vector contains a nucleic acid sequence encoding an input RNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the vector that encodes an mRNA switch is also called a switch vector.
  • 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.
  • a step of introducing the mRNA switch or the switch vector into a desired cell 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.
  • an input process 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.
  • 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).
  • the ON switch mRNA the translation of the mRNA switch is performed by the input process and the output protein is expressed (with output)
  • 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).
  • a system that independently controls the translation of two or more mRNA switches, that is, the expression of two or more output proteins.
  • 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.
  • 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.
  • the kit may include the input inhibitor protein itself, an mRNA encoding the input inhibitor protein, or a vector encoding the mRNA.
  • 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.
  • the mRNA encoding the input inhibitory protein may be an mRNA switch. This is referred to as an input-inhibiting mRNA switch.
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid targeting ability aptamer recognition ability
  • Fragmented input proteins can be designed based on the 25 Cas proteins listed above and other Cas proteins.
  • 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.
  • 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.
  • the heterodimerized domain is the isolated intein
  • 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. ..
  • 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.
  • heterodimerized domain is a domain that causes association in the presence of a drug consisting of a specific small molecule compound, and iDimerize TM Inducible Heterodimer System (Clontech) can be used.
  • 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.
  • a / C Heterodimerizer AP21967 ligand
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a drug for optionally promoting heterodimerization may be included in the kit.
  • the introduction step of the mRNA switch can be carried out in the same manner as in the aspect A.
  • 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.
  • 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.
  • 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).
  • 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.
  • An mRNA switch set consists of a plurality of different mRNA switches or vectors encoding the mRNA.
  • 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.
  • 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.
  • the kth protein and the (k + 1) th protein are different proteins, k is an integer from 1 to (n-1).
  • the nth mRNA switch included in the n kinds of mRNA switches is defined as follows.
  • 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.
  • 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.
  • 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.
  • the (n + 1) th protein can be designed to act as an input protein for the first mRNA switch. Details will be described later.
  • the first to nth proteins which can be input proteins, are preferably all different.
  • 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.
  • 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.
  • the mRNA switch set may be a switch set for configuring 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.
  • 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.
  • the mRNA switch set may be a switch set for configuring 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.
  • 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.
  • 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.
  • the mRNA switch can be used in RNA imaging methods in combination with the input protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • dSpCas9-VPR is exemplified as the input protein a
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the mRNA switch according to the present invention can be contained in cells to function.
  • 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.
  • an artificial circuit system can be constructed in which an mRNA switch is attached to a desired carrier, dried and supported.
  • 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.
  • the carrier include, but are not limited to, paper, plastic, porous material, fiber and the like.
  • 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.
  • 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.
  • TOYOBO KOD-Plus- Mutagenesis Kit
  • NEB Q5 Site-Directed Mutagenesis Kit
  • the plasmid for transfection into cultured cells was mass-purified using a commercially available Midiprep kit (QIAGEN or Promega).
  • Reference mRNA refers to mRNA that expresses a protein that is translated and encoded without being subject to translational control.
  • 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.
  • 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).
  • [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.
  • Opti-MEM In the split Cas9 test, a 4-fold amount of plasmid expressing each fragment was introduced into the switch.
  • a / C Heterodimerizer (Clontech) was added to the medium so that the final concentration was 0.5 uM at least 1 hour before transfection.
  • the iRFP670 expression plasmid was used as a transfection control in all experiments.
  • 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.
  • a nucleic acid sequence specifically recognized by an input protein
  • 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.
  • CRISPR RNA crRNA
  • sgRNA single guide RNA; chimeric RNA of crRNA and trans-activating crRNA (tracrRNA)
  • a unique crRNA / sgRNA embedded in mRNA which causes translational repression (Fig. 1).
  • 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.
  • the control switch plasmid No aptamer
  • 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.
  • SpCas9 response switch suppresses reporter expression by translation control
  • Many Cas proteins are originally used for genome editing targeting intracellular DNA.
  • 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 expression levels of the reporters were compared (Fig. 4A). As a result, no significant difference in reporter expression was observed under all conditions.
  • 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.
  • 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.
  • AsCas12a AsCpf1
  • AsCpf1 AsCas12a
  • H800A AsCas12a
  • 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).
  • RNA inverter [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.
  • PspCas13b 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.
  • the PspCas13b-crRNA complex is cleaved from the mRNA, resulting in an exposed mRNA at the 5'end.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • each split protein expression plasmid was prepared and co-introduced into 293FT cells with a switch plasmid and a reference plasmid.
  • a switch 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).
  • 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).
  • 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). ..
  • GFP the output gene
  • 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.
  • pHL-gRNA [TRE] -iRFP-RIH The gRNA expression plasmid pHL-gRNA [TRE] -iRFP-RIH was prepared as follows. First, pHL-gRNA [EGFP] -mEF1 ⁇ -mRFP-RIH was prepared from pHL-gRNA [DMD] -mEF1 ⁇ -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] -mEF1 ⁇ -mRFP-RIH to generate pHL-gRNA [EGFP] -mEF1 ⁇ -mRFP-RIH.
  • pHL-gRNA [TRE] -mEF1 ⁇ -mRFP-RIH was prepared from pHL-gRNA [EGFP] -mEF1 ⁇ -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] -mEF1 ⁇ -mRFP-RIH to generate pHL-gRNA [TRE] -mEF1 ⁇ -mRFP-RIH.
  • pHL-gRNA [TRE] -mEF1 ⁇ -iRFP-RIH was prepared from pHL-gRNA [TRE] -mEF1 ⁇ -mRFP-RIH.
  • 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] -mEF1 ⁇ -mRFP-RIH to generate pHL-gRNA [TRE] -mEF1 ⁇ -iRFP-RIH.
  • the primer sets used are shown in Table 11. Target sequences are underlined.
  • 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.
  • 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.
  • the plasmid backbone was first amplified by inverse PCR using pSp_IgRNA_a-CMVmin-Gluc_Sa_gRNA-tagBFP as a template.
  • the amplified plasmid backbone and double-stranded DNA containing MALAT1 triplet, HH ribozyme, SaCas9 gRNA, and HDV ribozyme were ligated using In-Fusion HD Cloning Kit (Clontech).
  • 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).
  • this plasmid was cleaved with SpeI and EcoRI.
  • the double-stranded DNA containing the SpCas9 gRNA binding site was amplified by PCR and cleaved by XbaI and EcoRI.
  • the plasmid backbone and DNA fragment were ligated.
  • 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.
  • 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.
  • the plasmid for transfection into cultured cells was mass-purified using a commercially available Midiprep kit (QIAGEN or Promega).
  • [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).
  • 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.
  • 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.
  • 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.
  • 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 ( ⁇ : The smaller the value, the more AND gate-like behavior. The result of summarizing) is shown.
  • 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
  • Nc is an abbreviation for NcCas9.
  • 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.
  • FIG. 20 is a fluorescence photograph showing the results of the orthogonality test.
  • 26 types of Cas response ON switch mRNA including the newly developed AaCas12b response ON switch mRNA were used.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'objet de la présente invention est de développer un commutateur d'ARNm qui permet la construction d'un circuit de régulation de translation précis et peut être utilisé en tant que module de régulation de transformation ayant des propriétés de versatilité et d'orthogonalité.　L'invention concerne un commutateur d'ARNm comprenant une molécule d'ARNm artificiel qui comprend (i) une séquence d'acide nucléique pouvant être reconnue spécifiquement par une protéine d'entrée comprenant la protéine Cas ou un de ses variants et (ii) une séquence d'acide nucléique codant pour une protéine de sortie, laquelle séquence d'acide nucléique (i) est située sur le côté 5' ou 3' de la séquence d'acide nucléique (ii) et ledites séquences d'acide nucléique (i) et (ii) étant liées l'une à l'autre de manière fonctionnelle.
PCT/JP2020/044905 2019-12-02 2020-12-02 Commutateur d'arnm et procédé de régulation de l'expression d'une protéine l'utilisant WO2021112136A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2021562691A JPWO2021112136A1 (fr) 2019-12-02 2020-12-02

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019218386 2019-12-02
JP2019-218386 2019-12-02

Publications (1)

Publication Number Publication Date
WO2021112136A1 true WO2021112136A1 (fr) 2021-06-10

Family

ID=76221693

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/044905 WO2021112136A1 (fr) 2019-12-02 2020-12-02 Commutateur d'arnm et procédé de régulation de l'expression d'une protéine l'utilisant

Country Status (2)

Country Link
JP (1) JPWO2021112136A1 (fr)
WO (1) WO2021112136A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114075572A (zh) * 2021-11-16 2022-02-22 珠海中科先进技术研究院有限公司 一种与门基因电路及获取该与门基因电路的方法
CN114085834A (zh) * 2021-11-16 2022-02-25 珠海中科先进技术研究院有限公司 一种癌细胞导向电路组及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015525562A (ja) * 2012-07-16 2015-09-07 国立大学法人京都大学 Ｒｎａ−蛋白質相互作用モチーフを利用した蛋白質翻訳量調整システム
WO2018164948A1 (fr) * 2017-03-09 2018-09-13 The Scripps Research Institute Vecteurs comprenant des commutateurs dépendant de cpf1 autodirigés
WO2019027869A1 (fr) * 2017-07-31 2019-02-07 Massachusetts Institute Of Technology Stabilisant de transcript induite par clivage de l'arn et ses utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015525562A (ja) * 2012-07-16 2015-09-07 国立大学法人京都大学 Ｒｎａ−蛋白質相互作用モチーフを利用した蛋白質翻訳量調整システム
WO2018164948A1 (fr) * 2017-03-09 2018-09-13 The Scripps Research Institute Vecteurs comprenant des commutateurs dépendant de cpf1 autodirigés
WO2019027869A1 (fr) * 2017-07-31 2019-02-07 Massachusetts Institute Of Technology Stabilisant de transcript induite par clivage de l'arn et ses utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BORCHARDT, E. K. ET AL.: "Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4", RNA, vol. 21, 2015, pages 1921 - 1930, XP055513753, DOI: 10.1261/rna.051227.115 *
LI, B. ET AL.: "Synthetic Oligonucleotides Inhibit CRISPR-Cpf1-Mediated Genome Editing", CELL REP., vol. 25, 2018, pages 3262 - 3272, XP055759227, DOI: 10.1016/j.celrep.2018.11.079 *
NIHONGAKI, Y. ET AL.: "A split CRISPR-Cpfl platform for inducible genome editing and gene activation", NAT. CHEM. BIOL., vol. 15, September 2019 (2019-09-01), pages 882 - 888, XP036865816, DOI: 10.1038/s41589-019-0338-y *
ZHONG, G. ET AL.: "Cpfl proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells", NAT. CHEM. BIOL., vol. 13, 2017, pages 839 - 841, XP055538474, DOI: 10.1038/nchembio.2410 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114075572A (zh) * 2021-11-16 2022-02-22 珠海中科先进技术研究院有限公司 一种与门基因电路及获取该与门基因电路的方法
CN114085834A (zh) * 2021-11-16 2022-02-25 珠海中科先进技术研究院有限公司 一种癌细胞导向电路组及应用

Also Published As

Publication number Publication date
JPWO2021112136A1 (fr) 2021-06-10

Similar Documents

Publication Publication Date Title
CN116590257B (zh) VI-E型和VI-F型CRISPR-Cas系统及其用途
Newby et al. A genetic tool to track protein aggregates and control prion inheritance
JP6890846B2 (ja) ｍｉＲＮＡの発現を指標として所望の細胞種を判別する方法
US11030531B2 (en) DNA recombinase circuits for logical control of gene expression
WO2021112136A1 (fr) Commutateur d'arnm et procédé de régulation de l'expression d'une protéine l'utilisant
Mattijssen et al. LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly (A) tail length protection
US20100197006A1 (en) Molecular circuits
JP5258874B2 (ja) Ｒｎａ干渉タグ
US20190359972A1 (en) Compositions and Methods for Scarless Genome Editing
JP7084033B2 (ja) ｍｉＲＮＡの発現に応答して蛋白質遺伝子を発現させる方法
US20240209357A1 (en) Dna constructs comprising alternative promoters
JP6877752B2 (ja) 細胞内在性タンパク質の識別方法
JP7318931B2 (ja) 高発現性ｍＲＮＡスイッチ
WO2023100955A1 (fr) Molécule d'arn
WO2024010028A1 (fr) Molécule d'arn circulaire, procédé de régulation de la traduction, système d'activation de la traduction et composition pharmaceutique l'utilisant
Na et al. Synthetic inter-species cooperation of host and virus for targeted genetic evolution
US20190112600A1 (en) Conditional protein translation switches, conditional gene expression systems and uses thereof
Mohl Pathogenic Tau Perturbs Axonogenesis via Loss of Tau Function
Cao Application of synthetic biology and optogenetics to controlling gene expression
Rajakumar Constructing RNA based genetic circuits for the detection of endogenous microRNAs
WO2024015383A1 (fr) Biocapteurs d'hypoxie modifiés et leurs procédés d'utilisation
CN116064597A (zh) 通过自主复制rna实现哺乳动物细胞中的定向进化和达尔文适应
Perli An integrated CRISPR-Cas toolkit for engineering human cells
Nagaraj et al. Design of a genetic differential amplifier
Keatings Maintenance of transcription factor networks in mature neurons

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20895196

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021562691

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20895196

Country of ref document: EP

Kind code of ref document: A1